EPA-6 6 0/2-74-041
JUNE 1974
                       Environmental Protection Technology Series
      Wastewater Use in  the Production
        of  Food and Fiber—Proceedings
                                Office of Research and Development
                                U.S. Environmental Protection Agency
                                Washington, DC 20460

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            RESEARCH  REPORTING SERIES
Research reports of  the  Office  of  Research   and
Monitoring,   Environmental Protection Aqency,  have
been grouped  into five series.  These  five  broad
categories  were established to facilitate further
development   and  application   of   environmental
technology.    Elimination  of traditional grouping
was  consciously  planned  to  foster   technology
transfer   and  a  maximum  interface  in  related
fields.  The  five series are;

   1.  Environmental Health Effects Research
   2.  Environmental Protection Technology
   3.  Ecological Research
   4.  Environmental Monitoring
   5.  Socioeconomic Environmental Studies

This report has been assigned to the ENVIRONMENTAL
PROTECTION    TECHNOLOGY   series.    This    series
describes   research   performed  to  develop   and
demonstrate    instrumentation,    equipment     and
methodology   to  repair  or  prevent environmental
degradation from point and  -non-point  sources  of
pollution.  This work provides the new or improved
technology  required for the control and treatment
of pollution  sources to meet environmental quality
standards.
                   EPA REVIEW NOTICE
This report has "been reviewed by the Office of Research and
Development, EPA, and approved for publication.  Approval does
not signify that the contents necessarily reflect the views
and policies of the Environmental Protection Agency, nor does
mention of trade names or commercial products constitute
endorsement or recommendation for use.

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                                               EPA- 660/2-74-041
                                               June 1974
           WASTEWATER USE IN THE PRODUCTION
            OF FOOD AND FIBER—PROCEEDINGS
            Proceedings of the Conference held at
                 Oklahoma City, Oklahoma
                     March 5-7, 1974
                     Cosponsored by:

         OKLAHOMA STATE DEPARTMENT OF HEALTH
                  Oklahoma City,, Oklahoma

         CALIFORNIA STATE UNIVERSITY, HUMBOLDT
         Humboldt State University Sea Grant Program
                     Arcato, California

    MIDCONTINENTENVIRONMENTAL CENTER ASSOCIATION
                      Tulsa, Oklahoma

        EAST CENTRAL OKLAHOMA STATE UNIVERSITY
     Oklahoma Environmental Information and Media Center
                      Ada, Oklahoma

         U.S.  ENVIRONMENTAL PROTECTION AGENCY
       Robert S.  Kerr Environmental Research Laboratory
                      Ada, Oklahoma

         BUREAU OF SPORT FISHERIES AND  WILDLIFE
              Fish Farming Experimental Station
                    Stuttgart, Arkansas

                 UNIVERSITY OF OKLAHOMA
         College of Health and Allied Health Professions
                  Oklahoma City, Oklahoma
                       Prepared for

          OFFICE OF RESEARCH AND DEVELOPMENT
         U.S. ENVIRONMENTAL PROTECTION AGENCY
                 WASHINGTON, D.C.  20460
For Mto by the Superintendent of Document*, 17.8. Government Printing Office Washington, D.C. 20402 - Price S5.J5

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                               ABSTRACT

        A conference was convened to bring together an interdisciplinary
group of investigators for the purpose of critically reviewing the present
base of scientific knowledge relating to benefits and constraints of using
wastewaters for production of food and fiber.  Those gathered represented
the fields of public health, engineering, agriculture,  aquaculture, and
other related scientific areas. There were 27 papers presented at the
conference covering technical restraints,  aquacultural approaches, agri-
cultural approaches, nontechnical constraints, and new or integrated
experimental systems.  In addition to those papers presented at the con-
ference, nine others have been included to make a total of 36 papers in
this conference Proceedings.

        Papers in the two sections on potential restraints cover topics such
as historical instances of disease transmission, possible transport of micro-
bial pathogens through  the food chain, legal implications, and sociological
reactions.  The aquaculture section deals primarily with experimental studies
including such diverse  approaches as  culture ofdaphnia, salmon smolts, and
water hyacinth.  The agriculture section emphasizes the use of waste water
for crop production and the papers presented include case histories for
long-term operating systems, as well as data from experimental studies.
                                    n

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TABLE OF CONTENTS
                                                                 Page
Abstract

Foreword

Sponsors

List of Speakers
11

vii

ix

x
PARTI   INTRODUCTORY SESSION

          Review of American Public Works Association Study of
          Facilities Utilizing Land Application of Wastewater
                  Richard H. Sullivan

          Wastewater Utilization in Integrated Aquaculture and
          Agriculture Systems
                  B. Hepher and C. L, Schroeder
PART 2   TECHNICA L RES TRAI NTS

          Diseases Transmitted by Foods Contaminated by Wastewater    16
                 Frank L. Bryan

          The Evaluation of Microbial Pathogens in Sewage and
          Sewage-Crown Fish                                        46
                 R. LeRoy Carptenter, Harold K. Malone, Ara F.
                 Roy, Aaron L. Mitchum, Herbert E.  Beauchamp
                 and Mark S. Coleman

          Morbidity Risk Factors from Spray Irrigation with Treated
          Wastewaters                                              56
                 Geoffrey B. Stanford and Rafael Tuburan

          Permissible Levels of Heavy Metals in Secondary Effluent
          for Use in a Combined Sewage Treatment-Marine
          Aquaculture System I.  Monitoring During Pilot Operation      65
                 W.  B. KerfootandS. A. Jacobs

          Permissible Levels of Heavy Metals in Secondary Effluent
          for Use in a Combined Sewage Treatment-Marine
          Aquaculture System II.  Development of Guidelines by
          Method of Additions                                        79
                  W.  B. KerfootandG. A. Redmann
                                   in

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Table of Contents  (continued)
          Calculated yield of Sewage Lagoon Biomass, A Plan for
          Production, and Some of the Problems Inherent in Using
          Biomass or Lagoon Water for Production of Food and Fiber
                  Karl Schurr and J,  M.  Colombek
Page


102
PART 3   A QUA CUL TURE
          Analysis of Sewage Lagoon Biomass Water Soluble Vitamins
          by Microbiological Techniques
                  Nancy M.  Bakaitis

          Feed and Fiber from Effluent—Grown Water Hyacinth        116
                  L. O.  Bagnall, T.deS.  Furman, J.  F.  Hentges, Jr.,
                  W. J.  Nolan and R. L. Shirley

          The Availability of Daphnia for Water Quality Improvement
          and as an Animal Food Source                             742
                  Ray Dinges

          Report on Pilot Aquaculture System Using Domestic
          Wastewaters  for Rearing Pacific Salmon Smolts              162
                  George H. Allen and Larry Dennis

          Aquaculture  as a Means to Achieve Effluent Standards        199
                  Mark S. Coleman,  James P. Henderson, H.  G.
                  Chichester and R.  LeRoy Carpenter
PART H   AGRICULTURE
          An Experiment in the Eutrophication of Terrestrial
          Ecosystems with Sewage:  Evidence of Nitrification in
          a Late Successionaf Forest
                  G. M.  Woodwell,  J. Bollard,  J. Clinton,
                  M. Small and E. V. Pecan

          Irrigation with Waste water at Bakers field, California
                  Ronald W. Crites

          Nutritive Value of Aerobically Treated Livestock and
          Municipal Wastes
                  D. L. Day and B. G. Harmon

          Grass Filtration for Final  Treatment of Wastewater
                  R. M.  Butler, J.  V. Husted and J. N. Walter
215
229
240
256
                                      IV

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Table of Contents  (continued)
          Use of Cattle Feed/of Runoff in Crop Production
                 C.  IV.  Wai ting ford, L. S. Murphy, W. L.
                 Powers, H.  L. Manges,  and L. A. Schmid

          Irrigation of Trees and Crops with Sewage Stabilization
          Pond Effluent in Southern Michigan
                 Jeffrey C. Sutherland, John H. Cooley,
                 Daniel C. Neary and Dean H. Urie

          Uses of Power Plant Discharge Water in Greenhouse
          Production
                 B.  J. Bond, W. K. Furlong, L. D. King,
                 C.  E. Madewell and J. B. Martin
Page

273
295
PART 5   NONTECHNICAL RESTRAINTS

          Legal Constraints on the Use of Wastewater for Food and
          Fiber                                                    330
                  William R.  Walker and William E. Cox

          Social,  Political, Regulatory and Marketing Problems of
          Marine  Waste-Food  Recycling Systems                      344
                  John E. Huguenin and Judy T. Kildow

          Recycling for a Purpose—But for What Purpose? A
          Sociologist's View                                        357
                  Lewis H. Irving
PART 6   NEW OR INTEGRA TED SYSTEMS

          The Michigan State University Water Quality
          Management Program
                  T.  G. Bahr, R. C. Ball and H. A. Tanner

          Experiences with a Marine Aquaculture-Tertiary
          Sewage Treatment Complex
                  John E. Huguenin and John H. Ryther

          Polycultural Wastewater Reclamation at California
          Polytechnic State University—An Academic
          Instructional System
                  Richard J. Krejsa
362
377
387
                                      v

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Table of Contents (continued)
PART 7   ADDITIONAL PAPERS CONTRIBUTED FOR PUBLICATION

          Coliform and Phytoplankton Studies in a Brackish Water
          Aquacu/ture Pond Fertilized with Domestic Wastewater
                 Robert F. Donnelly and Tommy T. Inouye

          Mineral Quality of Fish Pond Effluent Related to Soil
          Properties and Crop Production
                 L. H. Hileman

          The Dialectics of a Proposal on Biological Control of
          Eutrophication in Sewage Lagoons
                 S. V. Lin

          The Harvesting of Algae as a Food Source from Wastewater
          Using Natural and Induced Flocculation Techniques
                 Joseph L. Pavoni, Steven W. Keiber and
                 Gary T. Boblitt

          Critical Variables in Food-Item Population Dynamics
          in a Waste-Water Aquaculture System
                 Joseph E. Powers

          The Feasibility ofPenaeid Shrimp Culture in Brackish
          Ponds Receiving Treated Sewage Effluent
                 William L. Rickards

          Standing Crops ofBenthic Fauna in Marine Aquaculture
          Ponds Using Reclaimed  Water
                 Thomas R. Sharp

          Controlled Eutrophication: Sewage Treatment and Food
          Production
                 J. Glenn Songer, N. M. Trieff, Rodney F.
                 Smith and Dov  Crajcer

          Principles of Sewage Treatment Through Utilization in
          Fish Ponds
                 Erno Donas zy
                                                                  Page
404
472
477
435
497
504
573
529
546
PART 8   APPENDICES

          A. Program Committees

          B. List of Participants
557

559
                                   VI

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                              FOREWORD
The topic of using domestic wastewater in the production of food and
fiber is of great importance to three distinctly different groups in
our society:

    (1)  Those persons living in arid or semiarid regions where econom-
         ic growth, industry and agriculture hang on the thin thread
         of dwindling ground water supply.  Reclamation and reuse of
         wastewater, even if only for irrigation of agriculture crops,
         would not only free a large percentage of treated water for
         municipal needs but would also minimize contamination of our
         primary water sources and provide cost benefits compared to
         the cost of developing new sources of water.

    (2)  Those persons living in developing countries where protein is
         produced in insufficient quantities to sustain an increasing
         population with new demands for better nutrition.  The cost
         and the shortage of fertilizer have severely damaged the
         "green revolution."  Nitrogen and phosphorus of wastewater,
         all too often rushed out of sight like the proverbial step-
         children, are more and more looked upon as agricultural assets
         rather than municipal liabilities.

    (3)  Those persons who fall into the category of concerned taxpay-
         ers and those municipal officials who are charged with meeting
         federal and state water quality standards with precious tax
         dollars.  The beneficial use of domestic wastewater for eco-
         nomic gain is no longer a laboratory curiosity or the practice
         of poverty stricken foreigners.  There are hundreds of proj-
         ects throughout the United States and thousands  in Europe and
         Asia where wastewater in various degrees of treatment is used
         for irrigation of forage crops, hardwood and Christmas trees,
         fish and shrimp culture, powerplant cooling water, factory
         process water and even recreation.  The removal  of nitrogen
         and phosphorus by trees, crops, or fishes not only produces a
         saleable product but greatly reduces the size and operational
         budget of mechanical and chemical treatment plants.

The conference and the proceedings that follow have been  the result of
much  thought, planning and effort on the part of persons  of many diff-
erent professional, governmental, and business disciplines.  There were
173 participants from  27 states, Canada, Virgin  Islands,  and Israel.
                                      VII

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Many facets of the use of wastewater in the production of food and
fiber were discussed in a scholarly, critical manner.  The technical
and public health constraints were given extra attention.

The success of the conference was largely due to the efforts of Dr.
George Allen and his excellent program committee; Charles Newton and
his local arrangements committee; Lovena Eaker, my long-suffering sec-
retary; and especially Toni Morrow who wore not only the hat of pub-
licity chairman but that of details clerk, timekeeper, memory bank,
bookkeeper, complaint solver, all with a constant smile.  To these and
Dr. B. Hepher, who keynoted the conference with many constructive com-
ments, I extend my heartfelt gratitude.
                                   R. LeRoy Carpenter, M.D., M.P.H.
                                   Commissioner of Health
                                   State of Oklahoma
                                    VIII

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SPONSORS

Oklahoma State Department of Health
Northeast Tenth and Stonewall
Oklahoma City, Oklahoma  73105

Humboldt State University Sea Grant Program
Office of Academic Affairs
California State University, Humboldt
Arcata, California  95521

Midcontinent Environmental Center Association
Box 201
Tulsa, Oklahoma  74102

Oklahoma Environmental Information and Media Center
East Central State College
Ada, Oklahoma  74820

Robert S. Kerr Environmental Research Laboratory
Environmental Protection Agency
Box 1198
Ada, Oklahoma  74820

Fish Farming Experimental Station
Bureau of Sport Fisheries and Wildlife
Box 860
Stuttgart, Arkansas  72160

University of Oklahoma College of Health and
Allied Health Professions
University Health Sciences Center
P.O. Box 26901
Oklahoma City, Oklahoma  73190
                                  IX

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SPEAKERS
George H. Allen, Ph.D..
School of Natural Resources
Calif. State Univ., Humboldt
Arcata, California  95521

Larry 0. Bagnail
Agricultural Engineer
University of Florida
Frazier Rogers Hall
Gainesville, Florida  32611

Thomas G. Bahr, Director
Institute of Water Research
Michigan State University
East  Lansing, Michigan  48824

Nancy Bakaitis
Asst.  Prof,  of  Chemistry
Natural  Science Division
Findlay  College
Findlay, Ohio   45840

Frank L. Bryan, Ph.D.
Foodborne  Disease Activity
Health Agencies Branch
 Center for Disease Control
 Atlanta, Georgia  30333

 R. LeRoy Carpenter, M.D., M.P.H.
 State Commissioner of Health
 State Department of Health
 Northeast Tenth and Stonewall
 Oklahoma City, Oklahoma  73105

 James Clinton
 Project Biologist
 Brookhaven Natl. Laboratory
 Building 318
 Upton, New York  11973

 Mark S. Coletnan, Director
 Water Quality Monitoring  and
    Research Division
 Water Quality  Services
 Oklahoma  State Department of Health
 Northeast  Tenth  and  Stonewall
 Oklahoma  City, Oklahoma   73105
Ronald W. Crites
Metcalf & Eddy, Inc.
1029 Corporation Way
Palo Alto, California 94303

Donald L. Day
College of Agriculture
  and Engineering
University of  Illinois
Urbana, Illinois  61801

W. R. Dinges,  R.S.
Field Activities
Div. of Wastewater  Technology
  & Surveillance
Texas State  Dept. of Health
 1100 West 49th
Austin, Texas   78756

Dr. B. Hepher, Director
Agricultural Research
Organization
 Fish  & Aquaculture  Research
 Station
 Dor,  Israel

 E.  W.  Houser
 Manager-Engineer
 Santee County Water District
 P.O.  Box 70
 Santee,  California  92071

 John E.  Huguenin
 Research Associate
 Woods Hole Oceanographic Inst.
 Woods Hole, Mass.  02543

 Lewis H. Irving, Ph.D.
 Department of Sociology
 Central State University
 Edmond, Oklahoma   73034

 William B. Kerfoot
 Woods Hole Oceanographic Inst,
 Woods Hole, Mass.  02543

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SPEAKERS  (Continued)

Larry D. King
Agriculture Resource Dev. Branch
Tennessee Valley Authority
Muscle Shoals, Alabama 35660

Dr. Richard J. Krejsa
Supervisor Fifth District
San Luis Obispo County
Room 220 - Courthouse Annex
San Luis Obispo, Calif. 93401

Harry Manges
Associate Professor
Agricultural Engineering
Kansas State University
Manhattan, Kansas 66506

Dr."Karl Schurr
Biology Department
Bowling Green State Univ.
Bowling Green, Ohio 43403

John R. Sheaffer, Ph.D.
President
Bauer, Sheaffer & Lear, Inc.
20 North Wacker Drive, Suite 2510
Chicago, Illinois 60606

Dr. Geoffrey Stanford
School of Public Health
Holcombe Boulevard
Houston, Texas  77025

Richard H. Sullivan
Asst. Executive Director
American Public Works Assn.
1313 East 60th Street
Chicago, Illinois  60637

Jeffrey C. Sutherland, Ph.D.
Williams & Works
250 Michigan Street, N.E.
Grand Rapids, Michigan 49503
Rafael Tuburan
School of Public Health
Holcombe Boulevard
Houston, Texas 77025

William R. Walker
Virginia Polytechnic Inst.
225 Norris Hall
Virginia Water Research Center
Blackburg, Virginia 24061

Jack N. Walter
Dept. of Agricultural
Engineering
Penn State University
University Park, Pa. 16802
                                   XI

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   PARTI   INTRODUCTORY SESSION
        CONFERENCE CHAIRMAN

   R. LeROY CARPENTER, M.D., M.P.H.
       COMMISSIONER OF HEALTH
OKLAHOMA STATE DEPARTMENT OF HEALTH

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         REVIEW OF AMERICAN PUBLIC WORKS ASSOCIATION
STUDY OF FACILITIES UTILIZING LAND APPLICATION OF WASTEWATER
                         Richard H. Sullivan
                      Assistant Executive Director
                   American Public Works Association
                     Presented March 6, 1974 at a
               Conference on the Use of Wastewater in the
                     Production of Food and Fiber
                      Oklahoma City, Oklahoma

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               REVIEW OF AMERICAN PUBLIC WORKS ASSOCIATION
     STUDY OF FACILITIES UTILIZING LAND APPLICATION OF WASTEWATER

                    Richard H. Sullivan, Assistant Executive Director
                           American Public Works Association

    The American Public Works Association, in 1972, conducted an on-site field survey or
approximately  100 facilities in all climatic zones  where land application of community of
industrial wastewaters are  being applied  to the  land,  as contrasted  to the  conventional
method of treating such wastes and discharging them into receiving waters. The project was
sponsored  by the U.S. Environmental Protection Agency under  Contract No. 68-01-0732.
The report, entitled Survey of Facilities Using Land Application  ofWastewater, is available
from the Government Printing Office as EPA-430/9-73-006 at a price of S6.80.
    Additional data were gathered from many existing land application facilities across the
country by means  of a mail survey addressed to  responsible officials. Another survey was
carried out to  ascertain  the nature and extent of state health and water pollution control
regulations governing the use and control of land application systems. To augment informa-
tion on U.S. practices, a survey  was made of experiences gained  in many foreign countries.
In addition, an extensive  bibliography was compiled of literature on all pertinent phases of
land  application practices. The  bibliography has  been combined with that of three other
studies and will be published separately. It will have abstracts of almost  800 articles.
    The facilities surveyed  were relatively large with long established operations in order
that as much operating experiences as possible could be obtained. The  surveyed application
facilities utilizing community wastes were predominantly located in the western and south-
western portions of the United States, while industrial facilities were generally sited in the
northeastern section because this is where the majority of such installations are  in service.
    Table 1, Summary of Facilities Using Land Application of Wastewater, lists  pertinent
information concerning  the sites  for which information was obtained for the states of
Kansas, New Mexico,  Oklahoma and  Texas. Two sites were found in  Kansas, nine in New
Mexico, three in Oklahoma, and 50 in Texas. Texas and California were the two states with
the most facilities. However,  in all  of the  southwest,  only two industrial facilities  were
•found and both were in Texas.
    Land application of effluent involves many means of accomplishing a variety of objec-
tives. Among the more important objectives, the following were found:
    1.  to provide supplemental  irrigation water;
    2.  to give economical  alternative solutions  for treating wastes and discharging them
        into receiving waters, without causing degradation of rivers, lakes and coastal waters
        and;
    3.  to overcome unavailability of suitable receiving waters and eliminate excessive  costs
        of long outfall lines to reach  suitable points of disposal into surface water sources.
    Among the major means of accomplishing land application of  wastewaters are:
    1.  irrigation of land areas by spraying, with high-pressure or low-pressure devices, of
        either stationary or movable types of distribution systems;
    2.  ridge and furrow irrigation systems;
    3.  use of overland flow or flooding methods; and
    4.  use of infiltration  lagoon or evaporation ponds.
    Although facilities of all types were surveyed, the report is  primarily concerned with
irrigation-type facilities for supplying supplemental water to crop areas, forest areas and
unharvested soil cover acreages. The other types are not as widely used, inasmuch as many
factors such as climate or soil conditions have an adverse impact on  these means of land
application.

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Table 1. Summary of Facilities Using Land Application of Wastewater
State
Kansas
Scott City
Sublette
New Mexico
Almagordo
Clovis
Raton
Roswell
Santa Fe
Lovington
Los Alamos Co.
Raton
Silver City
Oklahoma
Duncan
Boise City
Hollis
Texas
Abilene
Dumas
Kings ville
La Mesa
Midland

Monohans
San Angelo

Uvalde
Coleman
Comanche
Cotulla
Dalhart
Denver City
Elsa
Goldthwaite
Idalou
Morton
Munday
Rails
Raymond ville
San Saba
Seagraves
Van Horn
Winters
Campbell Soup,
Paris
Anson
Azle
Crane
Crosbyton
Eldorado
Freer
Friona
Armour Food,
Hereford
Hondo
McLean
Information

Questionnaire
Questionnaire

Visited
Visited
Visited
Visited
Visited
Questionnaire
Verified
Verified
Verified

Visited
Questionnaire
Verified

Visited
Visited
Visited
Visited
Visited

Visited
Visited

Visited
Questionnaire
Questionnaire
Questionnaire
Questionnaire
Questionnaire
Questionnaire
Questionnaire
Questionnaire
Questionnaire
Questionnaire
Questionnaire
Questionnaire
Questionnaire
Questionnaire
Questionnaire
Questionnaire

Questionnaire
Verified
Verified
Verified
Verified
Verified
Verified
Verified

Verified
Verified
Verified
Population

4,325
1,332

25,000
28,000
2,300
40,000
45,000
10,000




20,000
1,980


100,000
9,770
30,000
1 1 ,400
62,000

8,000
64,000

9,000
5,608
5,000
3,900
5,700
4,200
5,000
1,700
1,800
3,760
1,700
2,100
7,986
2,555
2,500
3,000
2,907

Industry








Industry


•TM**^*
Flow - mgd

0.432
0.13

2.5
3.5
0.5
2-3
5.3
0.5
0.36
0.4
0.5

2.5
0.001
0.3

9.0
1.0
3.0
0.6
4.3

0.8
5.0

0.9
0.3
0.4
0.165
0.64
0.15
0.18
0.2



0.1
0.87
0.126
0.2
0.15
0.154

3.1
0.4
0.25
0.2
0.125
1.
0.25
<1

1.15
0.4
0.1
Crops Area

Rice


Alfalfa, corn, oats, sorghum
Irrigated
Acres
25
40

260
Milo, alfalfa, corn, millet, wheat 1 ,193
Alfalfa
Alfalfa, barley, corn, cotton
Alfalfa, apples





Wheat, Bermuda grass



Cotton, maize, Bermuda grass
Wheat, maize
Maize

Bermuda grass, alfalfa, milo,
cotton

Barley, milo, rye, oats, fescue,
alfalfa, Bermuda grass
Maize, oats



Hay
Cotton


Feed, cotton
Cotton




Cotton

Grain













200
770
740
160




180
40


2,019
585
606
192
1,000

40
740

150
106
12
12

200
4.5
100
10
60
20
160

80
160
10
10

500












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    Table 1 (Continued)
           Odessa         Verified                 1.075
           Ozona         Verified                 0.3
           Petersburg      Verified                 0.2
           Quitague       Verified                 0.06
           Rotan         Verified                 0.11
           Sabinal        Verified
           Santa Ana      Verified
           Seminole       Verified                 0.9
           Slaton         Verified                 0.37
           Sonora        Verified                 0.3
           Stanton        Verified                12
           Stratford       Verified                 0.01
           Sundown       Verified                 0.16
           Sweet water     Verified                 0.75

                   Summary of Land Application Sites (Non-Military)
                         For Which Information Was Obtained

                           Total          Total           Total
                          Visited     Questionnaires      Verified        Total
       United States        89             122             136           347
       Kansas                —               1               12
       New Mexico           51               39
       Oklahoma              11               13
       Texas                  8              17              25            50

    Irrigation-type facilities were found to be broadly used under a wide variety of climate
and soil conditions, with various  degrees of prior treatment of the applied wastewater and
various types of ground cover utilized.
    Each method of application has inherent advantages and disadvantages which  must be
evaluated for their feasibility and effectiveness.
    The land application of waste waters has been practiced extensively in many parts of the
world for many years with varying degrees of control and success.
degrees of control and success.
    As knowledge of wastewater treatment processes improved, and the possibility of con-
fining  in a relatively small area the entire process needed  to obtain a "treated" effluent for
disposal into surface water sources, land application in the united States was relegated in
most states to being an undesirable and unacceptable process.
    New concerns about preserving the quality and reuse of the  nation's water resources
have resulted in a reawakening  of interest in  land application as a viable alternative to
conventional wastewater treatment and disposal into receiving waters. Increasing volumes of
sewage and industrial wastes, growing complexity of such raw wastes, and mounting needs
for water to serve growing urban  and industrial processing needs, have created doubts about
the ability of receiving waters to assimilate effluents which do not meet high-quality  stan-
dards.  In addition, progressive evidences of eutrophication of non-flowing  receiving waters
have focused attention on the need to eliminate the presence of nutrients in wastewater
effluents. Further, the presence of toxic trace elements in  effluents is considered a threat to
the safety  of waters receiving all  but highly purified waste discharges.  Thus, advanced
treatment methods have been developed and utilized to avoid discharge of such objection-
able components.
    Inasmuch as land application appears to offer comparable or superior degrees  of treat-
ment by augmenting waste treatment with the  "natural" purification offered by soil con-

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tact, land application is again being considered as one of the acceptable means of achieving
full treatment of waste waters.
    However, a most important factor of the current land  application concept is that it
must be limited to the  use of treated wastes. Generally, effluents are being conventionally
treated to almost meet  secondary treatment quality criteria. In at least three observed
facilities, applied effluents have received tertiary treatment, to the point where the effluent
would fully meet the  generally prescribed, as well  as proposed, criteria for discharge to
receiving waters.  Thus, land application is being used to give a degree of advanced waste
treatment, including high degrees  of nutrient and bacterial removal. In this context, land
application  can be viewed as an alternative to physical-chemical processes  and other
methods of ultra-treatment which are designed  to achieve a "pure" effluent.
    Economics of construction cost, operating costs, energy  requirements, and efficiencies
of performance of land application systems must be balanced with the ability to acquire the
right to apply wastewater upon the required land areas. The cost of advanced waste treat-
ment by conventional means must  be weighed in the light of the cost and complexities of
land application systems.
    Two informative reports were published  on the subject  of land application in 1972.
Green  Lands -  Clean  Streams, a report  by Temple University Center for  the Study of
Federalism, is a frankly written advocacy of the land application of wastewaters and sludges.
Wastewater Management by Disposal on the Land,  by the  U.S. Corps of Engineers,  is a
thorough review of the physical, chemical  and  biological interactions involved in  land appli-
cation.
    The firm of Metcalf and Eddy, Engineers,  has prepared a companion report for USEPA,
concerned with engineering considerations of land application systems.  These three reports,
together with the report on the study conducted by APWA, should be considered in evaluat-
ing land application systems,  because they deal with somewhat different aspects  of the
common problem. The APWA study has made no  special effort to examine the specific
aspects covered in detail in the other reports. Rather, it is concerned with reporting upon
the  policies, practices  and performances of a representative group of the relatively larger
systems within the United States;  policies, or  lack of policies, of state  regulatory agencies;
and the experience with land application in some foreign installations.
     Systems which were under construction, such as Muskegon County, Michigan, and
several major domestic and industrial systems which were intimately known to Metcalf and
Eddy  project  personnel were  not  investigated for this report. However, Metcalf and Eddy
has supplied copies of their field interviews at such sites to APWA for evaluation.
     The following highlights from  the field surveys are presented in order that a composite
picture of observed facilities might  be obtained:
     1.  Communities  generally use their land application system on  a  continuous basis.
        Food processing  plants, the predominant industrial users of the  system, generally
        practice discharge to land systems for three to eight months per year.
     2.  Ground cover utilized for municipal  systems is divided between grass and crops.
        Industries generally use grass cover.
    3.  Land application systems  are generally used on a daily basis, seven days per week.
    4.  Application rates for crop irrigation  are very low in terms of inches of water per
        week. Two inches or less was commonly used. (Note: Two inches per week equals
        54,300 gallons per acre per week.)
    5.  Many types of soils were used; although sand, loam and silt were the most common
        classification given. Two systems using application  over many feet of sand were
        applying up to eight inches per week, and one system on clay was  applying only 0.1
        inch per day.
    6.  Most operating agencies, municipal and  industrial, are planning to either expand or
        continue their land application installations. The few examples  of systems which

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       had been abandoned were due to either the desire to make a higher use of the land,
       or because  of reported overloading and poor operation of the land application
       facilities.
   7.  Industries surveyed generally treat their total waste flow by land application. Prac-
       tices of municipalities varied from less than 25 percent to all waste waters produced.
   8.  Treatment greater than primary is generally provided by municipalities prior to land
       application, often times accompanied by lagooning. Industrial systems often treated
       their process wastes by screening only.
   9.  Spray  irrigation is the most frequently used (57 facilities) method of application,
       although most municipalities use more than one method. Ridge and furrow irriga-
       tion is used at 23 facilities and flooding irrigation by 34 systems. Industry generally
       used spray irrigation.
    10. Land use zoning  for land  application sites  is predominantly classified as  farming,
       with some residential zoning in contiguous areas.
    11. Wastewater generally is transported to  the application  site by pressure lines, al-
       though a number of municipalities are able to utilize ditches  or gravity flow pipe
       lines.
    12. Many  community land application facilities have  been in use for several years -
       more than half for over 15 years. Industrial systems are relatively more recent.
    13. Renovated  wastewater  is seldom collected by under-drains; rather, evaporation,
       plant transpiration, and groundwater recharge take  up the flow.
    14. Land application facilities generally do not make appreciable efforts to  preclude
       public access.  Residences are frequently located adjacent to  the  land application
       sites. No special  effort  is made to .seclude land application areas from recreational
       facilities and from those who use these leisure sites.
    15. Monitoring of groundwater quality, soil uptake of contaminants, crop uptake of
       wastewater components, and surface water impacts is  not carried out with any
       consistency.
    Among the most "advanced" facilities which we reviewed were the Tallahassee system
and the systems at Colorado Springs,  Colorado,  and the University of Pennsylvania at State
College.
    The largest agriculture venture was found  to be at Tula Hidalgo where the wastes of
Mexico City, D.F., are applied to the land to supplement the natural irrigation waters of the
area. Some 115,620 acres are irrigated and in 1971-72, 1,624,424 U.S. tons of crops were
grown. Table 2, Summary of Agriculture production, 1971-72, Tula Hidalgo, lists  the 31
major crops which are grown.
    Information which was received on crop research conducted at Debrecen, Hungary, is
also included, in our report. A comparison in yields obtained with the use of treated waste-
water as contrasted with normal irrigation practices is given in Table 3, Comparison of Crop
Yield, Hungary.
    A major concern has also been the boron concentration of wastewater and the relative
tolerance of plants to boron. Table 4, Units of Boron in  Irrigation Waters for Agriculture
Products with Different  Grades of Tolerance, was developed by Mexican  Agriculture offi-
cials.  The data contained in the report allows ready identification of where  a wide variety of
crops have been raised and an indication as to the physical conditions which exist at the site.
    In summary, land application of treated municipal effluent and industrial wastes should
be a viable alternative to conventional disposal or advanced tertiary treatment techniques in
many areas of the country.  Full evaluation of application should be made as planning of
new or upgraded wastewater facilities is undertaken.

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                 Table 2.  Summary of Agriculture Production
                            1971-72, Tula Hidalgo
    Crop
 Alfalfa Verde
 A jo
 Arvcjon
 Avcna Verde
 Calabacita
 Cebada Grano
 Ccbada Pago
 Cebolhi
 Cila/itro (seniilla)
 Co!
 Chichnro
 Chiles Verde
 Florcs
 Fiijol Grano
 Fiijol Ejotc
 Espinaca
 Frutalcs
 Givasol
 Haba
 Jitoniate
 Lechuga
 Mai?, Grano
 Maiz Rastrojo
 Maiz Verde
 Nabo Forraje
 Melon
 Pepino
Pradera
Tomatc
Trigo Grano
Sandia
    Crop
 Alfalfa
 Garlic

 Green Oals
 Squash (small)
 Barley Grain
 Barley (forage)
 Onioi)
 Parsley Seed
 Cabbage
 Peas
 Green Hot Peppers
 Flowers
 Navy Beans
 Am. String Beans
 Spinach
 Fruit Trees
 Sunflower
 Lima Beans
 Am, Tomato
 Lettuce
 Corn (kernels)
 Corn (forage)
 Corn (sweet)
 Forage Turnips
 Melon
 Cucumber
 Meadow Grass
Tomato
Wheat Grain
Watermellon
 Hectares
12,396.40
    94.50
    12.89
 2,998.75
  674.33
 1,865.43

    23.79
     3.53
    27.95
     1.00
  768.80
    10.41
 Metric Tons
1,181,376.920
     258.456
       20.820
   54,426.714
    7,282.764
    3,645.410
    4,059.874
     168.909
        4.589
     501.843
        7.900
    8,231.350
1,259.02
58.30
0.82
25.08
37.19
95.84
1,554.65
74.47
17,053.60

101.20
112.37
1.00
34.74
12.80
216.90
7,293.79
0.40
46,809.95
Acres
115,620.65
1,563.870
151.580
9.020
213.180
230.578
1,990.070
49,437.870
1,457.764
70,260.525
65,179.023
7,084.000
1,011.330
7.100
166.752
2,080.000
2,051.706
13,865.494
3.960
1,476,749.37)
U.S. Tons
1,624,424.30
Note:    1 metric ton =1.1 (U.S.) tons
        1 (U.S.) ton = 0.907 metric tons
        \ hectare    =2.47 acres
Note:    The crop hectares listed arc more than the hectares of land available since a second
        Crop in some instances lias been produced on the same land.

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                    Table 3. Comparison of Crop Yield, Hungary



Sugar beets
Dry grain maize
Fodder maize
Alfalfa
Sunflower seed
Industrial potatoes
Debrecen
Station
(Wastewater)
363.24 q/ha
58.5 q/ha
346. 19 q/ha
99.25 q/ha
14.87 q/ha
11 9. 8 q/ha
Gyozelen
Farm
(Control)
365.0 q/ha
22.49 q/ha
2 17/2 5 q/ha
46/58 q/ha
4. 17 q/ha
59.3 q/ha
      Table 4. Units of Boron in Irrigation Waters for Agriculture
               Products with Different Grades of Tolerance
       Tolerant
4 ppm
 Asparagus
 Palm Trees (Phoenix
  Canariensis)
 Date (Phoenix Tytiferia)

 Sugar Beets
 Beet
 Bctabel
 Alfalfa
 Giadiota
 Bean
 Onion
 White Radish
 Cabbage
 Lettuce
 Carrot
   Semi-tolerant
2 ppm
 Sunflower
 Potato
 Cotton
 Tomato
 Sweet pea
 Radish

 Olive
 Barley
 Wheat
 Corn
 Oats
 Calabash
 Sweet Potato
  Lima or Kidney Beans
 2 ppm
 1 ppm
     Sensitive
1 ppm
 Mexican Oak
 Black Oak, Persian
  or English
 Tuberous Sunflower
 White Beans
 American Elm
 Plum
 Pear
 Apple
 Grape
  Fig
  Nispero
  Cherry
  Peach
  Apricot
  Blackberry
  Orange
  Avocado
  Grapefruit
  Lemon
 0.3 ppm
 Source:  Analysis of the Black Waters of the Cuenca of the  Valley of Mexico and tin-
         Region  of lil  Mczquital,  Hidalgo-Bulletin  2 Hydraulic Commission of the
         Cuenca of the Valley of Mexico, Mexico, D.I-'., March 1965
                                        8

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                      WASTEWATER UTILIZATION
                               IN
            INTEGRATED AQUACULTURE AND AGRICULTURE SYSTEMS

                               by

                   B. Hepher and G. L. Schroeder*

INTRODUCTION

Israel approaches today a hundred percent use of its water resources.
Out of an estimated potential 2.0 X 10 J m3 of water, 1.7 X 109 m3 are
being already used.  In these conditions, it is obvious that recycling
of wastewater becomes mandatory.  The problem, while extreme in Is-
rael, is experienced in many countries.

A semi-arid country, such as Israel, has the highest quantitative de-
mand for irrigation water in agriculture.  On the other hand, the
qualitative criteria for this water is low.  These two considerations
make agriculture the prime potential sector for the reuse of waste-
water.  However, the following limitations restrict this reuse and
lower its efficiency:
a)  Irrigation is restricted to the dry season, the summer.  In win-
ter, there is little use for the wastewater in irrigation.  According
to a recent study1, about 5.2 X 10s m3 of potentially useful waste-
water are produced in Israel per day.  Of this, in the peak irrigation
season 1.7 X 10* m3, i.e., about 32% are used.  However, when calcu-
lating on a yearly basis, out of 1.9 X 10** m3 wastewater produced,
only 3.7 X 107, or less than 20% are used.  The difference is produced
mainly in the winter and disposed of, often in a manner that produces
ecological disruption.
b)  Irrigation with treated waste effluent is limited by sanitary con-
siderations.  Some crops cannot be irrigated by this water because of
its residual pathogenic organisms.
c)  High salt concentrations make some effluents not suitable for ir-
rigation.  The chloride concentration in Haifa sewage, for example,
is 480 mg/1 (Miron2).  This limits the agricultural applications to
low volumes and a small number of crops.
d)  Areas exist where the agricultural demands for water are less
than the volumes of treated waste effluent available.

It seems to us that the solution to handling all of these problems en-
countered in profitably using waste effluent lies in a system which
integrates waste treatment with aquaculture and agriculture.  The re-
sult is a flow of waste from treatment plant through fish ponds to
irrigated fields.

*Fish and Aquaculture Research Station, Dor,
D. N. Hof HaCarmel, Israel

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BENEFIT OF FISH PONDS IN WASTEWATER UTILIZATION

Fish culture can accept wider variations in environmental conditions
than can agriculture.  Fresh water fish can adapt to the high salini-
ties often encountered in waste effluent.  The growth of carp, for
example, is not inhibited even at salt concentrations as high as
2000 ppm chloride.  Tilapia and trout can grow in sea water.  The per-
missible salinity in irrigation water is considered to be 400 ppm
chloride, in Israel.  Soirquality in fish culture is also less of a
limiting factor than it is in agriculture.  In Israel, ponds are often
constructed on swampy soil and on sand.  Water loss by seepage from
sand ponds decreases rapidly because of clogging of soil interstices
by organic matter.  The use of wastewater in fish ponds can come in
addition to, or in place of, agricultural use.

It is  clear that only impoundment and storage of wastewater effluents
can be the answer to the seasonal variations and daily fluctuations in
the demand for irrigation water.  This impoundment is usually expen-
sive.   However, utilizing the impoundments for fish culture cannot
only justify the holding expense, but also produce a protein rich food
for human consumption bringing, an overall profit.  It is interesting
that the new methods of superintensive aquaculture being developed in
Israel are directly applicable to a system integrating usilization of
waste  effluent and aquaculture.  This approach basically combines
densely stocked ponds with the introduction of air.  The aeration acts
as a safeguard against deoxygenation and fish kill.  This aeration
also can satisfy the uptake of oxygen by the decomposition of the ad-
ded organic matter.  At the same time this organic matter acts as the
basic  link in the chain of natural foods on which the fish can graze.

The yield of a fish pond depends on the stocking rate of the pond
(number of fish per unit area) and the pond management, both of these
factors being tightly linked.  If the stocking rate alone is increased
the amount of natural food per fish will decrease.  Fish growth will
then decrease or cease entirely and only a small yield or no yield at
all will be obtained.  However, if parallel to increasing the stock-
ing rate more food is provided, the growth rate is maintained and the
pond's  yield will be directly proportional to the density of stocking
(figure 1).  Because the costs of construction and operation of a pond
are largely fixed, regardless of the amount of fish stocked, it is
clear that the higher the rate of stocking, the greater will be the
net profit.  The required increase in food to maintain this higher
fish density can be achieved in several ways: increasing the product-
ion of  natural food through the use of chemical and organic fertili-
zer, in our case waste effluent; better utilization of existing nat-
ural foods through polyculture, i.e., the introduction of carefully
selected species of fish, each feeding on a different natural food;
and, supplementing the natural food with added fish feed.
                                   10

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Supplemental feed is often the largest single operating expense in
fish farming.  A way should be found to reduce the need for supplemen-
tal feed, while at the same time maintaining the high fish yield.
This can be achieved by increasing the amount of natural food in the
pond.  Abundant production of zooplankton in aerated sewage ponds is
regularly observed3.  Benthic fauna are also increased.  Wirshubsky
and Elchuness^ found a 30-fold increase in the production of chirono-
mides in ponds receiving organic wastes as compared to non-fertilized
ponds.  This fauna represents a source of valuable protein readily ac-
cepted by fish.  We have studied the effect of the introduction of or-
ganic matter at high rates on the production of natural foods with
fish.  Liquid cow shed manure was added to non-aerated ponds with and
without fish, at rates up to 100 kg BODs per hectare every two weeks.
During winter months (November - February; pond temperature
15°C), we observed standing stocks of fauna as follows:
               no fish   no fish      fish       fish
pond type:     manured   no manure   manured   no manure


zooplankton    3.3-42.4  <0.055     0.34-1.3   <0.055   gm dry/m3

chironomides   79-215      1-7         1-4        0-2    100's/m3
The zooplankton reported are those retained on  150u  screen.
The effects of the added organic waste on  increasing  the standing
stocks of natural food are clear.  Comparing  the data from ponds
with and without fish clearly  shows  the  effect  of  fish grazing on
the additional natural food  resulting from the  added  waste.
                                    77

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feed; (2) fertiliser,
 feed; (4) fertilizer,
                                                  no  feed
                                                   poly-
(3) fertilizer, cereal feed; (4) fertilizer,
culture;  (5) fertilizer, protein-rich feed;
(6) fertilizer, protein-rich feed, demand feeders;
(7) fertilizer, protein-rich feed, polyculture;
(8) fertilizer, protein-rich feed, polyculture,
                                                   g
   20
 1
 •<
 6-
 O
 a
 OT
 55
 O
 Q
                           Yield to cover fixed costs
            1     	 	 	Yield to cover investment
                     5             10
           STOCKING  RATE  (THOUSANDS FSR HECTARE)

Figure 1. Effect of Stocking Rate on Yield in Fish Ponds
                              12

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WATER IMPROVEMENT BY PONDS

The sanitary limits placed on irrigation water can be largely over-
come by holding the water in fish ponds.  A pond, by its very nature,
purifies the wastewater by exposure to the high ozygen ^tension and pH
which are produced by phytoplankton.  Kisalt and Ilzohoferb demonstra-
ted that the reduction in total number of bacteria in sewage in ponds
reaches 99.6%, compared with 90% reduction in activated sludge method,
and 89% in trickling filters.  We found that the addition of fish to
the ponds receiving biological wastes in all instances further im-
proves the quality and increases the capacity of the pond for waste
treatment.  In the experiments mentioned above, of adding high rates
of liquid manure to ponds with and without fish, we observed the
following results:
pond type:     no fish, manured   fish;manured    fish; no manure

bacteria           17  - 27          1.6 - 6.7       0.7 - 4.3
 (1000/ml)
pH  (0900 hrs)       7.9 - 8.3       8.3 - 8.9       8.6 - 8.7
D.O.  (ppm)          0.7 - 9.5       9.0 - 15.9     10.0 - 13.8
temperature         9-15          9-15          9-15
    , 0900 hrs)
 Bacteria were counted as  colonies  grown in 0.5% yeast extract agar
 as  described in Standard  Methods^.

 One factor which can be attributed to the  lower bacteria counts ob-
 served in  ponds containing fish  is the consistently high pH developed
 in  these ponds.  Oswald7  reports an increased  rate  of disinfection
 from coliform with increasing pH.

 A further  benefit of increased pH  is an increase  in the efficiency
 with which ponds act as nutrient trap by fixing phosphorous in insol-
 uble compounds  and releasing nitrogen to the atmosphere (Hepher8;
 Oswald, ibid).   This higher pH appears to  be due  to the lower C02
 concentrations  observed in the ponds stocked with fish. The lower
 C02, in turn, probably results from lower  standing  stocks  of zoo-
 plankton and then bacteria in these ponds, and hence lower respira-
 tion volumes.


 PROBLEMS WITH EFFLUENTS IN FISH PONDS

 Two major  factors affect the possibility of using fish in an integra-
 ted waste  effluent-aquaculture system: dissolved oxygen concentrations

                                    13

-------
in the pond water; and, the presence of piosons, especially deter-
gents, in the waste effluent.

It is clear that the D.O. must be maintained above the lethal level
for the species of fish stocked.  Even a single case of anaerobic con-
ditions for several hours will result in the loss of the season's en-
tire fish crop.  We find that we are able to predict the rate of D.O.
depletion in the water by taking into account the BOD of the waste,
the pond temperature, and the naturally occurring diurnal D.O. cycle
in the pond water.  It is clear that in any case these parameters
must be carefully monitored.  However, having the capacity of aeration
as we mentioned above, greatly reduces the hazards of fish kill by
suffocation.

Industrial waste may contain lethal concentrations of metallic poisons
and must be considered separately.  A much more common problem is the
piosonous effect of detergents on fish.  Hard detergents can pass
through a treatment plant with no appreciable reduction in concentra-
tion and kill  fish.  We have worked with waste water from the city of
Haifa.  This waste often contained about 17 ppm ABS, while the lethal
ABS concentration for carp  is 10 ppm.  In this work we found that even
sublethal concentrations reduced the growth rate of the fish.  A
natural degradation of one  ppm ABS per week was observed in the open
fish ponds.  Based on this  our solution was to age the waste in ponds
prior to stocking with fish, and then replace daily evaporation and
seepage loss by fresh waste.  This amounted to an addition of about
100 m'/hectare/day.  The resulting high delution of fresh, by aged,
waste made the system operational.  With the change to biodegradable
soft detergents this problem is greatly reduced.

In considering the problem  of integrating aquaculture, agriculture,
and waste effluent treatment, we should look at the system as an  eco-
logical entity.  Fish fill  one of the important ecological niches  in
this  system.   By so filling this niche we make the system more balan-
ced, more efficient, and more beneficial for man and his environment.

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REFERENCES
1.  E.E.T. Company.  Accumulation, Treatment, and Utilization of
    Sewage in Israel.  1971

2.  Miron, A. Salt Sources in Haifa Sewage.  Tahal, 1964, 26p.

3.  Pillay, T.  Culture of Fish Food Organisms.  FAO Fish Culture
    Bulletin. 2(2):6, January 1970

4.  Wirshubsky, A., and M. Elchuness.  A Preliminary Report on the
    Breeding of CMronomides in Concrete Ponds.  Bamidgeh
    £(1/2):1-5, 1952.

5.  Kisalt, A., and H. Ilzhoffer.  Die Reinigung von Abwasser in
    Fischteichen.  Arch.  Hyg.  Berl 118^:1-66, 1937

6.  Farber, L.  Standard Methods for the Examination of Water and
    Wastewater.  New York, American Public Health Assoc., 1962,
    478-493 p.

7.  Oswald, W.  Complete Waste Treatment in Ponds.  6th Inter-
    national Water Pollution Research.  New York, Pergamon Press,
    1972, B/3/6/1.
                                   75

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     PART 2   TECHNICAL RESTRAINTS
            SESSION CHAIRMAN

   LELANDJ. McCABE, M.P.H., B.S.C.E.
   CHIEF, CRITERIA DEVELOPMENT BRANCH
   WATER SUPPL Y RESEARCH LABORA TORY
NATIONAL ENVIRONMENTAL RESEARCH CENTER
 U.S. ENVIRONMENTAL PROTECTION AGENCY
            CINCINNATI, OHIO

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                       DISEASES TRANSMITTED BY FOODS
                        CONTAMINATED BY WASTEWATER

                        Frank L. Bryan, Ph.D.,M.P.H.


In a world of limited resources and expanding human populations, an
ample and safe food supply is vital for populations to thrive.  New
food sources and improvements in agricultural and aquacultural methods
must be investigated and utilized; wastes generated by increasing
concentrations of people must be disposed of adequately.  Recycling
wastewater for watering and nourishing food crops or for growing fish
contributes to both food production and waste disposal.  But when waste-
water is used to irrigate crops or to provide water in aquaculture,
appropriate precautions must be taken to prevent the diseases that might
otherwise be transmitted by wastewater-contaminated foods.

Foods can become contaminated during production either on farms or in
watercourses, during processing in food processing plants, and during
preparation in food service establishments and homes.  The point at
which contamination occurs will depend on the natural sources of a
pathogen and on the opportunities for transfer at each stage of the
food chain.  Factors that contribute to reported foodborne disease
outbreaks are reviewed by Bryan1.

Many of the pathogenic organisms that infect man reach him by being
conveyed by more than one vehicle.  For example, eggs of some parasitic
organisms can appear in feces of infected persons,  reach water in which
they hatch into a form that infects a vector  (as a  snail), and, after a
period of development metamorphaze into a free-swimming form which pene-
trates tissues of fish or water vegetables.  Diseases caused by such
parasites and features of their transmission are listed in Table 1.
Few of these parasites are found in the United States, but they are
prevalent in some other regions of the world.

Other parasites, such as those that cause hookworm  infection, schisto-
somiasis, and leptospirosis, penetrate human skin in their infective
stage and may be acquired by agricultural or aquacultural workers who
work in wastewater-contaminated fields or ponds  (World Health Organiza-
tion2) .

Most of the organisms that are conveyed by wastewater-contaminated foods
have less complicated transmission cycles.  The organisms that have
potential of transmission by such a route, and the  diseases they cause,
are listed in Table 2.
                                     76

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The following circumstances must occur for persons who ingest wastewater-
contaminated foods to become ill:

   (1) The infectious agent must be present in citizens of a community
       or in animals on farms, or toxic agents must be used for indus-
       trial or agricultural purposes; and wastes from these sources
       must reach sewerage or drainage systems.

   (2) The agents must survive and pass through all wastewater treatment
       processes to which they are exposed.

   (3) The waste-treatment effluent or watercourse receiving the
       effluent must be used as irrigation water for crops or as a grow-
       ing environment for fish or watercrops or for washing or freshen-
       ing harvested foods.  Thus, the agents must survive in the
       receiving watercourse.

   (4) The agents must survive in the soil in which irrigated foods are
       grown.
   (5) The agents must contaminate a food product.

   (6) Then one of the following events must occur:

       (a) The agents must be present on the contaminated food in
           sufficient numbers to survive storage and preparation and
           still cause illness.
       (b) Bacteria on foods in insufficient numbers to cause illness
           must multiply and reach levels that are necessary to cause
           illness.
       (c) Bacteria, and perhaps other organisms, enter food preparation
           areas on raw foods, where they may be transferred to workers'
           hands or to equipment surfaces which if inadequately washed
           will then contaminate other foods that they subsequently
           touch.

   (7) Sufficient quantities of the contaminated food that contain
       enough of the agent to exceed a person's susceptibility threshold
       must be ingested.  Ingestion of foods contaminated to this level
       may result in sporadic cases of illness as well as epidemics.
       When insufficient numbers of pathogens to cause illness are
       ingested, the infected individuals may become carriers and
       subsequently contaminate other foods that they touch.

Each  step of this chain of events necessary for wastewater to contribute
to foodborne outbreaks of human disease is reviewed based on information
from  engineering and medical literature.

PATHOGENS IN SEWAGE

Numerous investigators have isolated pathogens from sewage.  These
investigations have been reviewed by Rudolfs et al. 3 *, Greenberg and
Dean5, Kollins6, and Grabow7.  Animal wastes contain many of the
same  pathogens as well as other pathogens.

                                    77

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SURVIVAL IN WASTE-TREATMENT PROCESSES

Enteric pathogens survive some stages and sometimes the entire process
of wastewater treatment.  Table  3 highlights the results of some typical
investigations of the effect of  various wastewater-treatment processes
in removing or killing pathogens.  Primary  sedimentation usually removes
less than 50 percent of  coliform and pathogenic bacteria from sewage;
it is relatively ineffective in  removing viruses and protozoa.  Acti-
vated sludge or trickling  filter processes  followed by secondary sedi-
mentation usually  removes  over 90 percent of coliform or pathogenic
bacteria remaining after primary sedimentation.  Viruses can be signi-
ficantly reduced by activated sludge but not by trickling  filter
processes.  Sand  filtration is required to  remove  amoebic  cysts and
Ascarls eggs.  Anaerobic digestion reduces  90 percent of pathogenic
bacteria from sludge but is less effective  for destruction of Ascaris
eggs.  Hepatitis  viruses,  Endamoeba  histolytica cysts, and tapeworm
eggs withstand the chlorination  treatment generally applied to waste-
treatment effluent. Thus,  many  waste-treatment processes  remove or kill
90 percent  or more of  the  pathogens  that are present in influent sewage,
but  some still remain  in the effluent.  If  a million pathogens, for
example, are  present in influent sewage before exposure to processes
which  remove  90 percent of them, 100,000 survive;  in the case of
 99 percent  destruction,  10,000 survive.  Kampelmacher and  van Noorle
 Janssen22  found that 1010  salmonella entered a trickling filter
 secondary  treatment plant  each hour  and that 109 left the  plant in the
 effluent each hour.

 SURVIVAL IN RECEIVING  WATERCOURSES AND USE  OF WASTEWATER FOR IRRIGATION

 Those  organisms  that survive wastewater treatment  must also survive in
 receiving waters.   Such survival has been  reviewed by Rudolfs3 "*,
 Dunlop et al.eo,  Clarke et al.81, Rollins6, and Grabow7.   Factors in-
 fluencing survival in  water include  temperature, amount of light, flow
 rate,  presence of oxygen,  pH,  dilution, amount of  dissolved material,
 and types  of  organisms in  the water.

 Irrigation  water  may be sprayed  over crops, flow through channels in
 fields and  seep  to roots,  or be  flooded over the  field.  The numbers of
 pathogens applied to soil  during irrigation can be of such magnitude
 that outbreaks can result.  During  an  epidemiologic  investigation,
 RenteIn and Hinman176  found that sewage plant effluent used to irrigate
 a field was channeled  by gopher  holes  to  a well pit  and entered  a
 community water supply through a defective well  casing.

 SURVIVAL OF PATHOGENS  IN SOIL

 After the wastewater or receiving water is  used  for  irrigation,  enteric
 pathogens  must survive long enough to  contaminate  crops.   Helminths not
 only survive  in  soil but must stay in  soil  for a period of several  days
 to develop  into  an infective stage.  Survival times  for many enteric
 pathogens  in  soil are  reviewed in Table 4.   Factors  that effect

                                     18

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resistance include number and type of organism, type of soil (structure,
moisture content, pH, amount of organic matter) temperature, amount of
rainfall, amount of sunlight, protection provided by foliage, and
competitive microbial flora.  The range of survival times suggests that
pathogens introduced into a field by irrigation with wastewater would,
despite considerable reduction in numbers, survive in the soil until
harvest under some agricultural conditions.  Pathogens in soil are more
likely to contaminate foods if the soil is kept moist by intermittent
application of irrigation water.  Also, continuous application of
wastewater to soil results in accumulation of pathogens in the soil.
Soil filters microorganisms and they often concentrate near the areas
where plants grow.

CONTAMINATION OF FOODS

Foods become contaminated from water during irrigation (flooding, spray-
ing, or seepage), from soil when the plant grows, when vegetables or
fruits fall on the ground, and when the crop is harvested.  They also
are contaminated by dust blowing over the crops or from workers, birds,
and insects that convey organisms from irrigation water or soil to the
foods.  Organisms contaminating foods in one of these ways remain on
the food surface until they succumb to dessication, exposure to sunlight,
starvation, or action of other organisms.  They do not penetrate into
the vegetables or fruits unless their skin is broken.  Survival times of
many enteric pathogens on foods are reviewed in Table 5.  These times
suggest ample opportunity for crops that become contaminated during
irrigation or during growth to remain contaminated until harvest.  Shell-
fish and watercress trap and accumulate enteric pathogens; thus, they
become particularly hazardous if grown in contaminated waters.

Pathogens are infrequently isolated from  foods in fields or after
harvesting.  When pathogens are found, they are found in a low percent-
age of samples and usually for only a short time after irrigation.
Studies in which isolations were made are summarized in Table 6.

SURVIVAL, CROSS-CONTAMINATION, AND MULTIPLICATION

Foodborne disease organisms must survive processing and preparation
steps and bacteria usually have to multiply to reach infective levels.
If wastewater-contaminated fruits, vegetables, or seafoods are cooked
so that the contaminated portions reach 165°F  (or even lower tempera-
tures for sufficient periods of time), vegetative bacteria  (but not
spores), protozoa, helminthic eggs, and most viruses will be killed.
All foods, however,  are not  cooked before serving, and the temperatures
reached during cooking may be too low to kill pathogens.

Contaminated raw foods bring pathogens into food preparation environ-
ments.  These organisms are killed in foods that are thoroughly cooked,
but before cooking they can  contaminate the hands of any worker who
touches them, or they can contaminate equipment that they contact.
Such cross-contamination is  commonly reported  in outbreaks of

                                    79

-------
salrnonellosis; the initial source is usually raw meat or poultry.  Some
food service workers and homemakers are aware of this danger, but they
are unaware that wastewater-contaminated vegetables present the same
hazard.  Citizens in the United States assume that the  fruits, vege-
tables, and shellfish they eat are free from fecal contamination.
But this is the case only if wastewater has not contaminated crops
or the waters in which shellfish grow.

Contaminated  foods which are to be eaten raw or used as a raw ingredient
in a salad can support the growth of foodborne disease  bacteria under
the following set of conditions:  the food must contain sufficient
moisture and  essential nutrients to support bacterial growth; the
foods must be held within a temperature range that permits the contami-
nating bacteria to multiply, usually near the organism's optimal tempe-
rature for growth; and the foods must be held at such temperatures for
sufficient time for enough organisms or toxins to be produced to cause
illness in those who ingest the contaminated food.

NUMBERS OF PATHOGENS REQUIRED TO CAUSE ILLNESS

Ingestion of  contaminated food does not always result in illness.  The
pathogens must be swallowed in sufficient quantity to exceed a person's
threshold of  susceptibility if illness is to result.  Human volunteer
feeding studies have indicated susceptibility threshold levels of
various enteric pathogens.  Table 7 reviews such studies.  Conceivably,
wastewater-contaminated  food could harbor 10 Shigella dysenteriae 1,
180 Shigella  flexneri 2a, or 1,000 Vibrio cholerae biotype ogawa
 (numbers found to cause  illness in adult volunteers) when they reach
the consumer.  Thus, ingestion of foods contaminated at these levels
could  result  in illness.  Such foods could also convey  1 Endamoeba
cola,  10 Giardia lamblia, and 1,000 Vibrio cholerae biotype inaba,
which  if ingested could  result in infection and carrier status.  Ten
thousand Salmonella typhi and 7ibrio cholerae biotype inaba could,
conceivably,  be present  on foods recently fertilized with night soil,
human manure, or raw sewage; and ingestion of this amount of these
pathogens could cause illness.  Certain waterborne outbreaks of these
diseases and  other diseases, such as salmonellosis, suggest that even
lower  levels  of organisms than those indicated by volunteer feeding
studies cause illness.  As few as 15,000 Salmonella cubana caused
death in infants who were given contaminated carmine dye for diagnostic
purposes (Lang et al.114).  All volunteer studies cited were conducted
on healthy adults.  Infants, elderly persons, malnourished persons,
and persons with concomitant illnesses would be more susceptible—
perhaps a one or more log reduction in dosage could result in illness
of such persons.  Most of the other organisms in which  human feeding
tests have been performed, as well as some of the organisms just cited,
usually require a period of time for multiplication before the large
numbers (100,000 or more) necessary to cause illness would be generated.
Thus, it would be unlikely for an infective dose of salmonellae to be
on lettuce or other raw vegetables, but it is possible  for the same
foods  to contain an infective dose of shigellae, Ascaris, or £ndamoei>a

                                   20

-------
histolytica.  One saving feature is that shigellae and Endamoeba histo-
lytica do not survive long in the competitive environment which occurs
in wastewater, soil, or foods.  Salmonellae and many of the other
bacteria which have been mentioned would become problems if contaminated
foods were allowed to stand at room temperatures or refrigerated in
large masses.

OUTBREAKS

On some occasions all circumstances described have occurred and
outbreaks of human illness have resulted.  Such outbreaks and source of
contamination and vehicle for each are listed in Table 8.  This table,
which is obviously biased by incompleteness, shows that foods grown in
water have been vehicles frequently.  Shellfish which were contaminated
in their growing area or during bloating after harvesting were vehicles
in 28 outbreaks; watercress was the vehicle in 10 outbreaks; fish were
vehicles in  3 outbreaks, and shrimp was the vehicle in 1 outbreak.
Vegetables contaminated by night soil, human manure, or raw or partially
treated sewage were  reported as vehicles in 21 outbreaks.  Fruits were
considered vehicles  in only 4 outbreaks.  Before 1960, typhoid fever
led the list of foodborne disease outbreaks attributed to wastewater
contamination.  The  foods incriminated in these outbreaks were found to
have been grossly contaminated with night soil or raw sewage.  Outbreaks
of infectious hepatitis are now reported most frequently, followed in
frequency by outbreaks of fascioliasis and cholera.  These findings
reflect, in part, improvement in epidemiologic technique.

The epidemiologic evidence presented  in many of the reports would not
stand up to  critical evaluation.  On  the other hand, a number of the
investigators proved their hypotheses with epidemiologic and laboratory
evidence.  There are enough of these  investigations to indicate that
wastewater  from mining, industrial, agricultural, community, and
household sources have contaminated foods that, when eaten raw, have
resulted in  outbreaks of  foodborne illness.  These outbreaks will
continue to  occur sporadically if raw or partially treated wastewater
is discharged into watercourses which are used  for irrigation or
aquaculture,  and the contaminating pathogens survive in the wastewater,
in soil, and then on contaminated foods in sufficient quantities.

-------
Table 1.  Parasitic Diseases Maintained by the Cycle:  Human Feces to
          Water or Soil; Water or Soil to Food; Food for Human
          Consumption	
AGENT
INTERMEDIATE
VEHICLE
 Fasciola  hepa-
 tica
 F.  gigantica
fresh water
Fasciolopsis
buski
Opisthorchis
felineus
Paragonimus
westermani
Taenia
saginata
Taenia
sollum
fresh water
fresh water
fresh water
soil
(grass)
soil
(grass)
INTERMEDIATE
HOST
                                                            FOOD
Clonorchis fresh water
sinensis
Diphylloboth- fresh water
riuzn la turn
Echinostoma fresh water
ilocanum
snail fish

copepods f i sh
snail snails,
clams,
limpets ,
fish,
tadpoles
snail



snail


snail


snail
watercress,
water vege-
tables

water vege-
tables

fish
                                                            crabs,
                                                            crayfish

                                                            beef
                                                            pork
                                    22

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Table  2.  Diseases and Causative Agents Transmissible by Food that Has
          Been Contaminated by Wastwater or by Soil that Contains Fecal
          Material
 DISEASE
AGENT
                              Bacteria*
Arizona  infection
Bacillus cereus  gastroenteritis
Cholera**
Clostridium perfringens  gastro-
  enteritis
Enteropathogenic Escherichia
  coli  infection
Paratyphoid fever**
 Pseudotuberculosis
 Salmonellosis**
 Shigellosis**
 Typhoid fever**
 Yersinia gastroenteritis
Arizona hinshawii
Bacillus cereus
Vibrio cholerae
Clostridium perfringens

Escherichia coli (certain serotypes)

Salmonella paratyphi A
Salmonella paratyphi B
Salmonella paratyphi C
Salmonella sendai
Pasteurella pseudotuberculosis
Salmonella (over 1,500 serotypes)
Shigella sonnei
Shigella flexneri
Shigella dysenteriae
Shigella boydii
Salmonella typhi
Yersinia enterocolitica
                                Viruses
 Adenovirus infection
 Coxsackie infection
 ECHO virus infection
 Poliomyelitis
 Reovirus infection
 Viral hepatitis**
 Winter vomiting disease
 Ascariasis**
 Trichiniasis
Adenovi rus e s
Coxsackie viruses
ECHO viruses
Polioviruses
Reoviruses
Hepatitis virus A
Norwalk agent
                               Helminths
Ascaris lumbricoides
Trichuris trichiura
                                Protozoa
 Amebiasis**
 Balantidiasis
 Coccidiosis (Isospora infection)
 Dientamoeba infection
 Giardiasis
Entamoeba histolytica
Balantidium .coli
Isospora belli, I. hondnis
Dientamoeba fragilis
Giardia lamblia
 *Other enteric bacteria which could conceivably be transmitted by foods
  but proof is inconclusive:   Streptococcus faecalis,  S.  faecium,  Proteus
  spp., Providencia spp., Klebsiella spp.,  Citrobacter freundii, Entero-
  bacter spp., Edwardsiella spp.,  Aeromonas spp., Pseudononas aeruginosa
**Reported outbreaks, see Table 8
                                    23

-------
    Table 3.  Survival of Pathogens During Various Stages  of Wastewater Treatment
                                                        Process
PATHOGEN    SETTLING
ACTIVATED   TRICK-
ANAERO-  SAND
                                                                         STABIL-   DISIN-
                       SLUDGE       LING FIL-L  BIG DI-  FILTRA-  DRYING  IZATION   FECTION
                                    TRATION    GESTION  TION             POND
                                                                                                   REFER-
                                                                                                   ENCE
    Salmonella
    typhi
                                                                               V-
N*
    Salmonella
     p&ratyphi
     Utrecht
                          -n-
                                                    (8)
                                                    (9)
                                                   (10)
                                                   (ID
                                                   (12)
                                                   (13)
                                                   (14)
                                                   (15)
                                                   (16)

                                                   (17)
                                                   (18)
                                                   (19)
                                                   (21)
                                                    (8)
                                                   (14)
                                                   (20)
                                                   (22)
    Shigella
    flexneri
                                                                                                   (8)
                                                                                                  (18)
    Vibrio
    cholerae
                                                                                                  (23)
                                                                                                   (8)
    Streptococ-
    cus faecalis
                                                                                                  (24)
                                                                                                  (25)

-------
Table 3.  Continued
Process
ACTIVATED TRICK- ANAERO- SAND STABIL- DISIN-
PATHOGEN SETTLING SLUDGE LING FIL_ BIC DI- FILTRA- DRYING IZATION FECTION
TRATION GESTION TION POND
Enterovir- + +
uses + +
-H- +
Polio vir- -
uses +
+ + +
+
-
++++ + +
+++ ++
++++ -H-+
-(++)
+-H-+ -l-f
+++
+
Coxsackie -H--H- +++ +/-
viruses +
+++
+
+ +
4--H-+ - + - +
++++ ++++ -H-++ ++ +
+++
ECHO +
viruses +++
REFER-
ENCE
(26)
(27)
(28)
(29)
(30)
(31)
(32)
(33)
(34)
(35)
(36)
(37)
(38)
(39)
(40)
(41)
(39)
(42)
(32)
(35)
(34)
(43)
(39)
(32)
(39)

-------
Table 3.  Continued.
                                                    Process
PATHOGENS   SETTLING
ACTIVATED   TRICK-     ANAERO-  SAND             STABIL-   DISIN-       REFER-
SLUDGE      LING FIL-  BIC DI-  FILTRA-  DRYING  IZATION   FECTION      ENCE
            TRATION    GESTJON  TION             POND
Hepatitis
virus A
Endamoeba
                                                                          (44)
histolytica +
++++ ++++ +++ +
+ + + +
+
CO 11 ++
Giardia +
lairiblia
Ascaris + + +++ + - +
lumbri- + + + + -
coides - +
eggs +++
+++ ++ -M- +++4
Tri churls +
trichiura +++ ++
Tapeworm ++++ ++
eggs
Taenia + + + +
saginata +++ ++++ +++ ++++
•H-++ (45)
-H-++ (46)
(47)
(21)
++ (48)
(21)

(46)
(47)
(49)
+++ (48)
(50)
(21)
(50)
++++ (51)

(47)
(52)
- destruction; + survival; ++ over 90% removal; +++ over 50%  removal; ++-M-  less  than  50%  removal

-------
Table 4.  Survival of Enteric Pathogens in Soil
PATHOGEN
Salmonella
typhi





















Salmonellae





Streptococcus
faecalis



Endamoeba
histolytica
Ascaris ova

TYPE SOIL
moist sand
peat
damp soil
soil surface (sunlight)
soil
soil
garden soil (in open)
sandy soil
garden soil (hot house)
sandy soil (hot house)
moist neutral soil
moist arid soil
dry soil
sand
muck
clay loam
sandy loam
adobe
adobe -peat
loam
sand
peat
loam sand
soil and pasture
soil (raw sewage)
soil
sandy soil
soil and pasture
soil
sandy loam
sand
loam
clay loam
muck
moist soil

irrigated soil
soil
SURVIVAL
(in days)
<12
<13
35-74
5-22
70-80
35
32-58
29-36
49-74
53
70
<10-30
<14
<5
12
12
5
21-105
28-100
21-120
2-15
1-2
14-60
>200
46
46-70
35-84
>280
147-259
28-77
33-35
26-63
33-49
40-77
6-8

730-1035
2190
REFERENCE
(53)



(54)
(55)
(56)



(57)


(58)



(59)





(60)
(61)
(62)
(63)
(64)
(65)
(59)




(66)

(67)
(68)
                                   27

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Table 5.  Survival  of Enteric Pathogens on Foods
PATHOGEN
Salmonella
typhi
VEGETABLE
vegetables (leaves and
steins)
radishes
lettuce
SURVIVAL
(in days)
10-31
28-53
18-21
REFERENCE
(55)
(56)
Salmonellae     vegetables                         40           (68)
                 vegetables                         28           (69)
                 beet leaves                        21
                 tomatoes (laboratory)              20           (70)
                 tomatoes (field)                     3
                 soil and potatoes                 >40           (71)
                 carrots                           >10
                 cabbage and gooseberries          > .5
                 tomatoes                            3           (72)

Shigella        clams and shrimp                  >60           (73)
sonnei  and      oysters                           >40
S.  flexneri     tomato (surface)                     2           (74)
S.  sonnei        tomato (tissue)                     10
                 apple (skin)                         8

Vibrio           vegetables                          5           (75)
choleras        dates                            
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Table 6.  Recovery of Pathogens from Foods Contaminated by Wastewater

PATHOGEN        FOOD            SOURCE OF CONTAMINATION      REFERENCE
Salmonella
typhi
leaf tips
radishes
lettuce
contaminated soil
contaminated soil
contaminated soil
(55)
(56)
Salmonella
(other types)
Salmonella,
shigella,
other bac-
terial path-
ogens
(serologic
 evidence)

Ente ropatho-
genic
Escherichia
coli
celery
vegetables
fish
green onions

white perch
fish
irrigation water
irrigation water
river water
irrigation water

river water near heavily
populated areas
(82)
(83)
(84)
(85)

(86)
river water
(87)
Enteroviruses   vegetables
(Polio, ECHO,   oysters
Reo, Coxsackie)
Ascaris ova
Helminth ova
lettuce
cabbage

vegetables
cucumbers
tomatoes
carrots
irrigation water
sewage-contaminated
sea water

irrigation water
irrigation water

irrigation water
irrigation water
irrigation water
irrigation water
                                               (88)
                                               (89)
(48)


(90)
                                    29

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Table 7.  Clinical Response of Adult Humans to Varying Challenge Doses of Enteric Pathogens
ORGANISM
(strain)
                       Challenge doses                                   REFERENCE
)°    101    102    103    10"   10s   10*   107   108   109   1010   1011
Shigella
dysenteriae
(1) (A-l)
(1) (M 131)
Shi gel la
flexneri (w)
(2a)
(2a)
Vibrio
choleras
inaba 569B
(unbuffered)
inaba 569B
(+NaHC03)
ogawa
(4NaHCO )
j
Salmonella
typhi
(Quailes) vi
(Zermat) vi
(Ty2V) vi
(0-901)
(Ty2W)
(Quailes)
(Quailes)


+ +4 (91)
4 44 444 4444 (91)

+++ (92)
4 +++ -H-++ +44 4444 (93)
4 444 444 444 (94)



++ 44 441 (95)

(4) 444 4441 4441 441 (95)

44 +41 (95)


44 44 4444 4444 (96)
++4 (96)
44 (96)
44 (96)
4 (96)
4 44 4444 (97)
++ (98)

-------
Table 7. Continued
ORGANISM Challenge doses
10° IO1 IO2 IO3 10* 10S IO6 IO7 10fl IO9 IO10 IO11 ^ra^NCE
(strain)
Salmonella
newport + ++
Salmonella
bareilly •+• ++
Salmonella
anatum(I) - +
(II) 4-
(III) --++ + +
* ++ 4- +
Salmonella
meleagridis (I) - - - ++
(ID - ++
(III) - + ++
* +++ +++
Salmonella
derby - ++
Salmonella
jpullortun(I) ______ ++++
(II) _____ ++++
(III) _____ +4.4.4.
(IV) -----++
(99)
(99)
(100)
(100)
(100)
(101)
(100)
(100)
(100)
(101)
(99)
(102)
(102)
(102)
(102)

-------
Table 7.  Continued
ORGANISM Challenge doses
10° 10* 102 103 10" 105 106 107 108 109 1010 1011
(strain)
Escherichia
coJi(Olll:B4) +-H- 444 444
(055 :B4) 444 444 4444
(O6:H16) 44 444
(O124:K72:H-) + (+>
(O143:K?:H-) (4) (4) 444
(O144:K?:H-) - 4 444
(O148:H28) 4 4444

REFERENCE


(103)
(104)
(105)
(105)
(105)
(105)
(105)
Streptococcus
faecalis var.
liquefaciens

Clostridium
perfringens
type A (Heat-
resistant)

Clostridium
perfringens
type A (Heat
sensitive)
(106)
(107)
(108)

-------
    Table 7.  Continued
U)
ORGANISM Challenge doses
Feces 10° 101 102 103 10** 105 106 107 108 109 10i0 1011
(strain)
Endamoeba
coli (+) (++) {++) - (++++)
Giardia
lamblia - (++++) {+-»•) (++++) (++++) (++++)
Norwalk
agent
( virus)! ++
** 2 +++
3 +
Hepatitis
virus A +++
(fecal ++
filtrates)
REFERENCE

(109)

(110)




(111)

(112)

(113)
    - = 0, + = 1-25, ++ =* 26-50, +++ = 51-75, ++++ = 76-100 percent of volunteers developing illness
    * Refeeding trials of volunteers who months before became infected by the same strain
    **1,2,3 refers to serial passages of stool filtrates
    (+)  Infections without illness
    1 = cholera-like diarrhea

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Table 8.  Outbreaks Associated with Foods Contaminated by Sewage  or
          Wastewater
DISEASE
Typhoid fever
Typhoid fever
Typhoid fever
Typhoid fever
Typhoid fever
Typhoid fever
Typhoid fever
Typhoid fever
Typhoid fever
Typhoid fever
Typhoid fever
Typhoid fever
Typhoid fever,
gastroenter-
itis
Amebiasis
Taeniasis
Typhoid fever
SOURCE OF CONTAMINATION
Sewage-polluted waters
Sewage sludge fertilizer
and irrigation
Sewage- contaminated
watercress beds
Sewage-polluted water
Sewage-polluted water1
Human manure
Privy-polluted water*-
cress beds
Sewage irrigation
Sewage irrigation
Sewage-polluted water
Sewage-polluted water
Sewage-polluted water
Fecal-polluted (privy
and boats) water
Sewage irrigation
Sewage irrigation
Sewage-polluted wastes
FOOD
oysters
celery
watercress
shellfish
oysters , raw
vegetables and
fruits
rhubarb
watercress
vegetables,
blackberries
raw vegetables
oysters
oysters
oysters
oysters
vegetables
beef
oysters
REFERENCE
(115)
(116)
(117)
(118)
(119)
(120)
(121)
(122)
(123)
(124)
(125)
(125)
(126)
(127)
(128)
(129)
 Typhoid fever,
 Paratyphoid
 fever
 Typhoid fever

 Shigellosis



 Amebiasis
(privies and raw sewage)

Secondary sewage treat-     vegetables
ment (activated sludge)2
plant effluent irriga-
tion and wash water

Sewage                      clams

Irrigation water            cabbage
(Primary treatment plant
effluent) 3
Night soil                  vegetables
(130)




(131)

(132)



(133)

-------
Table 8.  Continued
DISEASE
Typhoid fever

Ascariasis
Typhoid fever
Typhoid fever
Ascariasis
Tyhpoid fever
Salmonellosis

Typhoid fever

Typhoid fever

Typhoid fever
Ascariasis
Ascariasis
Hookworm
infection
Viral
hepatitis
Typhoid fever


Diphylloboth-
riasis
Fascioliasis
Viral
hepatitis



Typhoid fever
Viral
hepatitis

SOURCE OF CONTAMINATION
Sewage-polluted water-
cress beds
Sewage spray irrigation
Sewage manure
Night soil
Night soil
Sewage irrigation
Sewage irrigation and
sludge
Human fecal material
as manure
Human fecal material
as manure
Night soil
Night soil
Night soil
Sewage farming

Sewage-polluted water

Sewage l


Sewage-polluted water

Animal feces1
Sewage-polluted water
by primary treatment
effluent, raw sewage,
and septic tank dis-
charges3
Sewage-polluted water
Sewage-polluted water
(overtaxed treatment
plant effluents)
FOOD
watercress

vegetables
vegetables
vegetables
vegetables
apples
vegetables

endive

raw salad

vegetables
vegetables
vegetables
vegetables

oysters

vegetables,
fruits ,
shellfish
fish

watercress
clams




oysters
clams


REFERENCE
(134)

(135)
(67)
(177)
(178)
(179)
(136)

(137)

(137)

(68)
(68)
(138)
(139)

(140)

(141)


(142)

(143)
(144)




(145)
(146)


                                    35

-------
Table 8.  Continued
DISEASE
Viral
hepatitis
Viral
hepatitis
Viral
hepatitis
Fascioliasis
Cholera
Fascioliasis
Salmonellosis
Viral
hepatitis
Viral
hepatitis
Taeniasis
Minamata
disease
(organic
mercury
poisoning)
Salmonellosis
SOURCE OF CONTAMINATION
Sewage-polluted water
(treatment plant by-
passed)
Sewage-polluted water
Sewage-polluted water
Sewage-polluted water1
Sewage-polluted water
Sewage-polluted water1
Dead animal in polluted
water from which cows
drank
Sewage-polluted water
Sewage-polluted water
Human feces contaminated
trench silo
Industrial waste
Sewage-polluted water
FOOD
oysters
oysters
oysters
watercress
shrimp
watercress
raw milk
oysters
clams
rare beef
fish, shellfish
white fish
REFERENCE
(147)
(148)
(153)
(149)
(150)
(151)
(152)
(153)
(153)
(154)
(155)
(156)
(157)
 Fascioliasis


 Viral
 hepatitis,
 gastroenter-
 itis

 Gastroenter-
 itis

 Salmonellosis
 (Animal in-
 fection)
by untreated sewage
(culture positive)

Animal feces contami-       watercress
nated watercress bed

Sewage-polluted water       clams
by cesspool
Sewage-polluted water1      clams


Animal dung slurry used     grass
to irrigate pastures
(158)


(159)




(159)


(160)
                                     36

-------
Table 8.  Continued
DISEASE
Viral
hepatitis
Fascioliasis


Cysticercosis
(Animal in-
fection)
Ouch-ouch
disease
(cadmium
poisoning)
Fascioliasis

Viral
hepatitis
Cholera
Viral
hepatitis
Salmonellosis
(Animal in-
fection.
possible
human cases)
Viral
hepatitis
Viral
hepatitis
Cholera

Cholera
SOURCE OF CONTAMINATION FOOD
Sewage-polluted water1 clams
Animal feces contami- watercress
nated water used for
watercress beds
Sewage- contaminated water
irrigation water used
for cattle watering
Mining waste used to rice
flood rice fields


Animal feces contami- watercress
nated watercress bed
Sewage-polluted water clams
Sewage-polluted water1 shellfish
Septic tank effluent watercress
contaminated water
Human sewage flowing grass
over grazing land

Sewage-polluted water1 clams
Sewage-polluted water1 oysters
Sewage-polluted water mussels
for growing and freshen-
ing
Raw sewage irrigation vegetables
REFERENCE
(161)
(162)


(163)
(164)
(165)


(166)

(167)
(168)
(169)
(170)

(171)
(172)
(173)
(174)
(175)

(2)
 1 Implied
 2Secondary treated sewage
 3Primary treated sewage
                                   37

-------
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155.  Peavy,  J. E.,  A.  B. Rich, M. S.  Dickerson, G. Martin, and  E. Cox.
      1968.   Morbidity  Mortality Weekly  Rept.  (U.S.)  17(26):241.
156.  Kumamoto Study Group on Minamata Disease.  Minamata Disease.
      Kumamoto Univ., Japan.  1968.
157.  Gangarosa,  E.  J., A. L. Bisno,  E.  R.  Eichrer, M.  D. Treger,
      M.  Goldfield,  W.  E. DeWitt,  T.  Fodor, S. M.  Fish, W. J.  Dougherty,
      J.  B.  Murphy,  J.  Feldman, and  H. Vogel.   1968.   Am. J.  Public
      Health 58:114.
 158.  Anon.   1969.   Lancet 1:658.
 159.  Dismukes, W.  F.,  A. L. Bisno,  S. Katz,  and R, F.  Johnson.   1969.
      Am.  J.  Epidemiol. 89:555.
 160.  Jack,  E. J.  and P.  T. Happer.   1969.  Vet.  Rec.  84:196.
 161.  Ruddy,  S. J.,  R.  F. Johnson, J. W.  Mosley,  J. B.  Atwater,
      M.  A.  Rosetti, and  J. C. Hart.   1969.   J.  Am. Med.  Assoc.  208:649.
 162.  Ashton,  W.  L.  G., P. L. Boardman,  C.  J.  D'Sa, P.  H. Everall, and
      A.  W.  J. Houghton.  1970.  Brit. Med. J.  3:500.
 163.  Brown,  R. R.   1970.  Morbidity Mortality Weekly Rept. 19(9):100.
 164.  Emmerson, B.  T.   1970.  Ann. Internal Med. 73:854.

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165.  Kobayashi, J.  1970.  Adv. Water Pollu. Res. 1:25/1.

166.  Hardman, E. W., R. L. H. Jones, and A. H. Davies.  1970.  Brit.
      Med. J. 3:502.

167.  U.S. Department Health, Education, and Welfare.  Foodborne
      Outbreaks Annual Summary 1969.  Atlanta, Georgia, National
      Communicable Disease Center.  1970.

168.  Dutt, A. K., S. Alwi, and T. Velauthan.  1971.  Trans. Royal Soc.
      Trop. Med. Hyg. 65:815.

169.  Hutcheson, R. H.  1971.  Morbidity Mortality Weekly Rept. 20(39) :
      357.

170.  Bicknell, S. R.  1972.  J. Hyg. 70:121.

171.  Herman, H. A., G. Waterman, and N. J. Fiumara.  1972.  Morbidity
      Mortality Weekly Rept.  (U.S.) 21(2) .-20.

172.  MacLean, R., A. Randall, and M. S. Dickerson.  1973.  Morbidity
      Mortality Weekly Rept.  (U.S.) 22(44):372.

173.  Hamilton, V., T. McKinley, J. E. McCroan, F. S. Wolf,
      E. C. Prather, R. A. MacLean, M. S. Dickerson, and R. H. Hutcheson,
      Jr.  1973.  Morbidity Mortality Weekly Rept. (U.S.) 22(46):388.

174.  World Health Organization.  1973.  Morbidity Mortality Weekly
      Rept. (U.S.) 22(35):300.

175.  Baine, W. B., W. H. Barker, Jr., and E. J. Gangarosa.  1974.
      Personal communication.

176.  Renteln, H. A. and A. R. Hinman.  1967.  Am. J. Epidemiol. 86:1.

177.  Viehl, K.  1950.  Gesundh. Ing. 71:126.  (Cited by Kreuz, 1955).

178.  Anders, W.  1952.  Der Off Gesundheitsdienst 14(H. 9,5):360.
      (Cited by Kreuz, 1955).

179.  Romer, W.  1953.  Wass. Bod. 5:288.  (Cited by Kreuz, 1955).

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               THE EVALUATION OF MICROBIAL PATHOGENS
                  IN SEWAGE AND SEWAGE-GROWN FISH

                                by

         R. LeRoy Carpenter, Harold K. Malone, Ara F. Roy,
     Aaron L. Mitchum, Herbert E. Beauchamp and Mark S. Coleman*


A significant removal of pathogenic bacteria and viruses during the
process of the lagoon method of sewage treatment is well documented,
though not entirely understood!> 2, s, 4.  Stabilization ponds with a
30-day retention time can achieve a reduction of up to 99 percent3.
OkunS reported 99.99 percent reduction of coliforms; Drew^ found 99.6
percent reduction in summer and 96.8 percent in winter.

The use of the traditional coliform test to indicate the likelihood of
pathogenic bacteria and viruses has been widely questioned'7 since human
pathogens, unlike coliforms, tend to die when not in the temperature
and chemical environment similar to a host^.  The health risks involved
in the reuse of wastewater reflect the range of pathogenic organisms
found in the community producing the sewage.  For example, sewage irri-
gation of vegetable crops was found to be linked to an outbreak of
cholera in Jerusalem in 1970.  In India hookworm and gastrointestinal
infections are more common among workers on sewage farms than the gene-
ral population.  On the other hand, a follow-up study of the health of
workers at sewage treatment plants in the U.S.A. did not reveal any
excess risk of disease or disability3.

Public health officials have noted a mushrooming interest in beneficial
uses of wastewater ox^er the past two years.  This increased interest
is mainly due to the following:  (1) the realization that many parts
of this country are outgrowing or haxre outgrown their freshwater
supply^; (2) the demands for new sources of inexpensive protein to feed
an expanding animal and human popuJatior;  (3) the rising cost of ni-
trogen and phosphorus fertilizers to support agricultural needs^; and
last, but not least, (4) the desperate need of municipalities to off-
set the high cost of meeting the federal and state effluent standards
for sewage treatment facilities-^.
*R. LeRoy Carpenter, M.D., M.P.H., Commissioner of Health; Harold K.
Malone, Assistant Chief, Laboratory- Services; Ara F. Roy, Director,
Sanitary Bacteriology Division; Aaron L. Mitchum, Microbiologist II;
Herbert E. Beauchamp, Director, Virology Division; Mark S. Coleman,
Director, Water Quality Monitoring and Research Division, State Depart-
ment of Health, Northeast Tenth and Stonewall, Oklahoma City, Oklahoma
73105.                                                     y

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This new surge of interest in beneficial uses of wastewater has prompt-
ed the Oklahoma State Department of Health to look objectively at the
health hazards associated with human and animal exposure to sewage ef-
fluents.  Such a preliminary investigation was carried out from July,
1973 through February, 1974 at the Quail Creek Sewage Lagoon System
near Oklahoma City.  The system is made up of six cells approximately
six acres each as shown in Figure 1.  It serves a residential district
of approximately 10,000 persons producing approximately one million
gallons of sewage/day.  The first two cells are aerated with the con-
ventional Hinde Air-Aqua System; the four following are operated in
series at a level of four to five feet.  An engineering flaw in the
system is that a modification necessary to convert flow from parallel
to series caused a short circuit in cell #4 as shown by the arrows in
Figure 1.

In May, 1973, 25,000 two to four-inch channel catfish fingerlings were
stocked in each of cells #3 and #4; 35 pounds of adult Golden Shiners
(Notemigonus crysoleucas) were stocked in each of cells #5 and #6.  In
July, 175 three-inch Tilapia nilotica and five pounds of Fathead Min-
nows  (Pimphales promelas) were introduced into cell #3 only.

Despite what was considered as adequate screening between the test
cells which were dry when the study began and the proceeding two con-
ventional cells, wild fish including Black Bullhead  (Ictalurus melas),
Green Sunfish  (Lepomis cyanellus), and Mosquitofish  (Gambusia affinis)
contaminated the test cells.

MATERIALS AND METHODS

One hundred seventy-nine fish on 12 different dates and 77 water
samples on 11 different dates were collected over a seven-month period
beginning July 1, 1973 and ending January 28, 1974.  While there was
some variation, most water collections consisted of four liter catch
samples of the untreated sewage and similar samples of the effluent
from each of the six cells.  A total of 34 samples of five channel cat-
fish each were collected from cells #1 through #5 during 12 fish-
sampling visits.  Tilapia, Green Sunfish and Bluegill were collected
one time each from cells #3, #4 and #5 respectively.  Water samples
were processed according to the "APHA. Standard Methods of Examination
of Water and Wastewater."12

Water samples were examined for total coliform, fecal coliform, fecal
streptococci, and pathogenic enteric bacteria using  the membrane filter
methodology recommended by the APHA13.  For isolation of pathogenic
enteric bacteria, 100 ml portions of sample were passed through 0.45
micron pore size membrane filters.  The membranes were then placed
in Hajna's gram negative  (GN) enrichment broth and incubated overnight.

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•ft-
Qs
       QUAIL CREEK LAGOON SYSTEM
                   FIGURE 1

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The broth was then streaked onto Bismuth Sulfite Agar, Brilliant Green
Agar, XLD Agar, and MacConkey's Agar.  Suspicious colonies were con-
firmed by conventional biochemical and serological tests as described
by Edwards and Ewing1^.  GN broth was initially selected for its known
superior ability to yield shigellae species while it is also reported
to be suitable for enrichment of Salmonella species1^, ^.  Failure
to isolate Salmonella from the first nine samples examined led to the
addition of a second membrane which was enriched in Tetrathionate broth
with Brilliant Green added1^.  This broth was then used to streak addi-
tional plates of Brilliant Green and Bismuth Sulfite Agars.

Subsequent control studies in our laboratory showed that Tetrathionate
broth with Brilliant Green added can recover essentially all Salmonella
seeded at low levels into lagoon water while, by comparison, the yield
from Hajna's GN broth was reduced by a factor of greater than ten.

For virus studies, 50 ml aliquots of lagoon water were centrifuged at
2,000 rpm.  The supernatant was decanted, treated with antibiotics and
0.1 ml portions inoculated into Rhesus Monkey Kidney, Wi38 and, when
available, HEp2 tissue culture tubes.  Cultures showing cellular de-
struction were examined by conventional methods for viral identifica-
tion^.  Cultures not showing cellular destruction were passed once to
the same cell line before discarding as negative.

It was recognized that the catch sample method has a comparatively low
degree of sensitivity; but the elaborate concentration techniques em-
ployed by Wallis, Melnick and Fields17' 18 and Metcalf, Vaughn and
Stiles1^ were beyond the financial scope of this study.  Control
studies conducted by seeding of the lagoon water with low levels of
Coxsackie A9 enterovirus followed by the above procedure showed no
apparent interference to the recovery of virus by naturally occurring
constituents in the lagoon water.  Samples of fish were carefully dis-
sected and the skin, upper intestines, cloaca, and muscle were cul-
tured separately.  However, after the first two collection dates, skin
was combined with muscle to constitute one specimen, and, all viscera
were pooled to constitute a separate specimen.  After homogenizing,
an aliquot of each pool was used for both virus and bacterial study.

For isolation of human pathogenic enteric bacteria, a 2 ml portion of
the homogenate was enriched by overnight incubation in 10 ml of Hajna's
GN broth followed by streaking for isolation as indicated previously
for water.  Virus examination was accomplished by processing the homog-
enate according to the procedures in use for human feces and tissue by
the Oklahoma State Department of Health Laboratory16.  The processed
material was assayed by inoculation into Rhesus Monkey Kidney, Wi38
and, when available, HEp2 tissue culture systems.

-------
Results of the study are  shown  in Figure  2.  The  log count of fecal
coliforms per 100 ml is shown in the bars and  sampling  dates are shown
along the horizontal axis.   The top shaded area shows the log of number
of coliforms in  the raw influent to the system and the  lower portion
of the bars, shown by  the dotted area,  represents the treated effluent
as it passed out of the last of the six cells  into the  receiving
stream.

Inspection of these bars  readily shows  that  there is a  remarkable re-
duction of fecal coliform bacteria to  less than detectable limits in
a standard 50 ml portion  in three separate samples.

The  pathogens  isolated are  shown by the boxes  at  the bottom:

          August  13  there  was an isolation of Edwardsiella tarda.

          December  17  there  was  an isolation  of ECHO 1 enterovirus.

          January 14  there was an isolation of  Salmonella newport.

 Figure 3  shows the  average  log  of the  count  of indicator organisms per
 100  ml isolated  on the 11 water-sampling  dates.  As the wastewater
 flows through the  system  from raw influent on  the left  to effluent from
 the  last  lagoon  on the right, one can  see a  progressive reduction  in
 the  indicator organisms.  By the time  the effluent passes through the
 third cell,  it  is  found to  contain less than ten fecal  coliform organ-
 isms/100  ml,  and remains  at this low level as  the effluent passes
 through the  last three cells.   It is interesting from a public  health
 standpoint that  of the three pathogens isolated in water during the
 study,  two were  found in raw influent  and one  was in  cell #2.   No
 pathogens were found in wastewater beyond the  first two conventional
 cells;  nor in any  of the  cells  containing the  test fish; nor  in any
 of the 179 fish  sampled.

 In conclusion, it can be  said that this study  has confirmed the pre-
 vious observations  of others that indicator  coliform  organisms  are
 efficiently  removed in a  lagoon-method wastewater-treatment systeml*
 2, 3, 4.   xhis study  has  shown  that human pathogens are rare  in those
 wastewaters  tested  and in fish  grown  in those  wastewaters beyond  the
 raw  or first  two conventionally-operated cells.

 Though it was not within  the scope of  this study to evaluate  the  ef-
 fect of extended lagooning, per se, on removal of coliforms,  future
 investigators  should  design studies in such  a  way as  to simultaneously
 compare the  results  in lagoons  both with and without  the addition of
 fish.
                                   50

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LOG COUNT
PER 100ml


            i


                 i
                 i

QUAIL CREEK LAGOON SYSTEM
     FECAL COLIFORM
    INFLUENT VS EFFLUENT
            AND
    PATHOGENS ISOLATED
    JULY 1973-JANUARY I974



              I
              \
                                                  INFLUENT
                                                EFFLUENT
i
              PATHOGENS ISOLATED
              III       I       I
       1973  JULY JULY JULY   JULY   AUG
              2    9   16      30     13
              AUG
               27
                                 I
                               OCT
          NOV

                        $
                        I
                        1


JAN 1974
 28
                                                  FIGURE 2

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1
6
                            QUAIL CREEK LAGOON SYSTEM
                            INDICATOR BACTERIA
                            AVERAGE COUNT PER CELL
                                    AND
                             PATHOGENS ISOLATED
                                                        TOT ALGOL I FORM
    AVERAGE LOG COUNT PER 100 ml
                                                        FECAL COLIFORM
            PATHOGENS ISOLATED
        RAW
CELL 1
                                CELL 2
    I
    CELL 3
FIGURES
                                                        I
                                                        CELL 4
                                                CELLS
                                                             I
                                                             CELL 6

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Many economic and conservation-oriented pressures are prompting a surge
of interest in using domestic wastewater for production of crops and
fish.  This study supports this concept.  But, as Shuval so aptly
pointed out, much -work yet remains to be done in accurately determining
the public health hazards associated with such practice2^.

We should investigate the potential dangers to the handlers, harvesters
and processors of fish and shellfish grown in wastewaters and not limit
our attention to the consumer only.  We must sharpen our techniques for
isolating pathogenic viruses and bacteria from wastewaters, use them
more frequently in our monitoring programs and not rely solely on coli-
form indicators as we have in the past.  Parasites such as worms and
amoebae should not be overlooked.  And last, where possible, we should
use sound epidemiological methods to correlate the presence or absence
of disease in a community with those organisms found in that communi-
ty's wastewaters.

-------
1.   Berg, Gerald.  Virus Transmission by the Water Vehicle.
     II Virus Removal by Sewage Treatment Procedures.  Health
     Library Science.  _2_:90, 1966

2.   KLock, John W.  Survival of Coliform Bacteria in Waste-
     water Treatment Lagoons.  Journal of Water  Pollution
     Control Federation.  43:2071-2083, October  1971.

3.   The Reuse  of  Wastewater.  WHO Meeting of Experts on the
     Reuse of Effluents: Methods of Wastewater Treatment and
     Health Safeguards  (1973) Report, Geneva (World Health
     Organization  techn. Rep. Ser., No. 517). WHO Chronicle
     27:492-495, November 1973.

4.   Little, John  A., Bobby J. Carroll and Ralph E. Gentry.
     Bacterial  Removal  in Oxidation Ponds.   Second International
     Symposium  for Waste Treatment Lagoons,  June 23-25, 1970,
     Kansas City,  Missouri. Lawrence, Kansas, Ross E. McKinney,
     University of Kansas,  pp.  141-151.

 5.   Okun, D. A.  Experience with  Stabilization  Ponds in the
     U.S.A.   Bulletin World Health Organization  26:550, 1962.

 6.   Drew, R. J.  Field Studies  of Large  Scale Maturation Ponds
     with Respect  to their  Purification Efficiency.  Journal  and
     Proceedings of Inst. Sewage Purification 3_:1-16, 1966.

 7.   Northington,  Charles W.,  Shih L.  Chang  and  Leland J. McCabe.
     Health Aspects of  Wastewater  Reuse.  Water  Quality Improve-
     ment by  Physical and Chemical Processes.  Austin, University
     of Texas Press,  Ed. by Earnest  F. Gloyna and W. Wesley
     Eckenfelder,  Jr.

 8.   Berg, Gerald.  Integrated Approach  to Problem of Viruses
     in Water.   Journal of  Sanitary  Engineering  Division,
     American Society of Civil  Engineers,  p.  867-882,
     December 1971.

 9.   Heckroth,  Charles  W.   Reuse and Recycling:   What's Ahead?
     Water and  Wastes Engineering, p.  A-l -  A-11, January 1973.
                                 54

-------
10.  Is There a Protein Problem?  Protein Advisory Group of the
     United Nations System, Statement No. 20, March 1, 1973.
     WHO Chronicle, 27_:487-491, November 1973.

11.  Wall Street Journal, October 15, 1973.  Dallas, Dow Jones
     § Co.

12.  American Public Health Association.  Standard Methods for
     the Examination of Water and Wastewater, 13th edition.  New
     York, American Public Health Association, 1971, p. 657-660.

13.  ibid, p. 692-704.

14.  Edwards, P. R. and W. H. Ewing.  Identification of
     Enterobacteriaceae, 3rd edition.  Minneapolis, Burgess
     Publishing Company, 1972, p. 8 f.

15.  Hajna, A. A.  A new specimen preservative for gram negative
     organisms of the intestinal group.  Public Health Laboratory.
     13:59-62, 83, 1955.

16.  Lennette, E. T. and N. J. Schmidt, ed.  Diagnostic Procedures
     for Viral and Rickettsial Infections, 4th edition.  New
     York, American Public Health Association, p. 560 f, 1969.

17.  Wallis, Craig, and Joseph L. Melnick.  Mechanism of Enhance-
     ment of Virus Plaques by Cationic Polymers.  Journal of
     Virology.  American Society for Microbiology, 2_: 267-274,
     April 1968.

18.  Wallis, Craig, Joseph L. Melnick and Joseph E. Fields.
     Detection of Viruses in Large Volumes of Natural Waters by
     Concentration on Insoluble Polyelectrolytes.  Great Britain,
     Water Research, 4_: 787-796, 1970.

19.  Metcalf, T. G., J. M. Vaughn and W. C. Stiles.  The Occurrence
     of Human Viruses and Coliphage in Marine Waters and Shellfish.
     FAO Technical Conference on Marine Pollution and its Effects
     on Living Resources and Fishing, Rome, Italy, December 1970.

20.  Shuval, Hillel I., and Nachman Gruener.  Health Considerations
     in Renovating Waste Water for Domestic Use.  Environmental
     Science $ Technology, 7_: 600-604, July 1973.
                                  55

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              MORBIDITY RISK FACTORS

                        from

              SPRAY IRRIGATION WITH TREATED WASTEWATERS

                        by
              Geoffrey B. Stanford*    and    Rafael Tuburan*
              MRCS   HMR   FRPS               B.  A.
In this paper we present the interim conclusions of an ongoing
literature review.  We have tried to find out whether properly treated
wastewaters from a properly designed and correctly operated wastewater
treatment plant, which provides at least secondary treatment, are safe
from the health viewpoint.  In particular, we wished to find out whe-
ther aerosols generated from these treated wastewaters present a signi-
ficant pathogen risk.  If not, we believe, their use on land for irri-
gation and for their fertilizer value can and should be encouraged for
a variety of environmental, economic, agricultural, and social reasons.

In our search we have adopted the antagonist attitude.  We have sought
to prove to ourselves that there are valid reports which show that di-
sease has occurred from this practice in the past, and therefore that
the practice must be controlled.

Since we have not yet found any such reports, we continue to recommend
that the many advantages known to accrue from this practice outweigh
the apparent chances of a health risk,  and that the practice should be
encouraged.  We will now show our reasoning, based on the findings we
have examined so  far.
 • School of Public Health, University of Texas at Houston

                                   56

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FINDINGS

Raw Night Soil

There is no room for doubt that, in an area where an enteric infection
is endemic in a human population, one important factor by which ende-
micity is maintained is the practice of applying fresh raw sewage as a
fertilizer on to crops which are eaten fresh and raw by man.l  There is
continuing 3Jdscussion whether this same practice helps to maintain re-
sistance to the disease in the resident population.  But that is irre-
levant to the argument that the practice transmits disease to visitors,
and therefore is a public health risk.

We have not yet found documentation whether pulmonary infection can be
spread in this way.  There is published evidence that bathing in sur-
face waters which are contaminated by human fecal matter may have cau-
sed disease.2

Urban Wastewaters

In urbanized communities where the flushing toilet and sewer system
are in use, the human waste load is subjected to a dilution of some
200-2000 times - or more, if the storm drains are combined with the
wastewater sewers.  Further, in properly sewered urban societies, the
endemic morbidity, and hence the average pathogen load per person, is
reduced by a factor of some 10-100 times.  These two factors combined
produce a total pathogen reduction per unit volume of between 2 x 10^
and 2 x 105 when raw modern city sewage is compared with raw night
soil applied to crops or to land.  A further reduction in viable patho-
gen load from urban sewers is caused by the extended time-delay during
transport from source to crops.

Some cities have continued or have re-introduced^ the 'sewage farm1
procedure first practiced in the late l800's.  In this technique the
raw sewage is applied year after year to a limited area of land, which
is used for crops or for pasture.  In spite of the large quantities
which have routinely been applied, in some cases over many years conse-
cutively, there are no recent reports of disease resulting.^  Indeed,
one such city reports that the carcasses of cattle grazed on this land
attract better-than-average prices in the market because of their high-
er quality.5

Sedimentation - It is generally accepted that treatment by primary
settling reduces the general pathogen load in the supernatant waters.
There is evidence that primary settling carries most of the viral load
into the sludge, probably by adsorption;0 this effect is accentuated
by chemical flocculants;' the degree of inactivation of this virus load ^
is not yet agreed.  The supernatant waters retain a significant proper- c\j
tion of the initial nonviral pathogens, and the balance settles with    o
the sludge.  The ultimate degree of any reduction  	

                                  57

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of the total initial load depends on the time delay during travel in
the sewers and in the settling system, and can be attributed to
normal die-off, possibly accentuated by the bacterial activity of
metabolic byproducts of non-pathogenic micro-organisms.

Aerobic Treatment - It is generally accepted that properly conducted
aerobic treatment, whether by biological filter or by activated sludge
tanks, in a plant which is not being overloaded or mismanaged, can re-
duce the non-viral pathogen load by better than 99?, although this is
seldom achieved in practice.

'When the effluent waters are properly chlorinated to the required stan-
dards, they are generally accepted to be safe for discharge into a
waterway when  judged by the criteria in force.  There is satisfactory
evidence that  such waters are acceptable to the public for irrigation
of agricultural soil, parkland, and golf courses,0 since several states
have  set criteria to regulate this practice.^10  Indeed, in many areas
today the more expensive developments, which include a golf course as
one of their amenities, are routinely requesting permits to irrigate
their courses  with the effluent  from their treatment plants.11  We have
 found no evidence of disease reported from this practice.1

Anaerobic Sludge - While there are reports of  disease being acquired
 from  sludge,13 it is likely that  these may have been primary  settled
 sludge, since  the majority of treatment  plants in  the  U.S.A.  are still
 confined to this simple procedure.

 There is  satisfactory  evidence that  secondary  anaerobic  treatment of
 sludges, properly conducted in a properly designed plant which is not
 being overloaded, produces a sludge which is acceptable  as safe for
 public use  as  a  soil improver without  further  disinfection or sterili-
 zation.  This  sludge has been applied wet (fresh)  onto farmland and on
 to city parkland11*  and children's play grounds.15   We  have found no  in-
 stance of reported  disease  from  this  practice.

 Further,  such  sludge,  after drying,  is  saleable16  as  a profitable ven-
 ture  over long periods of time both retail  and contract10 when pro-
 perly marketed.   We have  found no instance of reported disease from
 this  practice.

 Irrigation  - We  have found no  instance of disease reported from spray
 irrigation with chlorinated secondary effluent that has been properly
 treated ('green water').   This  is not surprising.   If pathogens were
 a risk from drift, spray, or aerosols generated from sewage, surely
 the clinical risk would be greatest where the concentration is great-
 est - in the sewage works, near the aerobic treatment unit?  We have
 found no evidence that long-term or newly introduced operators have
 disease attributed to this; indeed some experts have suggested that
 the morbidity of sewage plant operators is less than the morbidity of
 the general public.  Perhaps a more sensitive indicator could be the

                                   58

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adolescent school children visiting the sevage plant as a science
course field trip; a letter from the National Science Teachers Associa-
tion tells us that "From inquiries to the U. S. Public Health Service
and the Montgomery (Maryland) Health Department, we have learned of no
reported illnesses to students following visits to sewage treatment
plants, nor could we find that any guidelines have been developed or
are in practice as precautions for such visits.  Also, the student tours
director for the Washington Suburban Sanitary Commission told us that
no special precautions have been observed in their tour program, nor
did he know of any illnesses to students following tours of that facili-
ty. "'19

CONCLUSIONS

The Published Evidence

In short, our review of the literature has not yet provided an instance
in which satisfactory evidence is given that a disease incident has
arisen from using treated sewage products.  By treated we mean that the
evidence provided shows that during the days preceding the incident:
the treatment plant was operating within design capacity; that the unit
processes in that treatment plant were being operated properly and were
functioning properly; and that the effluent quality was within the cri-
teria required.  In other words, we suggest that before an incident can
Justifiably be attributed to the use of treated waste waters, it must
be shown that those wastewaters were being fully and properly treated
during the period of time which could have been implicated.  Until that
has been demonstrated one would be calling in question not the health
risks of using these materials, but the effectiveness of the design and
the operation of that plant.  And that demonstration should not be dif-
ficult in the future, even though it may, on occasion, have been so in
the past.

THE MAGNITUDE OF THE RISK

Infectivity

In actual practice, what is the likely risk that pathogens originating
in a sick person or a carrier will gain entry to a susceptible person?
In the U.S.A. the pathogen endemicity load is now low.  Will the com-
bined effects of attrition by die-off over time, of dilution into a
large volume of water, of attenuation by spread over a large area of
land, of sterilization by solar radiation, and of degradation by soil
organisms - will all these effects, working in series, reduce the real
risk to below the level of risk in a city street20 to below that of the
risk from food obtained from commercial sources, 1 or to below that in
an office building?  We can suppose so, but this also requires evi-
dence, and we have not yet found that in the literature.

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Spread - Much has been published about the effect of the configuration
of the spray-nozzles  of fuel injectors for reciprocating and for jet
engines, on the size distribution of the droplets produced.  There is
also information about the design of applicators for agri-drugs in the
field, and about the factors concerned in reduction of drift and of wind
spread:  the smaller the droplet, the further it travels: to reduce
drift, droplet sizes should be larger than 500 microns.2^  There is
literature which suggests that droplets often nucleate around a bacter-
ium; and that the physical changes which occur in the droplets during a
short time after its formation are complex:  these include relative
change of concentration of solutes across the diameter, and dessicca-
tion; it may be speculated that theE changes may be so fast as to pro-
duce effects comparable to freeze-drying, and so may preserve a patho-
gen rather than destroy it.  The free-floating spherule so formed be-
haves like a dust particle.  Droplets and dust can travel a considera-
ble height into the atmosphere before coming down again, 3 or they may
be blown long distances near ground-level before reaching their final
resting-place - 50 miles has been recorded for an aerosolized agri-
drug."  These effects, recorded in  the  literature from other special-
ties, require to be related to our questions.

 Susceptibility -  If we assume that a pathogen has  survived these ordeals
 and has  gained entry to a human being, what  are  the  chances of  its
 causing  an infection?   Will that  infection be  sub-clinical and  cause no
 apparent loss of  health?  and  if  so  will it  produce  another carrier?
 Or will  it cause  recognizable  disease which  will affect the patient  or
 society  adversely?  There is evidence that mere  entry is  not  always
 sufficient to start an infection;  the person has a battery of defense
 mechanisms,  and the  entrant pathogen is  more likely to be destroyed
 than not.  But there  is evidence  that although it requires many bacteria
 to produce  infection,2U a single  viral TCIDjo can do so if it achieves
 a susceptible target  - for  example,  the lung alveolus; in this instance
 small droplet size (below 2.5  microns diameter)  is a required factor.
 There is agreement that although bacteria and perhaps parasitic cysts
 can confidently be removed by conventional sewage plants the same can-
 not be said with the same confidence about viruses.

 HEED FOR INFORMATION

 We can summarize as follows:-
 1).  A literature search has not yet revealed any incidence of disease
 from irrigation with properly treated sewage products.
 2).  Irrigation techniques should strive to eliminate the formation of
 any droplets that are smaller than  500 microns in diameter.
 3).  Researches should focus on determining the viability of pathogenic
 organisms through the several attenuation pathways to which they are
 normally subjected, so that we can  quantify the actual risks of infec-
 tion.  Then we can work to minimize those risks if they prove to be
 significant.

-------
DISCUSSION

We recognize that in this paper we have posed more questions than ve
have provided evidence or answers - and properly so.  It has been our
purpose to reveal those questions, and so, hopefully to cause the need-
ed investigations to start.

Nevertheless, the existing information does not warrant any restrictions
on the use of properly treated wastewaters for land application; "but it
does support a case for careful management of sewage plants whose pro-
ducts are to "be used in this way.  Both the "better management of waste--
waters, and the greater use of the treated end-products are commended
by government and professional2" agencies as a national goal; they are-
therefore likely to be achieved soon.  Our investigations so far conf:irm
the need for both.

This brief paper is not claimed to be exhaustive, nor are our findin/gs
conclusive:  it is designed more to share with you the large areas of
doubt that exist both for and against there being a significant patho-
gen risk.

At the International Conference on Land for Waste Management held iin
Ottawa last October, a group of conferees came to the following conclu-
sions :-
1.  NEED FOR COORDINATED INFORMATION CENTER
Nobody present knew of a coordinated bibliography or information refer-
ence center which meets the present needs of administrators, field  wor-
kers, and research workers, about the pathogen risks from use of treated
wastewater products.  This need is growing.

2.  NEED FOR COORDINATED RESEARCH
The information so far gathered by those who were present is inconclu-
sive and/or inadequate to support policy recommendations or decisions
at field, administrative, or guideline legislative  levels.  Basic  re-
search is urgently needed.  Over-restrictive legislation based on  cau-
tion might soon be introduced which could obstruct  trial of innovative
procedures unless such basic information quickly becomes widely avail-
able.

3.  SCOPE
These information deficiencies apply to concentration  and/or dissemina-
tion  of pathogens from municipal  sewage treated  effluents,  from .landf.'ill
operations, from resource recovery  systems,  and  from animal wast-es.  In-
formation from these  sources is  also lacking about  the health of  the:
operators who  are directly working  with the  materials  and proces ses ,
and of the health or  disease implications  for the public at  largo, for
domestic  animals, and for  game  and  wildlife;  furthermore,  concentration
and/or dissemination  of  plant pathogens should also be included  i'.n the
enquiry.

-------
Bibliography

It was agreed that it would be desirable to set up a central reference
bibliography, and to provide the information as required.  That biblio-
graphy is now being compiled from information still being sent in.   You
are invited to join in the endeavor by sending in the citations of your
own bibliography, preferably with your annotations or reviews.  Sending
your citations is one certain way of ensuring that you will receive
copies of the compilation.*

Research Program - Future research may well be conducted under the
following headings:-
1 .  Evidence of  morbidity from aerosols that have originated from trea-
te-d effluent wastewaters, established under the criteria we have sug-
gested.

2.  Information  on the travel of aerosols, and their spread or capture
under  field conditions.

3.  Information  on the survival of pathogens in aerosols, and of their
inactivity after  reactivation.

U.  Environmental  factors that perturb  these prime  findings.

Only when we have  a substantive body of information on at least those
points, will we  be able  to  proceed to the real tasks before us:

5.  Evaluation of  the risks; and,  finally

6.  Preparation of recommendations,  guidelines,  and model  codes.

A projjram of this  magnitude can only be accomplished by numerous teams
working together to an agreed schedule.  It is our hope that  the infor-
mation, flow into and from this  study group will  encourage  that co-ordi-
 nation ; we welcome your comments.

 This puper is a summary of one  section of a larger three-part work
 presently in preparation.   At this stage it must be regarded as an in-
 terim  clraft, which we have presented today for your comments and im-
provements.

Please send information to Dr.  Geoffrey Stanford, School of Public
 He.alth,, P. 0. Box 20186, Astrodome Station, Houston, Texas  77025,
 U.t».A.
                                    62

-------
 1.  Sepp, Endel, The Use of Sewage for Irrigation  - A Literature Review
     Bureau of Sanitary Engineering, Dept.  Pub.  Health,  State of Cali-
     fornia (19T1).

 2.  Hawley, H. Bradford, Morin, David P.,  Geraghty, Margaret E., Tomkow
     Jean, Phillips, C. Alan, Coxsackie Virus  B  Epidemic at a Boy's
     Summer Camp., JAMA 226: 1 (October 1,  1973)  33-36.

 3.  e.g., Braunschweig, Germany; where after  some  years on a local
     scale, the system has been extended to a  regional program.

 U.  Wilson, Harold.  Some Risks of Transmission of Disease During the
     Treatment, Disposal, and Utilization of Sewage, Sewage Effluent,
     and Sewage Sludge.  J. Proc. Inst. Sew. Purif. , 19kh. 21^-239.

 5.  Melbourne, Australia.

 6.  Lund E. (1971) Observations on the Virus  Binding Capacity of'Sludge,
     Proc. of the 5th Int. Water Pollut. Res.  Conf.   I-2U, 1-5.
     London.

 7.  Lund, E. (1973) On the Isolation of Virus from Sewage Treatment
     Plant Sludges.  Water Research 7, 863-871.

 8.  Bruvold, William C. and Ward, Paul C.   Using Reclaimed Wastewater -
     Public Opinion  JWPCF hk:9  (September, 1972).  1691-1696.

 9.  e.g., Texas State Department of Health.   In November, 1973, 123
     Texas sewage plants were supplying effluent treated water for irri-
     gation, and sludges were supplied for  fertilizer dry from. 25^
     plants, liquid from 12, and by commercial sale from 10.

10.  e.g. , California State Department of Health; Statewide Standards
    for the Safe Direct Use of Reclaimed Wastewater for  Irrigation and
    Recreational Impoundments; California Administrative Code, Title 17;
    Department of Public Health, Berkeley,  California.

11. Lewis Clarke, landscape architect; personal  communication.

12. e.g., Merz, Robert C., 1955.  A Survey  of  Direct Utilization of
    Waste Waters.  Calif. State Water Poll. Control Bd.  Publn. Ho. 12.
    and Merz, Robert C. , 1956.  Report on Continued Study of Waste
    Water Reclamation and Utilization.  Calif. State Water Poll. Con-
    trol Bd. Publn. Ho. 15.

13. Dixon ,  Fritz R. and McCabe, Leland J.   Health  Aspects of Waste-
    water Treatment, JWPCF 36:8 (August, 196U).   98^.989.
                                  63

-------
lA.  e.g., Mission Bay Park,  San  Diego,  California.

15.  e.g., Vinton Bacon, Metropolitan Sanitary District of Greater
     Chicago, 1970; personal  communication.

l6.  Committee on Sewage Disposal (Am. Pub.  Health Assoc., Pub. Health
     Eng. Sect.)  The Utilization of Sewage  Sludge as Fertilizer,
     Sewage Works Journal 9:6 (Nov.  1937)  pp. 861-912.

17.  e.g., Milorganite, sold  retail  by the City  of Milwaukee, Wisconsin.

18.  e.g., City of Houston, Texas.

19.  1973, personal communication.

20.  Wright, T. J., Greene, V. W., Paulus, H. J., Viable Microorganisms
     in an Urban Atmosphere,  J. Air  Pollution Control Assoc.  19:337
     (1969).

21.  Center for Disease Control:   Foodborne  Outbreaks, Annual Summary
     1972; DHEW #CDC 7^-8l85; November,  1973.

22.  Akesson, Norman B.; Yates, V7esley E., and Christensen, Paul, 1971.
     Aerial Dispersion of Pesticide  Chemicals of Known Emissions,
     Particle Size and Weather Conditions.


23.  Gregory, P. H., Microbiology of the Atmosphere, New York,  1973.

2U. "Plotkin, Stanley and Katz, Michael.  "Minimal  Infective Doses  of
     Viruses for Man by the Oral Route."  IN Transmission  of Viruses
     by the Water Route (G. Berg., ed.), New York,  1966, 151-166.

25.  Hatch, T., Pulmonary Deposition and Retention  of Aerosols, New
     York, 196U.

26.  Joint Resolution of the Water Pollution Control Federation,  and
     the American Water Works Association, November 7,  1973.

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   PERMISSIBLE LEVELS  OF  HEAVY METALS  IN  SECONDARY EFFLUENT FOR USE
      IN A  COMBINED  SEWAGE  TREATMENT-MARINE AQUACULTURE  SYSTEM I.
                 MONITORING DURING  PILOT OPERATION*

                                 by

                     W.  B.  Kerfoot  and S. A.  Jacobs
     Domestic wastewater  inherently  contains  a higher  load of metals
than the original  source  water due to  the  treatment  of water for algae
control, leeching  from  pipes, and solutes  added during household
use1*2.  While nutrient-rich  secondarily-treated wastewater can serve
as a fertilizer for aquaculture  of commercially valuable algae and
shellfish3»^>5> excessive levels of  metals in solution may be toxic
to the cultured organisms,  accumulate  in the  meat of food products to
an extent  to pose  a danger  to public health if consumed, or impair
the visual appeal  and taste of the meat.   The question arises, does
the elevated metal content  of a  secondarily-treated  wastewater render
it unsuitable for  aquacultural purposes?   If  not, what levels of
heavy metals in the effluent  are acceptable for aquacultural usage
since domestic effluents  from industrialized  regions tend to contain
more elevated concentrations  than those from  rural sources^?

     Within this paper  we address the  first question,  citing the
levels of  cadmium, lead,  copper, zinc, chromium, and nickel observed
in the effluent, seawater,  and oysters during operation of a pilot
sewage treatment-marine aquaculture  system.   Guidelines for acceptable
levels of  metal in the  effluent  for  use in an aquaculture system are
developed  later in the  companion paper.

        THE WASTEWATER  TREATMENT-MARINE AQUACULTURE  SYSTEM

     Secondarily-treated  wastewater, diluted  with seawater, makes an
excellent  medium for growing marine  phytoplankton.   The phytoplankton
is, in turn, removed from the water  by filter-feeding  shellfish, con-
verting the organically-fixed nutrients into  valuable  protein (figure
1).  Nutrients regenerated  directly  from the  excretion or indirectly
from faecal deposits of the shellfish are  removed by the growth of
macroscopic algae  in tanks  before discharge of the effluent to coastal
water.
                                                       3
     As described  in detail previously by  Ryther et  al.  and Goldman
et^ jil^7, the system has several distinct advantages  over previous
*
 Contribution No. 3305 of the Woods Hole Oceanographic Institution.     c\i
 This study was supported by NSF(RANN) 32140 and the Woods Hole         °
 Oceanographic Institution.

                                   65

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algal processes for nutrient  stripping.  First,  removal  of  algal bio-
mass is accomplished by using the  algae  as  a food  source rather than
an end product which must  be  disposed  of.   Secondly,  the herbivores
represent a highly desirable, and  economically important protein
source.  Thirdly,  the  process lends itself  to the  development  of a
multi-component  food-chain system  useful for the production of many
commercially  valuable  food crops.   For instance, a town  of  50,000
people with a 126 acre aquaculture-sewage  treatment system  could raise
an  annual  crop of over 900 tons of oyster meat,  worth in today's market
upwards  of  5  million dollars  as a  luxury table oyster or 1  million
dollars  as  canned or frozen products.

      The design of the system is fairly simple.  Effluent is added  to
 filtered seawater at a ratio of 1:4 (figure 1).   Phytoplankton cul-
 tured in this mixture flows into tanks of shellfish where it mixes
 with diluting seawater at a concentration of 1 part culture to 19 parts
 100/i-filtered seawater.   The remaining water flows full strength
 into the macroscopic algae tanks and then,  finally, is returned to
 the coastal waters.   Other ratios  of effluent:seawater ranging from
 1:10 to 1:0.5 and ratios of algae  culture:seawater ranging from 1:1
 to 1:10 have been used.  The above conditions applied to experiments
 performed during summer 1972 and winter 1973.

      During the operation of the pilot project, the dominant species
 in the culture vats were always diatoms - usually either Phaeodactylum
 tricornutum or Chaetoceros simplex, often interspersed with smaller
 numbers of assorted diatoms,  flagellates,  and green algae.   The oyster
 culture system was a 760 liter rectangular tank (265 cm long,  62 cm
 wide, 53 cm high), constructed of  fiber glass-coated wood.   The
 oysters, numbering 500 in total, were positioned on trays of Vexar
 mesh.  Overflow from a standpipe in the shellfish tanks was introduced
 into another rectangular wooden tank which held seaweed (Chondrus
 crispus and Ulva lactuca) for final removal of nutrients from the  water
 before discharge.

                     TRACE METALS IN THE SYSTEM

      The metals selected for this  study are capable of being concen-
 trated in the tissue of marine organisms:  cadmium, which has been
 shown to cause kidney failure in humans and the "Itai-Itai disease"
 if present in sufficient quantities in consumed products ;  lead, which
 may cause encepholopathy, anemia,  and renal damage if present in high
 concentrations in meat^;  copper, which may be toxic to algae10, lead
 to discoloring and bitterness of oysters11, or may be toxic to human
 consumers if present in high concentration in the meat12; zinc, which
 may interfere with growth or organisms in the culture system if
 present in high enough concentrations10, but does not present a danger
 to consumers1-*; chromium, which may be carcinogenic at elevated con-
 centrations in foodiS, and nickel, which may be toxic to algae and
        but whose effect on consumers of meat is uncertain".

                                    66

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'
 I
                                FILTERED SEAWATER 80%
                                '^
             SECONDARY EFFLUENT 20%
                                                        DAILY BATCH PHYTOPLANKTON
                                                                        HARVEST
               SECONDARY EFFLUENT
                   SUPPLY TANK
  MARINE PHYTOPLANKTON
      CULTURE SYSTEM

FILTERED SEAWATER- 95%
SEAWEED GROWTH
      TANK
                                               SHELLFISH TANK
              FINAL
         EFFLUENT TO COASTAL
         WATER OUTFALL
                                                      PHYTOPLANKTON
                                                         FEED TANK
                                                                PHYTOPLANKTON FOOD-5%
         FIG. 1   SCHEMATIC FLOW  DIAGRAM OF A COMBINED NUTRIENT REMOVAL-MARINE AQUACULTURE SYSTEM

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                               PROCEDURE

     The object of the study was to determine  the metal  content of
the secondary effluent,  the dilution expected  by seawater  in  the
system, and the rate  of  accumulation of metal  by the  (cultured)
oysters.  Oysters have frequently been used  as indicators  of  metal
pollution because of  their higher sensitivity  to elevated  levels  of
many heavy metals^,16^  compared to other species of  shellfish*?.  The
rate of increase of metal, if  present, will  indicate  the final content
of metal expected at  the termination of  culture, important for public
health considerations.

     Determination  of the content of metal dissolved  in  the secondary
effluent was  performed by direct atomic  absorption  when  the metal con-
tent was  sufficiently high,  as often  in  the  case of copper and zinc.
Analyses  of other  elements in  effluent and in  seawater were done  by
the APDC-MIBK extraction procedure  used  by Brewer,  Spencer and Smith18.
With effluent, more MIBK must  be used  because  of its  increased solu-
bility,  as specified  in  the  EPA standard methods manual19.

      The oysters (Crassostrea  virginica)  came  directly from the  shell-
 fish tanks which held 500 individuals.  Each tank  received daily  595
jag carbon/liter of algae contained  in  500 liters  of culture20.   To
 establish the initial concentration of metal in the oysters,  eight
 individuals were removed at the beginning of the  experimental period
 and analyzed.  Later samples of six individuals each were taken  from
 the shellfish tanks after 45 days,  78  days,  and 102 days of exposure
 to the sewage-grown algae.

      For wet-ashing,  the entire body  of  an oyster  was rinsed  with dis-
 tilled water, weighed and transferred  to an  aqua-regia-washed 125 ml
 Erlenmeyer flask.   Digestion was  performed by adding 10 ml of concen-
 trated nitric acid to the sample,  boiling to dryness, and allowing to
 cool.   Then 10 ml of 30% hydrogen  peroxide was added and the  sample
 re-dissolved in a 57. nitric  acid  solution made up  of a standard  volume
 for analysis by atomic absorption.

      All analyses  were done  on a Jarrel-Ash Model  800 Atomic  Ab-
 sorption Spectrophototneter (AAS),  a dual-beam channel instrument with
 recorder printout.   The  most sensitive channel was set to the principal
 analytical wavelength.  The  second  channel was set to a nearby non-
 absorbing wavelength, using  the "B/A"  mode of the  instrument21,  to
 provide correction for matrix  effects  and background scattering.

      Efficiency of the digestion  procedure was evaluated by determining
 recovery and reproducibility.   Known  portions of metal were added to
 fifteen five gram amounts of homogenized oyster tissue, with five un-
 treated samples of 5 grams each used  for background levels.  The metals
 were added as zinc acetate,  copper sulfate,  nickel sulfate, cadmium
 iodide, lead acetate, and potassium chroroate.   Recoveries of Zn,  Cu,
 Ni, Cd, Pb, and Cr were  97±2%, 95±5%,  98±3%, 98±5%, 96±2%, and 99±3%.

                                   68

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                                RESULTS

     Chemical analysis did not reveal high concentrations of poten-
tially dangerous metals in the effluent obtained from the Otis Air
Force Base Sewage Treatment Plant and used in culture (table 1).  The
metal content generally ran less than considered average for secon-
dary effluent", although the copper concentration infrequently rose
above .11 ug/ml (ppm) and occasionally above the range expected for
average effluent.  With the exception of nickel, the concentration of
each metal in the effluent generally ran 8 to 150 times the level in
the seawater used for dilution.  The nickel content of seawater was
similar to that of the Otis effluent.

     The concentration of metal in the cultured oysters showed a con-
tinual decline from the 45th day of culture onwards (figure 2).  The
apparent increase in concentration during the first 45 days of cul-
ture is probably due to our choice of large individuals for the back-
ground metal determinations.  The later random removal of individuals
being monitored for growth indicated that as weight increased, the
metal content decreased.  Although not premeditated, the mean weight
of the samples taken at the termination of culture was similar to the
mean of the original samples taken for background  (5.68 to 5.18 gms
wet meat weight, respectively).  A t-test of the difference between
mean concentrations at the start of exposure and at the finish re-
vealed no significant differences.  With zinc, copper, and lead the
total content of metal per individual remained essentially constant,
despite changes in weight, while a slight decrease in total metal con-
tent occurred with cadmium, chromium, and nickel.  Overall, the
oysters in the tanks showed slightly over a 50% increase in average
weight during the monitoring period, from an initial dry meat weight
of  .425 gms to a final  .676 gms20.

     The  original and  final concentrations  of metal in  the meat are
also compared with alert  levels discussed by the Massachusetts Division
of Water  Pollution Control16.  The alert level  concept was developed
to be used as a guide  or  indicator of metal pollution in  shellfish
growing waters and was not intended  to  reflect  the toxicity of metals
contained in  the shellfish.  Only  in the case  of  lead was  any  indica-
tion of contamination  given,  the baseline value being above the pro-
posed state alert  level  (table  3), presumably  occurring  before  ex-
posure to sewage-grown algae.

                              DISCUSSION

     Lack of  accumulation of metal  can be  traced  to  three  factors:
1) Dilution  of  the  effluent  by  seawater, 2)  the  relatively low metal
content  of  the  Otis  effluent,  and  3)  the  increased growth of  the
oysters.  Dilution plays  by  far the  most  important role  of these
 factors  although the mixing  of seawater and effluent was  determined

                                   69

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      Table 1.   Constituents of Otis secondary effluent compared

            with the Washington report "Average" effluent^
             Material
                                               Concentration (ppm)
                                               Secondary effluent
 Washington
   Report
   Otis
   Plant
Organic content (particulate carbon)
                (dissolved organic)

Nitrogen (total as N)
   Ammonia (as N)
   Nitrite (as N)
   Nitrate (as N)

Phosphorus
   Phosphate phosphorus (as P)

Trace metals
   Chromium
   Copper
   Lead
   Zinc
   Cadmium
   Nickel
    25
  10-30

    20
    10
.02-.14
.07-.14
.01-.03
.20-.44
.01-.03
.03-.20
   12-30
    40

    17.6
    15.2
      .4
     2.0
     8.2
.003-.040
.025-.300
.005-.020
.035-.095
.0005-.0023
.0005-.010
                                  70

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  2OOO
            20
                40
                  60
                             8O
                               1OO
                                         0
       - ZINC
i
16OO

12OO-


8OO*
g
•S* 4OO
r
i
i.
   2OO


   16O
*
K

  5O


   0

  2O


  15

  10
  8


  6


  4
        COPPER
LEAD
        MEAN WEIGHT
            2O     40    6O     8O

           TIME-DAYS OF CULTURE
                                            2O

                                        CADMIUM
                                                     4O
                                                    60
                                                                  80
                                                                 100
                                          CHROMIUM
                                          NICKEL
                                       MEAN GROWTH
                                                                            3 b
                                                                            i
                                                                             31K
                                                                            •t
                                    100       2O     4O    60     8O

                                          TIME-DAYS OF CULTURE
                                 0
                                                                 100
      Fig 2  Metal concentration //? tissue of oysters during 1O2 Days of
              culture.   Mean met a/ concentration in mg/gm (ppm) wet
              weight and one standard deviation are included.  The mean
              weight of the individual oysters sampled and their growth,
              as mean increase  of length of shell is also shown
                                        77

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by culture process and not  originally  intended  for lessening metal
contamination.  The dilution  of  effluent  to seawater for the algae
culture mixture was based on  earlier experimental  results which indi-
cated that yields  of  algae  were  roughly proportional to the concentra-
tion of sewage effluent  in  the enriched seawater medium up to  concen-
trations of about  20% sewage  and 80% seawater^.  The dilution  of cul-
ture mixture with  20  parts  seawater as it is delivered to the  shellfish
tanks was to provide  a sufficient volume  flow of seawater to maintain
a high level of dissolved oxygen to the shellfish-*.

     With a very  low  background  metal  content,  seawater substantially
dilutes the original  metal  levels in the  effluent  (table 2).   For
comparison, the amounts  of  dissolved metal often introduced inadver-
tently as contaminants in media  prepared  for algae culture with
artificial  seawater and  nutrients from analytical  grade reagents, is
equal  to  or greater than the  levels of metals present in the algae
cultured  prepared  from Otis effluent^.

     A total dilution of 1:200  occurs  before contact of the effluent
with shellfish.   This is almost  sufficient to reduce the levels of
metals in the  effluent to the background  levels in the seawater (table
2).  Even with the element  copper, which  at times  reaches a high con-
centration  in  the  effluent  (300  jag/liter), the  maximum level which
occurs in water reaching the  shellfish is 12 ^ig/liter, just slightly
above  the maximum level  of  copper (9 ,ug/liter)  recorded in the sea-
water used  for culture.

     Secondly, the metal content of the Otis effluent, resembling
domestic wastewater in character rather than an industrial source,
was  not particularly  elevated.   Only copper, of all the elements
studied, was as high  or  higher than the concentrations expected in
average effluent as assumed in the Washington report^ while the
chromium and lead  content occasionally fell within the average range,
zinc, cadmium, and nickel at  their highest observed levels were less
than the minimum  level anticipated in  average effluent.

     Finally,  the  increased rate of growth of the  oysters probably
contributed to the observed decline of metal content in the latter
days of culture.   For instance,  despite the change in lead concen-
tration from 4.2 + .2 >ig/gm wet  weight initially to 10.1 + .5  at 45
days, 5.4 +2.2 at 80 days, and  4.1 +  2.2 at 102 days, the total lead
content of the individual oysters,  was relatively  constant at  21.6 +
12.0 ug, 20.6 + 14.8, 19.0 ±  13.0,  and 24 ± 12.6,  respectively.
Copper, and to a lesser  extent zinc showed the  same  constancy  of in-
dividual metal content despite fluctuations in weight and concentra-
tion of metal.  In contrast, with cadmium, which previous studies had
indicated a very slow loss, if any,  within 30 days following exposure
to a high concentration24>  some  loss did  definitely  occur after 102
days of culture that  was not due to weight change.
                                   72

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Table 2.  Concentration of metals in solution in secondary effluent, seawater,
and subsequent dilution in algae culture and shellfish tanks.  For comparison,
the content of metal found in synthetic algae growth media is indicated next
                   to that expected in the culture mixture


                      Metal concentration jug/liter (ppb)
Element
Zn
Cu
Cd
Cr
Pb
Ni
Otis
Effluent
35-95
25-300
.5-2.3
3-40
5-20
.5-10
Seawater
5-12
2-9
.06-. 08
.3-. 5
.02-. 8
1-5
Algae
Culture Media
11-28
7-67
.15-. 52
.9-8.5
1.0-4.6
.9-6.0
Ri ley's
Media
150
34
nd
12
31
8
Shellfish
Tanks
5-16
2-12
.06-. 10
.3-. 9
.07-1.0
1-5

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     As concentration of a metal in tissue is a ratio between the
total metal content and total weight of the organism, even though a
steady increase in total metal content may occur, if the rate of in-
crease in weight is greater, the concentration of metal in the tissue
will decrease.  For example, the mean total copper content of in-
dividual oysters raised from 375, to 304, to 433, and finally to 438
ug on the successive days of sampling while the concentration in
ug/gm wet weight (ppm) declined noticeably from the 45 to the 102 day.
In the aquaculture system, where growth of the shellfish is acceler-
ated, new tissue is added at a higher rate than that observed normally,
serving as a mechanism to dilute the concentration of some metals which
may  continue to be accumulated at a regular rate regardless of growth.

     The 102-day study indicates that oysters can be cultured with
the  use of secondary effluent as a substitute fertilizer without
raising the danger of metal contamination.  While other potential
contaminants,  including pathogens and organic compounds, need further
investigation  to evaluate the advisability of substituting secondary
effluent for artificial media in aquaculture, the initial results
suggest that the inherent elevated metal concentrations in domestic
wastewater do  not appear to pose a threat to shellfish culture if
effluent of a  principally domestic source is used.

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Table 3.  Accumulation of metals in oysters of pilot
  tertiary treatment-aquaculture system fed sewage-
grown algae for 102 days.  Mean metal concentration
     and one standard deviation are indicated
          Metal content (ppm wet weight)
Element
Zn
Cu
Cd
Cr
Pb
Ni
Initial
818 + 446
72 + 18
2.00 + .30
.10 + .04
4.2 + .2
.29 + .13
Final
743 + 182
81 ± 18
1.30 + .26
.04 + .02
3.6 + 2.5
.21 + .10
State Alert
Level16
2000
175
3.5
2.0
2.0
Unspecified
                         75

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                             REFERENCES


 1.  Muskegon County  Board  and Department  of  Public Works.  Engineer-
    ing  feasibility  demonstration  study for  Muskegon  County, Michigan
    wastewater treatment-irrigation  system,  Program 11010FMU, Federal
    Water Quality Administration,  September, 1970.

 2.  Young, D.  R. , C-S. Young and G.  E. Hlauka.   In Cycling and  Con-
    trol of Metals;  Proceedings  of an Environmental Resources Con-
    ference.  National Environmental Research Center,  Cincinnati,
    1973, p. 21.

 3.  Ryther, J.  H., W. H. Dunstan,  K. R. Tenore and J.  E. Huguenin.
    Bioscience 22(3): 144, 1972.

 4.  Dunstan, W. M. and D,  W. Menzel. Continuous cultures of natural
    populations of phytoplankton in  dilute,  treated sewage effluent.
    Limnol. Oceanogr. .16(4):623-632, July,  1971.

 5.  Dunstan, W. M. and K.  R. Tenore. Intensive outdoor culture of
    marine phytoplankton enriched  with  treated sewage effluent.
    Jour. Aquaculture 1.: 181-192,  1972.

 6.  Jacobs, S. A. Some measurements of heavy metal content of
    sewage effluent  and sludge.  In  The Use  of Flowing Biological
    Systems in Aquaculture, Sewage Treatment, Pollution Assay,  and
    Food-Chain Studies.  J. H.  Ryther,  edit., Technical Report  WHOI
    73-2, December,  1972.

 7.  Goldman, J. C.,  K. R.  Tenore,  J. H. Ryther and N.  Corwin.   In-
    organic nitrogen removal in a  combined  tertiary treatment-marine
    aquaculture system.  I.  Removal efficiencies.  Water Research,
    In Press.

 8.  Kjellstrom, T.,  L. Friberg,  G. Nordberg  and M. Piscator.   In
    Cadmium in the Environment.  CRC Press,  Cleveland, Ohio,  1971,
    p. 140.

 9.  Kehoe, R.  A.   Metabolism of lead under  abnormal conditions.
    Archives of Environmental  Health 8:235-243, February, 1964.

10.  Bryan, C.  W.   The effects  of heavy metals (other  than mercury)
    on marine and estuarine organisms.  Proc. R. Soc.  Lond. Ser. B
     177:389-410,  1971.

11.  Roosenburg, W. H.  Greening and  copper  accumulation in  the
    American oyster, Crassostrea virginica,  in the vicinity of a
    steam electric generating  station.  Chesapeake Science  10(3&4):
    241-252, Sept.-Dec.,  1969.
                                   76

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12.  Pringle, B. H. and C. N. Shuster.  A Guide to Trace Metal Levels
     in Shellfish.  Shellfish Sanitation Tech. Rept.  USDHEW.  Public
     Health Service, 1967, 19 p.

13.  Browning, E.  Toxicity of Industrial Metals, Second Edition.
     Appleton-Century-Crofts, Butterworth & Co. (Publishers) Ltd.,
     London, 1969.

14.  Jones, J. R. E.  Fish and Water Pollution.  Butterworths,
     London, England, 1964.

15.  Huggett, R. J., M. E. Bender and H. D. Slone.  Utilizing metal
     concentration relationships in the eastern oyster (Crassos_tr_ea
     virginica) to detect heavy metal pollution.  Water Research 7_:
     451-460, 1973.

16.  Isaac, R. A. and J, Delaney.  Toxic Element Survey.  Progress
     Report No. 1, Massachusetts Water Resources Commission, Division
     of Water Pollution Control, Publication No. 6108, 1972, 25 p.

17.  Pringle, B. H., D. E. Hissong, E. L. Katz and S. T. Molawk.
     Trace metal accumulation by estuarine mollusks.  J. Sanit. Eng.
     Div., Amer. Soc. Civ. Engrs. 94:455-475, June, 1968.

18.  Brewer, P. B., D. W. Spencer and C. L. Smith.  Determination of
     trace metals in seawater by atomic absorption spectrophotometry.
     Atomic Absorption Spectroscopy, ASTM STP 443:70-77, 1969.

19.  Methods for Chemical Analysis of Water and Wastes, Environmental
     Protection Agency, Analytical Quality Control Laboratory, Cin-
     cinnati, Ohio, 1971.

20.  Tenore, K. R., J. C. Goldman and J. P. Clarner.  The bio-
     energetics of the oyster, clam, and mussel in an aquaculture
     food chain.  J. Exp. Biol. and Ecol. 12:157-162, 1973.

21.  Jarrel-Ash.  Atomic Absorption Methods Manual, vol. 1, Jarrell-
     Ash Corp., Waltham, Mass., 1970.

22.  Driver, C. H., B. F. Hratfiord, D. E. Spyridakis, E. B. Welch
     and D. D. Woolridge.  Assessment of the Effectiveness and
     Effects of Land Disposal Methodologies of Waste Water Manage-
     ment.  Seattle, Washington, R. F. Christman, Project Coordinator,
     Final Report, Contract No. DACW 73-73-C-0041, U.S. Army Corp.
     of Engineers, January, 1972.

23.  Riley, J. P. and I. Roth.  The distribution of trace elements in
     some species of phytoplankton grown in culture.  Jour. Mar.
     Biol. Ass. U.K. 51(l):63-72, 1971.

                                   77

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24.  Kerfoot, W. B. and S. A. Jacobs.  Cadmium accrual in a combined
     wastewater treatment-aquaculture system.  In Proceedings of the
     First Annual NSF Trace Contaminants Conference, W. Fulkerson,
     edit., Oak Ridge National Laboratories, Oak Ridge, Tenn., 1973.
                                    78

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  PERMISSIBLE LEYSLS OF HEAVY METALS IN SECONDARY EFFLUENT
 FOR USE IN A COMBINED SEWAGE TREATMENT-MARINE AQUACULTURE
SYSTEM II.   DEVELOPMENT 0? GUIDELINES BY METHOD OF ADDITIONS*

                             by

               W. B. Kerfoot and G. A. Redmann
     Shellfish can be cultured on algae raised with
secondarily-treated wastewater substituted for artificial
nutrients without any apparent accumulation of toxic metals
in their tissues^.  The effluent used was found to be rela-
tively low in background concentrations of metals, similar
to principally domestic sources using anaerobic treatment
procedures.  In many of our industrialized cities, however,
the effluent from a sewage treatment plant contains an array
of metals at substantially higher levels than that received
from solely domestic sources2»3.  Rather than test each
source individually, it would be worthwhile to develop
guidelines which serve to define suitable sources of
effluent for aquacultural purposes.  Within this paper we
attempt to determine the concentrations of six metals, zinc
(Zn), copper (Cu), lead (Pb), cadmium (Cd), chromium (Cr),
and nickel (Ni) that are permissible in a combined sewage
treatment-marine aquaculture system.

            TERTIARY TREATMENT-MARINE AQUACULTURE

     Nutrient-laden secondary effluent can become a nuisance
when discharged into coastal waters, stimulating algae blooms
with foul odor and contaminating shellfish beds.  On the
other hand, careful control of the quality of the effluent
to assure a pathogen-free high nutrient source will provide
a potentially valuable resource in the face of diminishing
supplies of food and artificial fertilizer.  Secondarily
treated wastewater, diluted with seawater, makes an excellent
medium for growing marine phytoplankton.  The phytoplankton
is, in turn, removed from the water by filter-feeding shell-
fish, converting the organically-fixed nutrients into
valuable protein.

     During the past three years, a prototype system of
tertiary treatment-marine aquaculture has been in operation
Contribution No.  3297  of the Woods Hole Oceanographic
 Institution.  This study was supported by NSF (RANN) 32140
 and the Woods Hole Oceanographic Institution.
                              79

-------
at Woods Hole Oceanographic Institution.  Various designs'
have been engineered to culture marine phytoplankton and
subsequantly raise shellfish, with any nutrients regenerated
by faecal material of the shellfish being removed by the
growth of macroscopic algae in tanks before discharge to
coastal waters^»5*°.

     A more detailed explanation of the system employed
during the summer of 1972, upon which these experiments are
based, is explained in the previous paper (Kerfoot and
Jacobs)''.  Effluent was added to filtered seawater at a
ratio of 1 :if.  Phytoplankton cultured in this mixture
together with diluting seawater was then introduced into
tanks of shellfish at a concentration of one part culture to
19 parts 100 u-filtered seawater.  At this dilution, the
carbon content of the seawater, an indicator of the food
value to the shellfish, is raised from a normal level of
100 ug carbon/liter to roughly 600 ug carbon/liter.  More
than 80/0 of the phytoplankton fed to the shellfish originates
from nutrients provided by the effluent, and about 1% of the
water reaching the shellfish is derived from the secondary
effluent.

PATHWAYS OF CONTAMINATION AND TOXICITY

     When metals are introduced with the effluent, the
algae nay accumulate metallic ions directly from solution by
absorption (here indistinguishable from adsorption).  Then
the algae mixture flows into the herbivore tanks, where the
shellfish may directly absorb ions from the considerably
diluted culture solution or through ingestion of contamin-
ated phytoplankton and detritus (figure 1).  In this study
the algae cultured was Phaeodactylum trieornutum, a. pennate
diatom commonly occurring during normal operation of the
aquaculture system.  The American oyster (Crassostrea
vir^inica) was chosen as the shellfish species because of
its potential economic value as a luxury food and the need
to assess its sensitivity to metals which may be added in
the effluent.

     The sensitivity of Phaeodactylum tricornutum to change
in metallic ion concentrations is well recognized.
Previously Hayward has shown that P. tricornutum initially
absorbs zinc rapidly, but the concentration in cells
decreases as their numbers increase?.  RLley and Roth found
that P. tricornutum under similar conditions accumulated
less tThan half the amount of metals that Hayward found°.
Nuzzi reported that mercury levels as low as .06 ug/liter
(ppb) were inhibitory to P. tricornutum, and that morpho-
logical abnormalities increased~with mercury concentration,
                              80

-------
                     METAL
                  IN   EFFLUENT
                   SEA WATER
         ABSORPTION
         INGESTION
ALGAE
            INGESTION
SHELLFISH
MAN
        ELIMINATION
       Figure 1.  Pathways of contamination in the treatraent-
              aquaculture system.

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the normally biradiate cells becoming highly vacuolated and
assuming an ovoid state9,

     Hannon and Patouillet noted  that growth inhibition by
a toxicant varies inversely with  the concentration  of
nutrients available.  They found  that the  exponential
growth of P. tricornutura was not  affected  by exposure  to
0.1 ppm lead or copper.  Exposure to 1.0 lead  had no effect,
while 1.0 ppm  copner  inhibited  the growth  rate to less than
half that of controls10.

     The ability  of the  American  oyster to accumulate  trace
metals from solution  is  also well-documented.   Soosenberg
 founr that greening of oysters  could be traced to excessive
uptake of copper  due  to  thermal effluent  from  power plant
activity'1.  Pringle  et. al. surveyed the  average trace
metal levels in  shellfish  from  the Atlantic and Pacific
 coasts and also  carried  out  studies on metal uptake and
 concentration  in  a  simulated natural system12.  They
 reported  that  lead  was absorbed by C. virginica in  direct
 proportion to  concentration  in  seawater,  accumulating  four
 ir.igrograms of  Pb  per  gram  v/et meat per  day when exposed
 continuously to  0.2 ppm  in seawater.  Average  metal back-
 grounds  in Atlantic oysters  were:  zinc,  14.28  ppm;  copper,
 92 ppm;  lead,  0.47  PPia;  nickel, 0.19 PP^J  chromium, 0.40 ppm;
 and cadmium, 3.10 ppm.   Kerfoot and Jacobs found linear
 cadmium  uptake by C virginica  from seawater in a deliber-
 ately  contaminated  sewage  treatment-aquaculture system'-?.
 The observed rate of  uptake  of  cadmium  (in ug/gm/day)  was
 17.8 times the concentration of cadmium  in solution (in
 ppm-ug/al).  With effluent as the source  of cadmium,  the
 principal means  of  uptake  was through  the ingested  algae.


                    MATERIALS AND METHODS
      Scale models of a sewage treatnent-aquaculture system
 were constructed in the seawater laboratory at V.'oods Hole
 Oceanosraphic Institution (figure 2).   Two-liter erlensiyer
 flasks were filled daily with a 4:1. mixture of one micron
 filtered seawater and filtered (.45 u) secondary effluent
 from the Otis Air Force ?iase sewage treatment plant.
 Effluent containing 10 ppm,  3 ppra, .3  PPE» -and .03 ppm
 equal concentrations of 3n,  Cu, Pb, ITi, Cd, Cr was added to
 the flasks v/ith one flask receiving unsupplemented effluent
 as a control.  The effluent-seav/ater mixture, as media, was
 dripped continually at 1.5 ir.l per minute or 2 liters per day
 into'4-liter flasks containing cultures of the diatom
 SDecies.  Water containing the cultured diatoms flowed at
 the same rate from the culture flasks  into trays holding

                               82

-------
  INFLOW COOLANT
      WATER
 FILTERED-7
 COMPRESSED AIR
 CONTINUOUS
 CULTURE
      FLASK
                 CULTURE MEDIA INLET
                        THERMOMETER
                        OUTFLOW COOLANT WATER
                                              SEAWATER INLET
                       COOLANT
                       CYLINDER
                           CULTURE
                           MEDIA
                            INLET
                                                        BAG
                                                        FILTER
                                                      .OVERFLOW

                                                       O WASTE
BUBBLER
STIRRING
     BAR
MAGNETIC STIRRER
         CULTURE MIXTURE
       SEAWATER INLET
WATER
OUTFLOW
                                            CONSTANT  HEAD>
                                               BOTTLE
                                   OUTFLOW TO SHELLFISH TANK
                                     OUTFLOW CULTURE MIXTURE


                                        AIR INLET
                            SHELLFISH TANK'

        Figure 2.  Scale rrodel treatment-aquaculture sy::ter
                            83

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oysters in two liters of water, where it was mixed with
25 nl per minute of filtered seawater supplied from a
constant head tank.

     The culture flasks were illuminated by a bank of
fluorescent bulbs providing approximately 500 ft-candles,
stirred by magnetic stirring bars, and maintained at
20* ±2* C by seav/ater flowing through glass tubing inserted
into the culture mixture.  The  culture also received air,
cleaned by a water trap and activated carbon, bubbled
continually through the mixture from an inserted pipette.
Four 20 liter plastic carboys were filled with ,/4-5 u-filtered
secondary effluent.  The stock  solutions were made up to
10.0, 3.0, 0.3» and 0.03 PP& (nig/1) with zinc from zinc
acetate (Zn fc^^Og 3-2H20) , copper from copper sulfate
( CuS04'5H20), cadmium from cadmium iodide (Cdl2), lead from
lead acetate (PbfC2H30p3»3H20),  chromium from potassium
chromate (J^CrOO, and nickel  from nickel sulfate (NiSO^.
6H£0).  Each day" of the study,  400 ml of effluent or metal-
enriched effluent  from the four carboys v/as mixed with 1600
ml of one micron-filtered seawater in the 2 liter media
flasks which supplied each algae culture flask.

     Fluorimeter readings were  taken daily on 10 ml samples
of the algae cultures.  Weekly  samples were taken of the
oysters and  frozen after being  placed in clean seawater to
void their intestinal t^cts.   Samples of the seawater and
effluent were removed periodically during the study for
analysis of netal  content.  At  the termination of the exper-
iment, two liter samples of the algae were removed  from the
culture, centrifuged, and dried on nuclepore  filters for
metal analysis.

     The procedure employed for wet-ashing was the same as
that reported by Kerfoot and Jacobs*»'-5.  The entire body
of an oyster was removed from the shell, rinsed with
distilled water, weighed, and transferred to aqua-regia
washed 125 ml erlenmyer flasks.  Digestion was performed by
adding 10 ml of concentrated nitric acid to the sample.  The
sample was allowed to stand in  acid for 12 hours at room
temperature, heated gently close to dryness, and allowed to
cool.  Then 10 ml of J>Q% hydrogen peroxide was added and the
sample heated to dryness.  After cooling, the sample was
redissolved in a 5% nitric acid solution, made up of a
standard volume for analysis, and aspirated directly into
the atomic absorption spectrophotometer.  All analyses were
done on a Jarrell-Ash Model 800 Atomic Absorption Spectro-
photometer, using a nearby non-absorbing line for background
absorption.
                                                              K

-------
     Phytoplankton samples on the filters were added to
10 ml of 56^ nitric acid in individual 20 ml test tubes.
After 48 hours digestion, the solution was filtered through
3 u nuclepore filters into 20 ml centrifuge tubes for direct
aspiration into the atomic absorption spectrophotometer.

     Metal analysis of the seawater and effluent was
performed using the techniques described previously by
Kerfoot and Jacobs'1 .  The samples were filtered and analyzed
directly by atonic absorption or were extracted and analyzed.
For solvent extraction,  four 500 ml samples of seawater or
effluent were taken, adjusted to a pH of 2.0, and then
extracted twice with MIBK-APDC.  Graded amounts of the
individual metal were added at levels 1,2, and 3 times
higher than anticipated  in solution.  The concentration in
solution could then be calculated following the method of
additions reported previously by Brewer, Spencer, and
Smith1^.  The concentration of background metals in the
effluent used for stock  solution was .080, .055, .005»  .002,
.010, and .005 PP^ for Zn, Cu, Pb, Cd, Ni, and Cr.  Metal
content of the seawater  and effluent supplied to the
cultures was reported earlier in the preceding article'.

     The culture system  was continued for 28 days, during
four of which the effluent media mixture did not flow,  for
varying reasons.  During this time the algal populations
fluctuated somewhat.  To maintain a suitable level, it was
sometimes necessary to supplement the experimental cultures
from stock cultures.

     To further define toxic effects of specific metals on
the phytoplankton, four  separate experiments were devised
with"batch cultures of P. tricornutum.

     First of all, eight 250 ml  flasks were prepared by the
addition of SO mis filtered seawater and the effluent  stock
solutions, two flasks at each multiple metal concentration.
These were autoclaved and aseptically inoculated with
6 x 105 cells for  each flask from a parent culture in
exponential growth.  All batch cultures were maintained in a
growth  chamber at  1 Ff C,  If50 ft-candles, and a light:dark
regime  of 13:11.   Samples of 1 ml were taken every other
day for 10 days, preserved in Lugel's solution and counted
in either a Speirs-Levy  or Palmer-Mahoney  counting chamber.

     A  second series consisting  of twenty-four 125 ml  flasks
with  50 ml of effluent-seawater  media vras  prepared with 6
different 5-metal  mixtures at 3  concentrations:  5 ppm,
.5 pern, and  .05 T>pm metals in the effluent.  Each  flask was
inoculated with k~.k x  10° cells, producing 8.8 x  10^ cells/
ml.  The  five-metal solutions were added  to  the  cultures

                              85

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three days after inoculation and samples counted at 5, 8,
and 1? days after inoculation.

     A third series to determine relative toxicity of each
metal consisted of eight 250 ml flasks, using 100 ml of the
£4.:"! seawater/effluent media, with six receiving a single
metal at a concentration of .5 ppn in the effluent and two
controls.  These were inoculated with 4.4 x 10^ cells.
Sanples were taken 4, 6, and 7 days after inoculation.

     A fourth experiment tested the relationship between
cell density and metal inhibition.  Six 250 si flasks
holding effluent/seawater media and the 3 ppm mixed metal
solution added to yield a solution concentration of .6 ppm
mixed metals.  Then .1, 1.0, and 10.0 ml of inoculum were
added to each pair of flasks, experimental and control.
The cells were sampled and counted 1, 3» and 4 days after
inoculation.
                           RESULTS
PHYTOPLANKTON TOXICITY
     During  the course of the study, the levels of algae
varied greatly (figure 3) in the scale-model treatment-
aquaculture  systems.  The concentration of metals in the
effluent media had little influence on the algae abundance.
The mean density of P. tricornutum in fluorescence units
(fu) was 1060, 1157,"1090, T360, and 11?8 fu, respectively,
for 10.0, 3«0, 0.3» 0.03 and control.  Reduction in the
P. tricornutum cultures occurred primarily from predation
by flagellates which passed through the 1.0 micron filters
and grazed down the diatoms.  However, the cultures supplied
with higher  concentrations of metals in the media exhibited
a more rapid decrease in cell concentration once a drop
began.  Also, the cultures receiving 10.0 and 3»0 ppm mixed
metals in their effluent would not recover without supplemen-
tal additions of culture.

     With a  series of experiments, the relative contribution
of each metal to toxicity was isolated (figure 4).  When
£• tricprnutum was cultured in a mixture of all six species
of metals, growth was inhibited when concentrations exceeded
0.3 ppra.  In all series with five-metal combinations, all
showed reductions in algae growth in cultures of low density
at .05 ppn and above concentration, except when copper was
removed from the combination (figure 4» middle).  The
remaining metals (Zn, Pb, Cd, Cr, and Ni) did not cause a
drop in algae growth until .5 ppra concentration and above in

                              56

-------
           tV.jC
kj  500
Uj 1500
S*  5OO
O  1500-t
                         10       15      20
                        TIME(DAYS)
        ripure 3.  Concentration of phytoplonliton (in fluorescence
                units) in culture flaskc durin~ operation of
                experimental systcns.  The concentration of netals
                supplemented in the effluent indicated for each
                culture.  Arrows mark additions of a 1,^,36 from
                stock culture.
                               87

-------
the effluent.  The metals having the principal toxic effect
in the five-metal combinations (minus copper) were Cd and,
to a lesser extent, Ni.  When metals were added individually
at .5 ppm concentration in the effluent, copper (Cu),
cadmium (Cd) and nickel (Ni) had a pronounced depressing
effect on algae growth.

     From the toxicity series, levels of metal above .3, .5,
• 5» .5» 1.0, and 1.0 ppm, respectively, for Cu, Cd, Ni, Cr,
Pb, and Zn may inhibit algal growth if present in the
effluent and would appear undesirable.  The limit of
toxicity found for copper agrees with the report of Mandelli
(1969) that the growth of four different marine diatoms was
inhibited by Cu levels in seawater greater than .05 ppm,
equivalent to .25 ppm Cu in effluent before dilution^?.

     Other effects observed on the diatoms were certain
morphological variations.  Nuzzi previously described
abnormalities of P. tricornutum induced by mercury9.  At
2.0 ppra in the culture media, corresponding to the use of
10.0 ppm mixed metals in the effluent, misshapen chromato-
phores and double nuclei occassionally occurred, as well as
"fat" or foreshortened cells of this normally biradiate
pennate diatom.  Somewhat shrunken cells and paired nuclei
were found down to the .3 multiple metal level.  A bifurca-
tion at one end of the cell or "fishtail" was often found in
all the cultures, including the controls fed with effluent
unsupplemented with metals.

     The influence of cell density on the expression of
toxicity was also apparent (figure 5).  Initial cell concen-
trations ranging over three orders of magnitude were added
to media prepared with 3«0 ppni each of the six metals in the
effluent.   As the cell concentration rose, the toxicity
decreased.  This supports the observation that the high
density of cells being cultured in the aquaculture system
suppresses toxicity until a drop in cell number occurs.  The
drop is then accentuated by elevated levels of metal intro-
duced in the effluent.  Concentrations of metals exceeding
the toxic levels suggested have an important effect in
decreasing the stability of the culture and its ability to
recover from any slumps in production.

OYSTER TOXICITY

     Few oysters died during the operation of the scale
model treatment-aquaculture systems.  In no case could the
isolated deaths be attributed to metal concentrations.  This
is likely due to the high dilution (lOOx) of effluent
before exposure to the oysters.


                              88

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£
5000

3000

 1OOO

   30

   20

    10

    0
        \O.5ppm SINGLE METALS
  |io<
  *»*j
  a
6 METAL MIXTURE (Zn,Cu,Pb,Cd,Cr,Ni)
                 • 0.3 ppm
                  CONTROL

                 -3.0 ppm

                 -10.0 ppm
                              CONTROL
                                 ,.05 ppm
         5METAL MIXTURE(Zn,Pb,Cd,Cr,Ni)
                        CONTROL
                        Cr
                        Zn
                        Pb
                        Ni
                        Cu
                        Cd
                       1	1	1	1	1	1
     0
                    5            IO
                    TIME (DAYS)
                                            J	L
                                      15
    Figure k-  Effect o
                               on diaton growth.
                          89

-------
ABSORPTION BY PHYTOPLANXTON

     All of the elements examined were acumula.ted by the
phytoplankton (Table 1).  At the highest concentration of
metals added in the effluent (10.0 ppm), equivalent to
2,000 ug/liter since it is diluted five-fold with seawater,
the respective percentages of metal removed from solution
by the algae (.1 gin/liter dry weight) were 59.0, 16.9,
14-9, 9.6, 4.6, and 2.5% for Pb, Zn, Cu, Cd, Ni, and Cr.
Five of the metals remained relatively stable in their
order of magnitude of accumulation, Zn>Cu>Cd>Ni>Cr. However,
lead (Pb) showed a rapid increase in percentage absorption,
ranging from undetectable at .03 ppm concentration to 59/S
of the total lead added in solution from the effluent
concentration of 10.0 ppm.

     Of interest to those who may feel that the use of
effluent as a media will cause high background levels of
metal in the cultured algae, the levels of metal in the
controls raised on unsupplemented effluent were generally
lower than those found by Riley and Roth for P. tricornutum
cultured in artificial media containing low levels of trace
elements .  Riley and Roth found background levels of 325,
110, 46.3, 6.2, and 4.4 ppm in dry olankton for Zn, Cu, Pb,
Ni, and Cr.  We found 120.8, 60.4, 14.6, 12.5, and 4.03 ppm,
respectively, for the same elements.  While it should be
kept in mind that Riley and Roth used a dc arc spectro-
graphic technique for analysis, thereby likely analyzing
some additional metal in the silicaceous skeleton of the
diatom, the levels are quite comparable.

ACCUMULATION BY OYSTERS

     An increase in the level of metals in the seawater/
effluent media, in general, caused a progressive rise in the
content of metal in the tissue of the oysters being cultured
(figure 6).  The points represent the mean metal analyzed
from three or four oysters.  The clearest pattern of
accumulation occurred in the elements of lesser affinity for
the oyster tissue, Cr, Ni, and Cd.  The concentration of
lead showed a more abrupt rise in level than the other
metals.  Cadmium was the only element to show detectable
accumulation at .03 ppra, hut also appeared to saturate at
the highest level (10.0 ppin).  Some accumulation of copper
occurred, but no definite uptake of zinc could be discerned.
With zinc and copper, to some extent, the amount absorbed,
even though it may have been large in quantity compared to
the other elements, was obscured by the high background
metal content of the tissue.
                              90

-------
10'
                            control
10°  :
I04
^ control


 x3.
0          1
                         2          3
                           TIME (DAYS)
                   4          5
     Figure r^.  Tnflunr.ce  oe cell density on  the  expression of
                toxicity.

-------
               Table 1.   Metal content of phytoplankton (utf/gm dry weight).
fle"tal Added
to Effluent
Control
.03
.3
3.0
10.0
5Sn
121*8
113*3
396*13
592*18
3510±290
Cu
60±4
63*8
159*13
634*79
3050*450
Pb
14. 6*. 7
11.2*1.8
57*21
551*127
11,800*1366
Cd
3. 4*. 6
14.2*1.1
124*3
574*104
1930±260
Cr
4.0±1.4
9.7*1.2
48*2
67*15
498*59
Ni
12.5*9.0
2. 6*. 8
• 82±7
161*19
932*53
Background
(Rileynand
 Roth)0
325
110
46.3
n.d.
4.4
6.2
n.d. - not determined

-------

^
• ->»
i^h
20
15
10

5
Oi

140
100
60
9n
cU
800

600
<
400
CADMIUM
0
.
--O ^x
^^-^**^*--* ^ ~~&
Q^z^~'^'_&-~^ ""
i-fT^^a*- — • ••IB-- 	 B
I 	 	 •^ 	
COPPER
\
"-«^z4^rT-Tffl . . -a


. ZINC
-A

L friir^ ,
NICKEL
X
X
x O
X ^,***^
x° °
^X^ A— — — A 	 A
t^...g 	 g 	 £)
	 5 	
CHROMIUM /X
.^—^ ^^ ^ rt
^8f^&:°^-'-fa '
' ~ ~ffl IsJ 1=

LEAD
•
^*^
, ^^— ,"
2.0
1.5
1.0




.3
.2
I


3

2
n
          0
10    15
0
10    15    20
           •-lOOppm in EFFLUENT
           o—30ppm in EFFLUENT
           A—.3ppm in  EFFLUENT
             D —,03ppm in EFFLUENT
             A—CONTROL
                                               ^
                                               MN
vi-'ure
                                    tion or r^etnls :rj oysters.

-------
     When the rate of increase was plotted versus the
concentration of metal added in the effluent, a disposition
of uptake similar to that encountered with the phytoplankton
occurred (figure 7).  Each point represents the mean rate of
uptake from each series in figure 6.  The order of affinity
for the four metals with roughly parallel slopes was Cu>Cd>
Ni>Cr.  The rate of uptake of lead (Pb) was over twice the
rate of the other metallic species and undetectable below
3.0 ppm. in the effluent.  The abruptness of accumulation
was similar to that observed with the phytoplankton.

     Although Cu, Cd, Ni, and Cr exhibited similar rates of
accumulation, the fitted lines all had a slope of less than
1 .0.  This indicates that the magnitude of metal uptake
decreases v/ith increasing concentration.  A previous detailed
study of cadmium uptake in the treatment-aquaculture system
indicated a relatively  fixed ratio of uptake by oysters over
a  wide range of metal concentration in both effluent and
seawater as sources.  If the combination of metals acted
synergistically, the effect was to impede uptake of metallic
ions  at  the higher  concentrations.
                       DEFINING  A LIMIT
      Ideally,  the  permissible  level of  a metal should be one
 which allows  the organisms  of  the  culture  system  to be  free
 from  acute  or chronic  toxicity and the  food  products to be
 entirely  safe for  human consumption.  With this in mind a
 series of limits were  prescribed based  on  phytoplankton
 toxicity, shellfish  toxicity, acute toxicity  to human
 consumers,  chronic toxicity to human  consumers, and finally,
 pollution alert levels.  The results  are presented in
 Table 2.

      First  of all, the levels  of concentrations of metals
 which bring about  inhibition of growth  of  Phaeodactylun
 tricornutum are listed in the  first row.   These are based
 on the continuous  culture and  batch culture  tests performed
 on 6-metal, 5-metal, and single-metal additions in the
 effluent  as reported in this paper.

      Secondly, since no toxic  effects of metal additions
 were  observable with the oysters,  the permissible levels
 were  set  at 10 ppm.  Further experiments conducted for
 periods of  time" longer than 28 days may v/ell show chronic
 toxic effect  as metals accumulate  in  the tissues  of the
 oysters.  However, no  acute effects were noticeable.

-------

       10
   ki
       0.1

     .001
                               Cu
'  I  I I Hill	1	1  I I I Mil
ki   ^^:oi          o.i          i.o          10


    METAL  CONCENTRATION IN EFFLUENT

                    (ppm-/u.g/ml)

           V\rt\\TQ 7. ••.-.-Jte of -increase of rr.etal concentration in
                  tissue as ,T function of the concer.trat j on of
                  •r.etal added in the effluent.
                           95

-------
10
CT>
               Table  3.   Concentrations of metal  permissible  in solution in
               secondary effluent  for use in a combined tertiary treatment-
               marine aquaculture  system using a  1 :if dilution of effluent for
               algae  media and a 1:19 dilution of algae culture for raising
               oysters.


                                Concentration of  Metal in Effluent   (ppm)
Based on:
Algae Acute Toxicity
Shellfish Acute Toxicity
Human Acute Toxicity
Human Chronic Toxicity
Pollution Alert Level
Cu
.3
10
.2
N.S.
.36
Cd
.5
10
.080
.010
.012
Ni
.5
10
N.S.
N.S.
.05
Cr
.5
10
>10.0
N.S.
3.0
Pb
1.0
to
N.S.
6.0
2. if
Zn
1.0
10
N.S.
N.S.
N.S.
     Suggested Guideline         .2     .010   .05    «5      1.0    1.0


     N.S.  - Hot Specified

-------
     A number of the metals studied are known to have toxic
effects when present in food in high amounts.  Copper has
been reported to cause bitterness and gastroenteritis when
present in high levels in oysters^'»'".  The excessive
copper also imparts a greenish tinge to the oyster meat.
As a result, Roosenburg has suggested a 100 ppm level of
copper in tissue be set for adult oysters for human consump-
tion.  Browning has reported that one to two grains of
copper as sulfate will produce severe abdominal pain, vomit-
ing, and diarrhea when ingested17,  One grain is equivalent
to a dose of 6.6 mg.  For a meal of 40 gm of oyster meat,
equivalent to eight 5-gram oysters,  this would represent a.
concentration of 180 ppm in their tissue.  The acute
toxicity level is computed in Table 2 on the basis of a
100 ppm limit on concentration, which would necessitate a
daily increase of .5 ug.

     To reach a concentration of 100 ppm in a 5 gm oyster
after 3 years of culture (roughly 1000 days - the average
time needed) the oyster v/ould have to accumulate 500 ug of
copper, requiring an uptake rate of .5 ug/da.y.  From figure
7, this would occur with a .2 ppm concentration of copper
in the effluent used for culture.

     Schwarze and Alsberg found that the maximum amount of
cadmium in food that could be tolerated without producing
emesis was roughly 400 ppm1t-j.  However, reported instances
of poisoning since then have lowered the emetic threshold
concentration to 13 ug/gm cadmium'9>20B  This level requires
an uptake rate of .065 ug cadmium/day by the cultured
oysters.  A concentration of .080 ppm in the effluent would
be necessary to reach that level after 3 years (1000 days)
culture.

     No acute toxicity from ingestion of nickel has been
reported for the levels likely to be encountered in shell-
fish culture.  Systemic poisoning from nickel salts is
almost unknown, although dermatitis has occassionally been
noted''.  Nickel carbonyl poisoning has been well-documented
but is unlikely to occur in the effluent used for aquacul-
ture purposes.

     By oral administration, elevated levels of chromate
(as potassium) cause stunting of growth.  Gross and Heller
reported that the maximum amount that can be added to feed
without causing toxic effects was 1/8 of 1 percent, a
concentration of 1250 ppm Cr^'.  To reach this level would
require a rate of uptake in excess of 6.2 ug/day, requiring
concentrations in the effluent greater than 10.0 ppm,
unlikely to occur.
                              97

-------
     There is little point in computing limits based on
acute toxicity for lead or zinc, the tv/o remaining metals.
Very little ingested lead or zinc is absorbed in the intes-
tines.  Intakes of zinc in the form of chloride or carbonate
equal to 2,500 ppm of the diet shov-r little effect on
animals' '.

     Of the six metals, cadmium and lead are known to cause
chronic toxicity from long-term accumulation from ingested
food in humans.  ;Ve ^re not avrare, at present, of any
reported cases of poisoning \7hich can be traced to concen-
trations of either of these elements in oyster meat.
However, the critical levels in meat are known or have been
estimated  for each of the metals and can be calculated.
t
     Kerfoot and Jacobs, using a model similar to tha
employed by Kjellstrom to set cadmium standards in air,
computed that  concentrations of cadmium in oyster meat above
3.0 ppm would  lead  to a critical level in the kidney cortex
if JfO gm of oyster  meat were invested daily  for 50 years1 3.
This would require  a daily rate of uptake of .015 ppra for
the 1000 day culture period.  A concentration of  .010 pprr.
in the effluent would be necessary to reach  this  level.

     According to Kehoe, any level of lead greater than a
total of .6 mg/day  is potentially dangerous22.  Using the
generous estimate of 40 gm of oyster meat per day as the
diet, a concentration of 15-0 ppm would be undesirable.
This requires  a rate of uptake of .75 ug/day for  the 1000
day culture period.  A concentration of 6.0  ppm in the
secondary effluent  would bring about this eventual level.

     Finally,  a series of permissible levels were computed
on the basis of alert levels at one time considered  -or_,
indicators for pollution for the State of Massachusetts2-?.
The alert levels for Zn, Cu, Cd, Cr, and Pb  were, respect-
ively, 2000, 175, 3-5> 2.0, and 2.0 ppm wet  meat  weight.
Nickel was not specified.  The alert levels  represent the
upper value three standard deviations from the mean  level
 found in unpolluted areas.  This v/as not meant to indicate
a significance of public health but to serve only as signal
of an unnatural source.  Since the standard  deviation of
 nickel content in oysters is about ±.13» the alert level
based on a  .29 ppm  natural concentration in  the tissue would
 be about  .70 ppm.

      Permissible levels, based on the alert  levels,  were
 calculated  in  the  final row of Table 2.  Since zinc  was not
 accumulated  significantly during  the study,  no limit v/as
 calculated  for it.  As before, the culture period was
 assumed  to  be  1000  days, resulting in a  5  grain oyster.

                              98

-------
     The final permissible level suggested as a. guideline
was taken to be the lowest value occurring in the column
for each metal.  Effluent containing a concentration of
metal in solution above or at this level is not recommended
for aquacultural purposes.  In three cases, Cr, Pb, and Zn,
the suggested level represented the concentration toxic to
algae.  For copper, the level was based on acute toxicity
to humans.  With cadmium, it reflected chronic toxicity of
the metal in humans.  Oddly, nickel was the only element
whose recommended concentration in the effluent was based
on alert le\rels, representing a level three standard
deviations above that normally found.

     Even though the levels of metal in the effluent for use
in the aquaculture system were calculated against standards
suggested for food regulations, many questions are left
unanswered as to whether consumption of the shellfish is
completely safe from a public health point of view, consid-
ering only metals.  Total metal content may be misleading
since the structure of the compound is not elucidated.
Organometallic species of metals may be present in effluent
due to industrial discharge or possible reactions encountered
during the sewage treatment process.  These compounds, if
present, may have toxic effects not commonly associated with
their inorganic counterparts.  Also, the possible carcino-
genic, niutagenic, and teratogenic effects were not consider-
ed here and should be investigated.

     The many difficulties which beset the definition and
validity of standards should not delay progressing towards
guidelines, despite their tentative nature.  Much will be
gained by establishing the proper limits for metals in
effluents to be used for aquaculture, although the defini-
tion of precise limits to cover all possible toxic effects
may take a long time to develop, particularly since many
oysters sold commercially have been unintentionally exposed
to discharge of municipal effluents, retaining elevated
levels of metals despite depuration for possible pathogenic
organisms.

-------
                         REFERENCES
1.  Kerfoot, W. B. and S. A. Jacobs.  Permissible levels of
    heavy metals in secondary effluent for use in a combined
    sewage treatment -marine aquaculture system I.  Monitor-
    ing during pilot operation.  (Preceding paper)

2.  Jacobs, S. A.  Some measurements of heavy metal content
    of sev/age effluent and sludge.  In The Use of Flowing
    Biological Systems in Aquaculture, Sewage Treatment,
    Pollution Assay, and Food-Chain Studies,  J. H. Ryther,
    ed., Technical Report V/HOI 73-2, December, 1972.

3,  Young, D. R. , C-S. Young and G. E. Hlauka.  In Cycling
    and Control of Metals; Proceedings of an Environmental
    Resources Conference.  National Environmental Resaarch
    Center, Cincinnati, p. 21, 1973.

4.  Ryther, J. H. , W. H. Dunstan, K. R. Tenore and J. E.
    Huguenin.  Controlled eutrophication - Increasing food
    production from the sea by recycling human wastes.
    Bioscience 22(3), 144, 1972.
5.  Goldman, J. C. , K. R. Tenore, J. H. Ryther and N. Corv/in,
    Inorganic nitrogen removal in a combined  tertiary
    treatment -marine aquaculture system. I. Removal
    efficiencies.  Water Research, in press.

6.  Huguenin, J. E.  Experiences with a marine aquaculture-
    tertiary sewage treatment complex. (In  this volume)

7.  Hayward, J.  Studies on the growth of Phaeodactylum
    tricornutum V. The relationship to iron,  manganese, and
    zinc.  J. Mar. Biol. Assoc. U. K. 49:439-446,  1969.

8.  Riley, J. P. and I. Roth.  The distribution of trace
    elements in some species of phytoplankton grovm in
    culture.  J. Mar. Biol. Assoc. U. K. 51(1):63-72, 1971.

9.  Nuzzi, R.  Toxicity of mercury to ohytoplankton.
    Nature 237:38-40, 1971 »

10. Hannon, P. J.  and C. Patouillet.  Effect  of mercury on
    algal growth rates.  Biotechnol. Bioeng.  14:93-101, 1970,

11. Roosenburg, J.  Greening and copper accumulation in the
    oyster Crassostrea virginica near a thermal electric
    generating station.  Chesapeake Sci. 10:241-252, 1969.
                              100

-------
12.   Pringle, 3. H., D. E. Hissong, E. L. Katz and S. T.
     Molawk.  Trace metal accumulation by estuarine inollusks.
     J. Sanit. Eng. Div., Amer. Soc. Civ. Engrs. 94:455-
     475, June, 1968.

13.   Kerfoot, V/. B. and S. A. Jacobs.  Cadmium accrual in a
     combined wastewater treatment-aquaculture system.  In
     Proceedings of the First Annual NSF Trace Contaminants
     Conference, W. Fulkerson, R. I. VanHook, W. D. Schults,
     eds., Oak Ridge National Laboratories, ORNL-NSF-SATC5,
     Oak Ridge, Tenn., March, 1974.

14.   Brewer, P. B., D. W. Spencer, and C. L. Smith.
     Determination of trace metals in seawater by atomic
     absorption spectrophotometry.  Atomic Absorption
     Spectroscopy, ASTM STP 443:70-77, 1969-

15.   Mandelli, E.  F.  The inhibitory effects of copper on
     marine phytoplankton.  Contr. Mar. Sci. 14:47-57, 1969.

16.   Pringle, B. H. and C. N. Shuster.  A Guide to Trace
     Metal Levels  in Shellfish.  Shellfish Sanitation Tech.
     Rep., USDHEW, Public Health Service, 19 p., 1967.

17.   Browning, E.  Toxicity of Industrial Metals, second
     ed., Anpleton-Century-Crofts, Butterworth and Co.
     (Publishers)  Ltd., London,  1969.

1(3.   Schwarze, E.  \V. and C. L. Alsberg.  Pharmacology of
     cadmium and zinc.  J. Pha.rma.col. Exp. Ther. 21:1, 1923-

19.   PHS, Public Health Service  Drinking Water Standards,
     Revised 1962, Public Health Service Publication 0.
     956.  U.S. Govt. Printing Office, Washington, D.C.

20.   Frank, S. and I. Kleeman.   "Food Poisoning". J.A.M.A.
     117:86, 194L

21.   Gross, V.r. G.  and V. G. Heller.  Chromates in animal
     nutrition.  J. Industr.  Hyg. 28:52, 1946.

22.   Kehoe, R. A.  The metabolism of lead in man in health
     and disease.  Arch. Envir.  Health 2:418-422, April,
     1961.

23.   Issac, R. A.  and J. Delaney.  Toxic element survey,
     Progress report no.  1, Massachusetts Water Resources
     Commission, Division of  Water Pollution Control,
     Publication No. 6108, 25 p.,  1972.
                              707

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CALCULATED YIELD OF SEWAGE LAGOON BIOMASS, A PLAN FOR PRODUCTION, AND
SOME OF THE PROBLEMS INHERENT IN USING BIOMASS OR LAGOON WATER FOR
PRODUCTION OF FOOD AND FIBER

                                  BY
                          *.**                   ***
               KARL SCHURR     and J. M. GOLOMBEK
INTRODUCTION

Lagoon treatment of sewage is rapidly expanding both in the United
States and throughout the world.  It is certainly opportune and
appropriate that we should be meeting to exchange information about
our difficulties and successes in the application of research to
sewage lagoon operation and technology.  The focus of this paper will
be on the Deshler, Ohio terminal sewage lagoon system.  This is an
aerobic lagoon which is 5.4 hectares in area and has an even depth of
0.8 to 0.9 m at the present time.  A preceding anaerobic lagoon
receives sewage from the village of Deshler.  Two thousand residents
and the town drainage contribute from 1.2 million liters to 2 million
liters of sewage per day.  An engineering or construction firm would
evaluate the Deshler lagoon system negatively because it obviously is
designed to accept a much larger volume.  Retention of sewage in the
system is from one to several months.  The reason for such a capacity
may have been a group of city fathers who were over-optimistic about
the population growth of their village, or architects who were
extremely conservative.  Be that as it may, this lagoon is not subject
to the regular ills and misfortunes of lagoon systems whose capacities
are constructed close to the margin of input volumes.

The question of efficiency in lagoon systems needs to be debated no
longer.  A lagoon system with sufficient capacity can produce effluent
of the highest quality (there are several studies which document this,
but we will cite Schurr  and Schurr and Raum ).  In comparative
efficiency, a properly designed lagoon system will convert nutrients
better than standard activated sludge or trickling filter systems.
Lagoons are appropriate treatment methods for small towns, villages,
*  Professor of Biology, Professor of Health and Community Services,
   Bowling Green State University, Bowling Green, Ohio 43403.  Adjunct
   Professor, Medical College of Ohio.
** This research was supported in part by grant RDl(Bl) an endowment
   from the estate of Margaret Yocum:  "Research in Aquatic Pollution"
   K. S. Grant, Director.  The plan for a "Comprehensive Wastewater
   Study" was designed with funding from Bowling Green State University
   Dr. Hollis Moore, President.
***President of Inviron Inc., environmental specialists, P. 0. Box
   3119 Cleveland, Ohio 44117.

                                  702

-------
animal feed lots and similar industrial  uses.   However, they are  the
system of choice only where land use and cost  of land will  permit
their construction.  Benefits of lagoons are low construction cost  and
low operational costs.

There are two products of lagoon sewage treatment:  the water, and  a
huge volume of plant and animal material.  Both are subjects for  our
conference.  Plant and animal biomass is quite innocuous from a public
health standpoint.  It is not offensive to the senses.  If this biomass
is allowed to leave with the effluent, however, it will eventually  die
and decay will release the nutrients downstream.  We can not tolerate
this eutrophication.  Algal blooms in Lake Erie have nearly destroyed
it for swimming and recreational boating because they are so negative
aesthetically.  With severe eutrophication, the algae supersaturate
the water with Oo from photosynthesis in the day, but there is zero
oxygen because of respiration at night.  Few fish can tolerate such
conditions and even fewer eggs or embryos will survive.

The logical  solution to this difficulty would be to remove the biomass
and find a use for  it.  Many researchers have tried to utilize single
celled algae from primary lagoons, but their efforts have been plagued
with  problems.  The high yields of algal cultures tantalize physiolo-
gists.  Verduin and Schmid3 could document approximately 7000 grams of
protein produced by Chlorella,  per m2 of surface, as compared with
about 150  grams for traditional terrestrial crops.  The difficulties
in use of  primary  lagoon algae  have  been with harvesting where they
clog  pumps and filters.  This would  not  be an insurmountable  technical
problem, but the population dynamics of  the algae vary through the
seasons and  many species taste  badly or  are toxic.  Dohms4 has
summarized the efforts to use  algae  for  food.   Since outstanding
scientists have found  so many  problems with unicellular algae, we have
studied the  possibility of  using multicellular  forms,  the flowering
plants and invertebrates that  grow  in  the  terminal  lagoon at  Deshler.
A following  paper  at  this meeting by Dr. Bakaitis will evaluate
vitamin content of biomass.

YIELD AND  USE  OF  BIOMASS

Schurr5 and  Schurr6 describe potential  nutritional  value of  the
invertebrates,  while  Dohms  and  Schurr?  document the protein,  lipid and
carbohydrate content.   Living  biomass  in the  Deshler  terminal  lagoon
is dominated by  Potamogeton foliosus,  a  flowering  plant which has
several growth forms.   In  the  terminal  lagoon,  the  stems break and the
plant blooms in  large floating masses  which continue  to grow through
the  summer to a  maximum  crop in August,  September and October.   It
forms a  thick mat that eventually  shades the  bottom of the  pond.
Water content of the  biomass was determined by lyophilization of pre-
weighed  samples  (Dohms^  &  BakaitisS).   This rigorous  drying  of biomass
 showed  an  average percentage of water to be 87% (standard  deviation
0.25) for  1971,  1972  and 1973.  We  took two  samples in 1968, three

                                    703

-------
sets in 1969, two sets in 1971 and one sample in 1973.  The samples
were blotted in newspaper, and air-dried in sunlight.  The final
consistency was the same as well-cured hay.  Water content calculated
from this method of drying was slightly above 84%.  The method of
drying is much less accurate than lyophilizing, but it more closely
approximates the results from potential commercial treatment.  For
this reason, we will use 85% water in estimating productivity.  Samples
were raked into a plastic container, excess water was drained, and the
biomass was packed into plastic bags to be frozen for later chemical
analysis or determination of volume.  Measurements of the floating
biomass mat gave a calculated yield of wet weight comparable to sample
volume.

A single crop for the terminal Deshler lagoon will yield 1596 cubic
meters of dry biomass per hectare.  Proper removal of the floating
material will benefit growth, so that no shading of the undersurface
can occur.  We expect this would increase yield to over 2000 m3 per
hectare.  Nutritional content of the biomass has been documented over
the growing period by Dohms^.  A typical sample for October was
composed of 22% protein, 4% lipid, 40% carbohydrate and 34% ash
(fractions rounded to nearest percent).  Oxygen bomb calorimitry
measured energy content per gram of the biomass at 3.86 Kcal to 3.92
Kcal with a standard error of 0.047 on five replicates.  Sewage lagoon
biomass, therefore, has considerable potential as a food resource.  The
high protein content becomes particularly significant because of the
general shortage of protein.  Foss9 has summarized the information,
from the international symposium on "New Sources of Protein Foods,"
as a world requirement for increased protein.  This is 3,630,000 metric
tons of new dry weight protein each year for the foreseeable future.
Conventional agriculture and our normal fisheries will not produce this
quantity (SchurrS).  Biomass from sewage lagoons may offer another
source of protein.

PROPOSED METHODS OF HARVEST

Methods of handling biomass have yet to be evaluated.  The standard
alfalfa drying plant should be able to dry and pellitize biomass.  The
consistency of biomass and wet alfalfa cut at night is similar.
Removal from the lagoon is possible with a floating rake pulled to one
end by cable and two electric motors.   This would remove floating mats
yet leave attached stems for continued growth.  Biomass could be
loaded for transportation with the same pick-up choppers used for wet
alfalfa.  These suggested methods of removal would enable harvesting
at the best rate to promote plant growth.  They also utilize the
existing technology for alfalfa drying.

ALTERNATIVE USES

The actual disposal of biomass will be determined by the research
which  is in progress or has yet to be done.  If there are no public

                                   704

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health problems, sewage lagoon biomass could be used as a component in
feed for several domestic animal  species.  It could even serve as a
fractional part of meat substitutes in the human diet; I have eaten it
and the taste is bland.  Feeding of biomass would be particularly
advantageous for animal feed lots, where it could be added to more
conventional feeds.  Animal manure could be converted to biomass in
lagoons on the farm and then recycled through the animals again.  If
there should be evidence of pathogenic bacteria or viruses in the
biomass, it may be necessary to mix the biomass with corn as ensilage.
As is usual with this feed, the mixing of molasses with the biomass
and corn would certainly increase its nutritional value.  Fermentation
and heating would probably destroy pathogens in the silo.

Should some unforeseen problem prevent feeding of biomass, then the
material could be used to generate methanol as is described by Reed
and LernerlO.  Mixed-grade methanol can be produced cheaply from
garbage, plant refuse, paper pulp wastes and biomass.  Methanol can
be used as a fuel additive to gasoline, where about 15£ will increase
octane, reduce air pollution and extend mileage in standard automobile
engines.  Methanol converters could generate fuel, from wastes and
biomass, to be used in municipal  vehicles and farm tractors.

A final possible use of biomass would be as a soil conditioner.  Such
use would not give as much monetary return as those mentioned above,
but it would dispose of the material.  Sweet clover is frequently
planted and then ploughed under in order to improve soil fertility and
texture.  Whatever the use, it is necessary to remove biomass from
lagoons so that it will not go out with effluent, die, decompose and
release the nutrients in down-stream areas.

PROBLEMS

The use of biomass or water from lagoons will depend on their freedom
from toxic substances.  Pesticides, herbicides and industrial
chemicals must not be present in significant levels.  Cyanides are
often released from factories as are lead, mercury, cadmium and
selenium.  Unexpected contaminants may be found; Robinson, Draper and
GelmanTI showed that copper sulfate fed to pigs resulted in sufficient
copper in feces to inhibit nitrogen degradation.  Radioactive elements
are another possible hazard.

A serious public health concern is the fate of waterborne virus
particles and pathogenic bacteria.  Hicks!2, Saslaw"l3 and Kurowski14
show the potential danger.  Several studies have been completed on
lagoon systems and the pathogens they may harbor.  There has been no
well-controlled study of virus particles in a lagoon system having a
reasonable retention time.  We expect several scientific papers which
speak to this problem because there is new commercial equipment now
available for concentrating virus particles from large volumes of
water (Wallis, Homma and MelnicklS).  The Deshler system seems to

                                   705

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dispose of bacteria rather well  (Schurr1).   Kott16 has stated that
virus particles are attenuated in the lagoon system that he is testing.
Nevertheless, it is necessary to have exaustive studies completed  on
lagoon systems which are properly loaded.   It is appropriate that
similar research be done on activated sludqe and trickling filter
systems as a comparison.

Problems May Be Caused By_ Members Of Our Profession

A lagoon system which is "efficient" from the standpoint of land use
and cost, is frequently the type of system that concerns the public
health officer, offends the senses and is an ecological abomination.
We can evaluate lagoons as efficient in cost or loading as opposed to
those which are efficient for their purpose.  Our definition of
efficiency is the second type; sewage treatment should convert wastes
to materials or chemicals which can be removed during or after treat-
ment so that the effluent is water of highest quality.  Absolute
standards of water quality can be met by trickling filter or activated
sludge systems combined with flocculation and chemical treatment.   The
system which is in operation at Lake Tahoe would be an example of
efficiency in our view (Abelson'7).  Cost of such a system is quite
high.  We differentiate efficiency of treatment from cost in our
discussion because there is a tendency of some designers to mix apples
and oranges, in their calculations, and the product is something that
smells badly.  It is wrong ethically and wrong from an engineering
viewpoint to present a plan that mixes circa 60% waste conversion  with
construction costs that seem what the traffic will bear.  It is also
wrong to view the sewage system as a microcosm and then reject any
responsibility for what leaves it.  The public health officer who  is
only concerned with heavy chlorination of effluent is in error.
Nothing may be alive in the outflow, but there is an indication that
reactions may take place which produce chlorinated hydrocarbon
residues that will be of public concern in food chains further down
in the watershed.  Chlorinated effluent will destroy stream ecology
for miles.  There are individuals with tunnel vision.  If all bacteria
and viruses are destroyed, they don't concern themselves with the
chlorine, nitrates, nitrites, phosphates, etc. being dumped in our
waterways.  Ozone (actually ozonized air) would be a reasonable
alternative to chlorination if the chemical were removed by aeration.

PLAN FOR INTEGRATED LAGOON AND STANDARD SEWAGE SYSTEMS

Many small towns have overloaded sewage plants.  They may also have
mixed storm drains and sanitary sewers.  This places an intolerable
burden on those responsible for proper treatment of sewage.  Bowling
Green State University responded to such conditions by offering land
at no cost for the construction of 400 acres of lagoon system.  The
university also financed a comprehensive waste water study (E.C.CJ8).
Bowling Green has mixed storm and sanitary sewers, with a trickling
filter sewage plant that is overloaded during rainfall.  The proposed

                                   106

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sewage lagoon system would receive all effluent during rainfall  and
treatment would progress through a chain of lagoons.  Biomass would
be removed from terminal lagoons.  Effluent could be pumped back
through the trickling filter system during dry periods.  High loadings
could be sent to the lagoons and then brought back to the standard
plant.  An efficient removal of phosphates could be achieved most
economically.

The alternatives to this proposed combination of lagoons and trickling
filter sewage treatment plant are as follows.  The town must construct
a greatly expanded sewage treatment plant.  Design figures on plans
show that even this expanded capacity will be unable to accept the
volume during heavy rains and it will be necessary to bypass.  Storm
and sanitary sewers must be separated.  We predict that federal  regula-
tions will become more stringent and storm water will be treated within
a few years.  Better quality standards on effluents will be mandated
by the government.  This will require a flocculation system being added
to the new treatment plant.

It is difficult to estimate construction costs within the next few
years because of inflation, energy shortages and land expense.  However,
we think the total cost of a new sewage treatment plant, plus separa-
tion of storm and sanitary sewers, plus treatment of storm water, plus
special treatment required as standards increase, may reach the level
of twenty million dollars.  No useable product will be produced by
this type of construction.  Alternatively, with free land contributed
by the university, a lagoon system could be added to the sewage plant
at a cost of $400,000 to $600,000.  A compromise treatment system
composed of a new activated sludge system and a lagoon system would
cost about $7 million and could meet the highest water quality
standards.  The town has 20,000 residents listed on the last census,
but this included all students living off campus.  Actual residents
are about 15,000.  Those with taxable incomes are much fewer.  It
becomes obvious that there must be alternatives to standard sewage
treatment because the average American community simply doesn't have
the tax base to build an activated sludge or trickling filter system
that will meet the highest water quality standards.  Increased welfare
costs, fire protection, police protection, education, mental health
programs, etc., etc. will all have priority over sewage treatment.
Clean water will be an impossible goal unless we can find ways to
treat sewage at moderate cost.

SUMMARY

We suggest that the proper prescription for optimum sewage treatment
with reasonable costs is as follows.  Villages, small towns and
animal feed lots would be appropriate for lagoon systems if land cost
is reasonable.  Loading should be moderate so that terminal lagoons
will be aerobic and will produce biomass from plant and animal growth.
The biomass should be removed and commercially used as described in

                                   707

-------
this paper.  Whenever possible, the lagoons should be combined with
standard sewage treatment systems in order to obtain the highest water
quality standards in treatment.  The biomass should be recycled close
to the site of production.  This may be used as animal feed.   We do
not discount the use for humans if it could meet pure food and drug
standards.  We may find the proper fate of biomass would be a conver-
sion to methanol to be used as an additive to gasoline.  Biomass may
be disposed of as a soil conditioner.  Its removal from lagoons,
however, is a necessity if we are to reach the ideal of pure water as
effluent.
                             REFERENCES

1.  Schurr, K.  A Comparison of an Efficient Lagoon System With Other
    Means of Sewage Disposal in Small Towns.  2nd International
    Symposium for Waste Treatment Lagoons.  1:95-100, 1971.

2.  Schurr, K., and W. Raum.  Comparative Efficiency of Well-Operated
    Trickling Filter and Lagoon Sewage Systems.  Second National
    Symposium on Societal Problems of Water Resources.  April, 1973
    (Proc.  in press)

3.  Verduin, J., and W. Schmid.  Evaluation of Algal Culture as a
    Source  of Food Supply.   Developments  in Industrial Microbiology.
    7:205-209,  1966.

4.  Dohms,  J.   A Nutritional Analysis of  Sewage Lagoon Biomass.
    Master  of Arts Thesis,  The Graduate School.  Bowling Green State
    University, 1972,  77 p.

5.  Schurr, K.  Insects as  a Mjaor Protein Source in Sewage Lagoon
    Biomass Useable as Animal Food,  Proc. N. C. B. Ent. Soc.  Amer.
    27:135-137, 1972.

6.  Schurr, K.  Insect Protein in Food Recycled from Sewage Lagoon
    Biomass (abstract  only; invitational  paper).  Symposium on
    Agricultural Entomology, XIV  International Congress of Entomology.
    Canberra, Australia p.  303,  1972.  (This paper  is cited in new
    directions  for  life sciences  by  Encyclopaedia Britannica Yearbook
    for 1973  on p. 422.)

 7.  Dohms,  J. and  K.  Schurr.  Biochemical  Evaluation of Sewage Lagoon
    Biomass to  be  Used for  Animal Food as Recycled  Nutrients.   Second
    National  Symposium on Societal  Problems of Water Resources.
    April,  1973 (Proc. in press)

 8.  Bakaitis, N.  M.   Water  Soluble  Vitamins  in  Sewage Lagoon Biomass.
    Doctor of Philosophy Thesis,  The Graduate  School.  Bowling Green
    State University,  1974, 47  p.
                                   108

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 9.  Foss, J. G.  New Sources of Protein Foods.   European  Scientific
     Notes (ONR) 25:85-87, 1971.
10.  Reed, T. B., and R. M. Lerner.   Methanol:   A Versatile  Fuel  for
     Immediate Use.  Science.  182:1299-1304,  1973.
11.  Robinson, K., S. R. Draper and A. L. Gelman.  Biodegradation of
     Pig Waste:  Breakdown of Soluble Nitrogen Compounds and the  Effect
     of Copper.  Environmental Pollution.  2:49-57,  1971.
12.  Hicks, A.  Waterborne Hepatitis-A Outbreak-Alabama.  Morbidity
     and Mortality.  22:118, 1973.
13.  Saslaw, M. S.  Typhoid Fever in Florida.   Morbidity and Mortality.
     22:77-78, 1973.
14.  Kurowski, J.  Shi gel!a dysenteriae 1 in Colorado.  Morbidity and
     Mortality.  22:101-102, 1973.
15.  Wallis, C., A. Homma and J. L. Melnick.  Apparatus for
     Concentrating Viruses from Large Volumes.  Jour. Amer.  Water
     Works Assoc.  64:189-196, 1972.
16.  Kott, Y.   Personal communication.  Technion, Haifa, Israel.
     6 p.  1971.
17.  Abel son,  P. H.  Long-Term Efforts to Clean the Environment.
     Science.   167:1082,  1970.
18.  Environment Control  Corporation.  Comprehensive Wastewater Study
     for  Bowling Green  State  University.  Painesville,  Ohio, 1973,
     89 p.
                                   109

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        PART 3   A QUA CUL JURE
          SESSION CHAIRMAN

       KENNETH O, ALLEN, Ph.D.
         FISHERIES BIOLOGIST
BUREAU OF SPORT FISHERIES AND WILDLIFE
        STUTTGART, ARKANSAS

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                 ANALYSIS OF SEWAGE LAGOON BIOMASS WATER

             SOLUBLE VITAMINS BY MICROBIOLOGICAL TECHNIQUES

                                  by

                          Nancy M. Bakaitis*


Eutrophication of surface water resources, due to enrichment from
fertilizer runoff, municipal sewage effluent and other sources, has
led to great economic losses and environmental damage.  If the vast
growths of vascular plants and algae, which occur under these con-
ditions, could be harvested economically, mechanical removal would be
an attractive alternative to the chemical treatments now employed.

Water weeds have been suggested as a potential source of nutrition for
livestock by Boydl and Little.2  Recent work on cultivation of aquatic
plants 1n sewage lagoons to remove pollutants and produce a clean ef-
fluent has had considerable success in the United States and in Asia,
according to Oswald and Golueke^ and McGarry and Tongasamer   This con-
cept of sewage treatment and food production has also been tested suc-
cessfully by RytherS using aquatic plants in marine systems.

The sewage lagoon system of Deshler, Ohio was studied from 1971 to 1973
to determine the nature of the life forms commonly present and their
nutritional value.  The possibilities of sewage treatment and water
purification with the production of a useful product were explored by
several workers including Dohms6, Schurr7"8, and Yerdirtn.

The Deshler sewage  lagoon system consists of two ponds with a depth of
approximately one meter.  The system handles all of the sewage from a
community of 2000.  Pond One receives raw sewage through a submerged
line at the rate of from 300,000 to 500,000 gallons per day, depending
on rainfall.  Anaerobic digestion of sewage occurs  1n this nine acre
pond.  Pond Two  1s  thirteen acres  in area and is aerobic.  The water
leaves Pond One  through two corner outlets and enters Pond Two, which
in turn, discharges Into a  small stream.  Material  from  Pond Two was
studied.


  *Chemistry Department, Findlay College,  Findlay, Ohio.
**This research  was supported,  1n  part,  by funds  from grant RDI(BI)
   "Research  in Aquatic Pollution", an endowment from  the estate of
   Margaret Yocum.
                                    770

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Pond Two biomass consists of floating plant material in which
Potamogeton foliosus Raf. predominates and has associated with 1t many
aquatic insects at various life cycle stages, aquatic annelids, mites,
and snails.  The plant material forms a thick floating mat extending
many feet from the shore toward the center of the pond.  Sampling of the
biomass was done by standard random sample techniques with samples being
of 5 to 10 liters in drained volume.  Samples were taken at various
intervals during August and September each year when the material was
present in significant quantities.  Samples were lypholized and frozen.

Analysis of the water soluble vitamin content of the biomass was con-
ducted to determine if it was comparable to common animal feeds as a
vitamin source.  Thiamine, riboflavin, niacin, pantothenic acid, Bi2,
total B6, biotin, choline, and beta-carotene were selected for study
based on the requirements of chickens and swine.   Choline was deter-
mined using the method outlined by  Sebrell and Harris.'0 Beta-carotene
was found to be present using the procedure of Gyb'rgy and Pearson.'!
All other vitamins were assayed using the preferred microbiological
techniques of the Association of Official Agricultural Chemtstsls the
Association of Vitamin Chemists13,  or the United States Pharmocopiae.I*
The content of each of the vitamins studied did not vary significantly
during the growing season or from one year to the next so that the data
was pooled and a mean vitamin content for each vitamin  is presented.

Results indicate that the sewage lagoon biomass vitamin content compares
favorably with common feeds, such as, alfalfa, ear  corn, dried barley,
sugar beet pulp, and solvent extracted soybean hulls.  The amount of
riboflavin present in the biomass is superior to these feeds; and, the
amounts of biotin, choline, niacin, pantothenic acid,  and thiamine are
comparable.  The total B6 content of the sewage lagoon biomass is low
in  comparison to these feeds and would need to be augmented  if it was
the sole B6 source in chicken  rations.    A summary of these findings
is  presented in Table I.

Analysis of subsamples of biomass material taken in 1971 were completed
by  Dohms6  for protein,  carbohydrate, lipid, and mineral content.  The
amounts of carbohydrate  present was reported as 3.89  Kcal/g  dry weight
of  material; and, the protein  content was found to  be  17.9 per cent.
These values also compare quite favorably with dried  alfalfa meal,  corn,
barley, soybean  hulls, and sugar  beet pulp protein  and  carbohydrate
content.  The mineral content  of  the  biomass was also  found  to   compare
favorably with  these feeds being  higher  in calcium  and zinc, but  lower
1n  manganese content.  A summary of these findings  is  in Table II.

Therefore,  the  sewage lagoon  biomass  from the Deshler, Ohio  aerobic
sewage  lagoon  compares favorably  in protein,  fat, carbohydrate,  mineral,
and vitamin  content with commercial feeds.   Regardless of  the  source,

                                    777

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   Table I.   Vitamin Content  of  Feeds Compared  to Sewage
   Lagoon Biomass Mean  Vitamin Content  for  1971 to 1973a

Vitamin
Biotin
B12
SLB*>
0.36
0.10
Choline 1020
Niacin
Pantothenic
Acid
Total B6
Riboflavin
Thiamine
30.00
13.18
1.35
19.81
5.00
Feed C
::
1550
41.90
20.90
6.50
10.60
3.00
Feedd
0.20
1030
57.40
6.50
2.90
2.00
5.10
Feede
^ ^
829
16.30
1.50
0.70
0.40
Feedf
0.05
Feed9
0.32
550 2743
20.00
5.00
5.00
1.10
—
26.80
14.50
8.00
3.30
6.60
a. Expressed as mg/kg dry weight of material.
b. Sewage Lagoon Biomass mean assayed value based  on  1971,  1972
   and 1973 samples.
c. Alfalfa, Medicago sativa, aerial portion, dehydrated.
d. Barley, Hordeum vulgare, dried grain.
e. Sugar Beet, Beta saccharifer, dehydrated pulp.
f. Corn, Zea mays, ground ears.
g. Soybean, Glycine max, seed meal , whole, solvent extracted and
   ground, 7 per cent fiber.                                 ..
   Data for c.-g. from NRC, Nutrient Requirements  of  Poultry,IS
   1971. pp. 24-37.  Table 13. Composition of Some Common
   Poultry Feeds.
                                772

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      Table II.  Mineral Content of Feeds Compared To
      Sewage Lagoon Biomass Assayed Mineral Content a
Mineral
Sodium
Phosphorous
Calcium
Potassium
SLBb
0.
1.
3.
1.
Manganese (ppm)464
Magnesium
Iron (ppm)
Zinc (ppm)
Copper (ppm)
0.
1601.
50.
10.
29
03
44
39
.0
41
00
00
00
Feedc
0
0
1
2
29
0
0
20
10
.07
.22
.23
.33
.00
.29
.03a
.00
.40
Feedd
0
0
0
0
16
0
0
15
7
.02
.42
.08
.53
.30
.12
.Ola
.30
.60
Feed6
0.
0.
0.
0.
13.
0.
0.
9.
7.
01
28
04
53
00
15
Ola
00
70
Feedf
0
0
0
2
45
0
0
45

.01
.62
.26
.02
.50
.28
.02*
.00
—
a.  Expressed as per cent dry weight unless otherwise indicated.
b.  Sewage Lagoon Biomass, Dohms,61972.   A Nutrient Analysis  of
    Sewage Lagoon Biomass.  Table VI.
c.  Alfalfa, Medicago sativa. aerial portion,  dehydrated.
d.  Barley, Hordeum vulgare. dried grain.
e.  Sugar Beet, Beta saccHarifer, dehydrated pulp.
f.  Corn, Zea mays, ground ears.
    Data for c.-f. from NRC, Nutrient Requirements  of PoultryJ5
    1971.  pp. 24-37.  Table 13.   Composition of Some Common
    Poultry Feeds.
                                113

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if nutritionally valuable feeds can be found, they will be employed in
animal feeds.  The only significant limiting factors are cost and public
health.  The cost of sewage lagoon biomass will be determined by the
expense of harvesting and transport, since it is a "waste product" of
sewage treatment at the present time.  A positive economic factor is the
present necessity of removing this material from effluents in order to
release clean, treated water.  Such removal prevents eutrophication in
down-stream areas of the watershed.  Since lagoons are the least expen-
sive method of treating sewage from villages, small towns, and animal
feed lots, the removal of biomass will be the final requirement of la-
goon treatment in the future.  With removal of biomass, lagoons also
become the most efficient method of sewage treatment.  Removal of bio-
logical oxygen demand, nitrates, nitrites, and phosphates is consider-
ably  better than trickling filter or activated sludge systems operating
at optimum efficiency.

Removal and utilization of biomass in feeds would enable our society to
reap a triple benefit.  Lagoon treatment could be employed more general-
ly in small communities and for animal feedlots.  Secondly, the biomass
could be used for animal feeds near the location of production.  Actual
recycling of nutrients would be possible for small geographic areas and
for individual farms.  Thirdly, we could approach the ecological ideal
of confining nutrients to the areas where we want to promote growth,
while avoiding dispersal of nutrients to locations where we consider
them harmful to the habitat.

At this time, all studies conducted on the Deshler sewage biomass have
produced results which indicate that biomass would be an excellent feed
supplement. Before a final evaluation of biomass as a feed source can be
made, information must be obtained on the fate of virus particles and
bacteria in a well-operated sewage lagoon system.  The combinations of
sewage lagoon biomass with other components of livestock rations and the
effect on growth need to be documented in actual feeding experiments.
It will also be necessary to design methods of harvesting and drying the
biomass.
                                   774

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Literature Cited

1  Boyd, Claude E.   Freshwater Plants:  A Potential  Soruce  of
   Protein.  Economic Botany,  22_: 359-368,  1968.
2  Little, E. C. S. (ed.).  Handbook of Utilization  of Aquatic
   Plants, A Compilation of the World's Publications.   F. A. 0.,
   U. N.f Rome,  1968,  122 p.
3  Oswald, William J. and C. G. Golueke.  Harvesting and Processing
   of Waste-Grown Microalgae. p. 371-387. In D.  F. Jackson  (ed.).
   Algae, Man and Environment.   Syracuse University  Press,  Syracuse,
   New York,  1968.
4  McGarry, M. G. and C. Tongasame.   Water Reclamation and  Algae
   Harvesting.  J. Water Pollution  Control Fed., 43_: 824-835,   1971.
5  Ryther, J. H.  The Status and Potential of Aquaculture,  Parti-
   cularly Invertebrate and Algae Culture.  A. I.  B. S. Gov. Print.
   Off., PB 177767, Washington, D.  C.  1968.
6  Dohms, John.  A Nutrient Analysis of Sewage Lagoon Biomass.  Un-
   published Master's Thesis, Bowling Green State University, Bowling
   Green, Ohio, 1972.
7  Schurr, K. M.  Insect protein in food recycled from sewage lagoon
   biomass.  Symposium on Agricultural Entomology,  XIV International
   Symposium of Entomology, Canberra, p. 303.  1972.
8  Schurr, K. M.  Insects as a Major Protein Source  in Sewage Lagoon
   Biomass Useable as Animal Food.   Proceedings North Central Branch
   E. S. A., 27_:135-137,  1972.
9  Verduin, Jacob.  Phytoplankton energetics in a sewage-treatment
   lagoon.  Ecology, 52(4);621-631.  1971.
10 Sebrell, W. H., Jr. and R. S. Harris (ed.).  The  Vitamins. Vol.  II
   Second Ed., Academic Press, Inc., New York.  1967,  pp.  52-55.
11 Gyb'rgy, P. and W. N. Pearson (ed.).  The Vitamins. Vol.  VI.  Second
   Ed., Academic Press, Inc., New York.  1967,  pp.194-196.
12 Association of Official Agricultural Chemists (ed.).  Official
   Methods of Analysis of the Association of Official Agricultural
13 Association'of Vitamin Chemists  (ed.).  Methods  of Vitamin Assay.
   Second Edition.  Wiley-Interscience, New York, 1965,  301 p.
14 U. S. Pharmacopeia.  Revision XVI.  Mack Publishers, Easton,  Pa.,
   1960,  pp. 42-48.
15 National Research Council, Subcommittee on Poultry.  No. 1,  Nutrient
   Requirements of Poultry.  (6th Edition).  National Academy of Sciences
   Washington, D. C., 1971, 54 p.
                                   775

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          FEED AND FIBER FROM EFFLUENT - GROWN WATER HYACINTH

                                   by

             L.O. Bagnall, T.deS. Furman, J.F. Hentges,Jr.,
                      W.J. Nolan and R.L. Shirley*
SUMMARY
Water hyacinth was used to remove nutrients, primarily nitrogen and
phosphorous, from secondary treated sewage effluent; some of the plants
were subsequently ensiled or dried and fed to sheep and cattle and some
were pulped to make paper.  The irregularly harvested pond removed 10%
of the nitrogen and phosphorous from the effluent, only 10% of which
could be accounted for in the plant tissues.  Cattle and sheep readily
ate processed water hyacinth in complete diets and remained in good
health, but did not utilize the nutrients as well as nutrients in a
land forage, coastal Bermudagrass.  The primary feeding value is as
sources of energy, mineral elements and roughage for ruminants.  Paper
can be made from water hyacinth, but production cost is uneconomically
high.  Compost may be the best use, having the highest value and lowest
processing cost.

INTRODUCTION

Fertilization by wastewater often encourages excessive growth of some
plant species in waterways, reducing the availability of the water for
more desirable uses.  The focusof this conference is on how to manage
these fertilizing effluents to some beneficial ends.  One possible
means to this end is to manage the growth and utilization of one of the
problem species, water hyacinth (Eichhornia crassipes).

Water Hyacinth

Holm, Weidon and Blackburn  estimate water hyacinth caused losses of
$43 million in the Southeastern United States in 1956 and that control
 •Assistant  Professor, Agricultural  Engineering; Professor, Environ-
  mental  Engineering; Professor, Animal Science; Professor, Chemical
  Engineering;  Professor, Animal Science.  University of Florida,
  Gainesville,  FL.
 **The  projects  on which  this  paper  is  based were supported by the Flor-
  ida  Agricultural  Experiment Station, University  of Florida Engineer-
  ing  and Industrial  Experiment Station,  Florida Department of Natural
  Resources, Florida  Game and Fresh Water Fish  Commission, Southwest
  Florida Water Management  District, and  the  Office of Water Resources
  Research,  USDI, through the Florida  Water Resources Research Center.

                                    116

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programs cost millions of dollars.  Though not the only cause of this
undesirable excessive growth, over-nutrification of the water is a
contributing factor.

Water hyacinth is a large free-floating plant with an attractive laven-
der flower and shiny bright-green leaves on long petioles.   Uncrowded
plants, particularly in shallow water and full sunlight, have bulbular
float petioles about 8 inches long, whereas crowded plants  produce  elon-
gate pwtioles up to 50 inches long .  The plants Reproduce  from stolons
and, less importantly, seeds.  Penfound and.Earle  report that the  num-
ber of water hyacinth plants doubled every 11.2 to 15.0 days in field
observations and that the edge of a water hyacinth mat extends 2 feet
per month.  Bock^reported a 50% weight increase in 13 days, and Knipling
West and Haller  observed mass growth rates of 50% in 10-4  days. Stand
densities of 125 to 184 tons per acre have been reported.
                                                                      4
Water hyacinth grows most rapidly in water temperature from 28 to 30 C
and pH from 4.0 to 8.0 , and ceases to grow when water temperature  is
above 40 C or below 10 C.  Water hyacinth is killed when the tip of the
rhizome is frozen .  The plant has little tolerance for salt water  and
will only grow in fresh to faintly brackish water.  The plants are
normally free-floating but, if stranded by receding water,  will root in
mud and survive.  Phosphorous is limiting to growth in concentrations
belowfi0.1 ppm and is consumed in luxury amounts at higher concentra-
tions .  Plants usually exhibit lower root-to-shoot ratios as water
nutrification increases.

Because of their habit of vegetative reproduction from stolons, individ-
ual plants remain tied together in a floating mat.  In confined waters,
the mat spreads until it covers all the water.  Penfound and Earle
report that dissolved oxygen under mats ranged from 0.1 to 1.5 ppm,
depending on mat density, when nearby open water contained 4.0 ppm oxy-
gen.  The oxygen depression is caused primarily by decay of detritus
from the mat and in turn causes dramatic changes in the types and
quantities of life  in adjacent areas.

Wastewater and Water Hyacinth

Sheffield7 passed extended aeration effluent through a water hyacinth
pond with a detention time of 10 days and observed 81% reduction of
ammonia nitrogen.   No reduction in nitrate nitrogen was observed until
anaerobiosis was established at 42 days, after which reduction rose to
81%.  Orthophosphates were reduced 51% during the first month of opera-
tion, but reduction fell to 20% after that, as decaying plant detritus
released accumulated phosphorous  back to the water.

Clock8 found that a water hyacinth system removed 39 to 94% of the total
nitrogen from extended aeration effluent when the detention time was
five days, with best reduction coming during the period of most vigor-
ous growth.  Average nitrate reduction was 61%.  Phosphates were re-
duced by 60% during periods of vigorous growth, but increased during

                                   777

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dormancy.

Steward9 projected that an acre of water hyacinth can take up the nitro-
gen production of 130 to 595 people from secondary sewage effluent when
growth rate ranges from 294 to 1340 tons per acre-year.  At the same
growth the phosphorous production of 40 to 180 people could be absorbed.

Miner, Wooten and Dodd   grew water hyacinth on anaerobic swine lagoon
effluent in Iowa and found the system to reduce Kjeldahl nitrogen 95%,
ammonia nitrogen 96%, phosphate 82% and COD 88% in 102 days detention
in pools 18 inches deep, and recommended that depth for application.

Food, Feed, Fiber and Water Hyacinth

Boyd11 found  the composition of naturally growing water hyacinth to be
as shown in Table 1.  He noted that the composition varies considerably
with  location and season, but concluded that, chemically, it was suit-
able  for cattle feed.  Water hyacinth would be unsuitable for human food
because of its high fiber content.  It could be fractionated to yield
leaf  protein  and other non-fibrous components.

Cattle have been observed eating  water hyacinth leaves, especially when
other types of forage were scarce.  Hentges   reported  that cattle ac-
cepted limited amounts of fresh and sun-dried chopped water hyacinth,
but refused some plant parts and  stale feed.  He  fed dehydrated water
hyacinth to cattle at high levels of  their diets  and found that, though
the animals only maintained their weight, the material  was not toxic;
he noted that the quality of the  feed was poor due  to a combination of
source and processing.

Most  paper is made from wood, but other plant materials have been test-
ed as pulp sources.  Paper-making from water hyacinth has been suggest-
ed and tried  in the past, but thorough, scientific  data is not in the
literature.

Processing

The water hyacinth must be removed from the effluent to remove the  nut-
rients and put them to use.  For  most uses the plants must be processed
 to  reduce bulk and weight, retard or  enhance decomposition, and  to  im-
 prove acceptance and utilization. Growth on an artificial pond  should
 be more  consistent  in quality and quantity than at  natural sites, and
 controlled harvesting should be easier, Important factors  in commercial
 application.  The non-conventional harvesters required  are available.

 The harvested plants' characteristics and  some of its  proposed uses re-
 semble  those  of  some traditional  land forages.   Forage processing prac-
 tices,  including chopping, ensiling,  drying and  pelleting, should be
 applicable with modification.  Casselman,  et. al.   explored  the use of
 presses for  reducing the  high  initial moisture content of  some tropical
 forages.  Pirie1   applied maceration  and  pressing to extraction  of  pro-
                                   118

-------
Table 1.  Composition of Water Hyacinth'
                                               %, dry basis
                                             (except as noted)
Dry Matter, % wet basis
     Crude Protein
          Nitrogen
     Cellulose
     Available Carbohydrate
     Ether Extract
     Ash
          Phosphorous
          Calcium
          Potassium
          Magnesium
     Energy, kcal/g dry
 5.9
16
 2.5
28
 7.8
 3.5
17
 0.42
 1.0
 4.4
 1.1
 3.8
a.  from Boyd
             11
                              779

-------
tein from freen plants.  In both cases, the resulting juice was rich in
protein, minerals and pigments.  Pirie describes ways that these valua-
ble components can be recovered, including pH adjustment, aging and
heating which cause coagulation or precipitation.  The required tech-
nology for utilization is available and needs only to be adapted to the
crop.

OBJECTIVES

How much nitrogen and phosphates can water hyacinth remove from second-
ary treated sewage effluent?  How do pool geometry, season, and deten-
tion time affect removal?  How should plant harvest be managed?

What is the cost of producing compost?  What is its value?  Is there a
market?

Will cattle readily consume processed water hyacinth?  Can they utilize
the nutrients the processed water hyacinth contain?  Will they perform
well on processed water hyacinth?  Are there health-related problems
from eating water hyacinth?  What is the cost of producing processed
water hyacinth feed?  What is its value?  Is there a market?

Can paper be made from water hyacinth?  What are the production re-
quirements of such paper?  What are the characteristics of the pulp and
paper?  What is the value of the paper and the cost of its production?

What product is most feasible, economically?

PROCEDURES

Water hyacinth was grown on extended aeration secondary treated sewage
effluent, harvested, and processed to produce a range of products as
shown in the overall system schematic in Figure 1.  Loosely coordinated
individual projects in nutrient removal, paper-making, processing and
animal feeding used the same pool of water hyacinth.

Water hyacinth was established on one of the oxidation ponds at the
University of Florida Campus Sewage Treatment Plant in early April,
1972.  Seven bushels of plants were collected from Lake Alice, on the
University of Florida campus about a half mile downstream from the
sewage plant, and placed in the 7400 ft  pond, where they initially
covered an estimated 75 ft .  Surface coverage was determined period-
ically until the pond was covered.  In late May, nine 3x3 ft pens were
built in an adjacent pond and plants were placed in them for mass
growth rate determinations.

Influent and effluent were sampled automatically and analyzed for Kjel-
dahl nitrogen, ammonia nitrogen, nitrate nitrogen, orthophosphate, and
total phosphate.  Samples for dissolved oxygen determinations were tak-
en  near the center of the pond at 6 inches below the surface, mid-depth


                                  720

-------
  CAMPUS
  SEWAGE
     I
SEWAGE
TREATMENT
PLANT
     1
                                        COMPOST
•COMPOST
                                         PULP
•PAPER
OXIDATION
POND
(WATER
HYACINTH)


WATER
HYACINTH
HARVEST -
PROCESS


ENSILE
                                                     'SILAGE
     1
  EFFLUENT
  TO
  LAKE ALICE
 DRY FEED
                                                      JUICE
                                                  CONCENTRATE
 Figure 1.  Water Hyacinth Effluent Nutrient Removal -
             Utilization Scheme
                             727

-------
and 6 inches above the bottom.

The pond was operated throughout most of the test with a 15-hour deten-
tion time, much shorter than any reported previously in nutrient uptake
tests with water hyacinth.  During the last 2% months of the test, from
early February to mid-April, 1973, detention times of 6 to 24 hours
were used, to establish the effect of detention time in this range.

The system shown in Figure 2, combining conventional and unconventional
forage processes, was used to convert aquatic plants to a variety of
products.  It consisted of harvesting, chopping, pressing, ensiling,
drying, pelleting and juice solids recovery operations and produced
compost, pulping stock, silage, dry feed and a feed concentrate.

Plants were harvested irregularly throughout the summer and fall of
1972 by hand or with a small conveyor.  Harvested plants were chopped
with a slightly modified forage harvester.

Compost was produced from whole and chopped plants by storing wet
plants aerobically for 1 to 6 months, then drying and grinding.  Com-
post was evaluated by mixing with other soil materials and growing
plants in pots.

Large quantities of chopped plants for feed production tests WCKC
pressed in the 12-inch Vincent press used by Casselman, et.al.  .
Smaller lots were pressed in the eight-inch and nine-inch presses built
by Bagnall  .  Methods of recovering nutrients, particularly protein,
from the juice were explored.

Most of the eff 1 u.ent-grown water hyacinth was ensiled.  The pressed
water hyacinth was mixed with up to 4% dried citrus pulp or cracked
corn and up to 1% standard cane molasses as free carbohydrate sources
prior to ensiling.  Lots of 220 Ib were stored for 21 to 60 days in
polyethylene-lined barrels with trapped drains.  Lots of about 3000
pounds were stored in four-feet diameter by 8-feet long asphalt-lined
currugated culverts set on end.

Some of the pressed water hyacinth was dried in a static bed at 125 F
to 140 F and stored as coarsely ground feed.  Water hyacinth from other
sources was dried on other types of dryers, including an instrumented
high-temperature rotary dehydrator.  Drying characteristics and costs
can be projected from the combined data.

One lot of processed water hyacinth was pelleted through a 3/8x2-inch
die in a flat plate pellet mill.  Densities and durabilities were
found and observations made regarding production rate and energy
requirement.

Whole plants, chopped plants, pressed plant residue, pressed juice,
dried plant products, silage and mixed feeds were analyzed for moisture
and nutrients, primarily crude protein and ash.  In some cases, more
                                  722

-------
                    WEEDS
                  HARVEST
     PROCESS





    TRANSFER




/\ STORE
    USE
       WASTE
CONCENTRATE
COMPOST
                                           COMPOST
                                           PULPING STOCK
                                                    SILAGE
                                                    FEED
                       PELLET
         FEED
 Figure 2.   Aquatic Weed Harvesting - Processing System
                              123

-------
detailed analyses, primarily of the ash, were made.

Animal acceptability of water hyacinth products was established by plac-
ing them before the animals for several days, either as the only feed
available or in competition with other feeds, and observing order of
choice and quantity consumed.  Feed was weighed before feeding and left-
overs weighed back and replaced each day to minimize effects of deteri-
oration, especially with silage.

Yearling steers were fed dried water hyacinth, dried hydrilla (Hydrilla
verticillata) and coastal Bermudagrass  (Cynodon dactyl on) as 33% of the
organic matter in complete pelleted diets to determine acceptability,
as measure by voluntary  intake, and utilization of nutrients, as meas-
ured  by balance of intake and collected excrement.  Sheep and cattle
were  fed water hyacinth  silage from the culvert and tower silos, alone
and with supplementary dietary components, to determine voluntary intake
and nutrient utilization.  Coarsely ground dried water hyacinth press
residue was included as  10%  of cattle  diets for comparison with cotton-
seed  hulls and sugarcane bagasse pellets as roughage sources in a 112-
day feeding trial.

Health of all  animals was  observed during all  trials and selected ani-
mals  were slaughtered and  examined in  detail.

Whole plants  and  elongate  petioles only were  chopped and used for pulp-
ing    The pulping system is  shown  in  Figure  3.  The chopped  plants were
passed  through an attrition  mill to reduce the plants  to very  small
particles and  break  the  physical bond  between fiber bundles  and  pith.
The  pulp was  washed  on a travelling screen by high pressure  water spray,
and  the clean  fiber  bundles  were dried and stored.  The fiber was pulped
by cooking  in  experimental  digesters  using four different  processes
shown in Table 2.  Pulp  fiber strength was developed  and fibers  separat-
ed by beating.   The  pulp was tested for freeness,  a measure  of  drainage
rate, and  test handsheets  were formed.  Tear, burst and  breaking
strengths were found.

 RESULTS AND DISCUSSION

An area growth curve of  water hyacinth in the oxidation  pond is shown in
 Figure 4.   Time for doubling of area  in the  central  part of the curve,
 following  establishment of the stand  and before crowding,  is 6.2 days.2
 This rate of growth is about twice that observed by Penfound and Earle .
 When 60% of the water hyacinth was harvested in August,  recovery was
 very slow until  the remaining mat was broken up;  peripheral  length  must
 be maximized to assure maximum growth.

 Time for 50% increase in wet weight was 7 days, indicating^ growth rate
 43%  higher than that observed by  Knipling, West and Nailer  and 86%
 faster than that observed by BockJ.   Fertilization and other conditions
 existing in the  pond are conducive to  higher than normal growth rates.


                                    724

-------
                       CHOPPED WATER  HYACINTH
                        ATTRITION
    WASTE
WASH
                        DRY
CHEMICALS
DIGEST
                        BEAT





                            FREENESS SAMPLE




                        FORM SHEET










                        PAPER
 Figure 3.  Water Hyacinth Pulping  Process
                     725

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Table 2.  Water Hyacinth Paper-Making Processes
Chemical concentration
70 g/1 sodium sulflte
25 g/1 sodium hydroxide
36.6 a/1 sodium bisulfite
Liquor
Ratio
7:1
7:1
12:1
Temperature
C
135 -
121 -
156 -
172
148
162
        10-35  g/1  sodium sulfite

                                          12:1           121 - 156
35 g/1 equivalent Na«0, 25$ sulfldlty
     sodium hydroxide + sodium sulfide
     (kraft process)
                                     726

-------
Q
LJ
OH
LU
§
UJ
or
Q

2
LL
O
    100
50
     20
10-
      2-
                             POND  AREA : 7361 FT
                       20
                                  40
60
                             DAYS
       Figure 4,  Water Hyacinth Area Growth  in Sewage Treatment
                   Plant Effluent Oxidation  Pond
                            727

-------
Dissolved oxygen concentrations in the pond ranged from 0.95 ppm near
the bottom to 3.46 ppm near the surface; anaerobiosis was not an impor-
tant factor in nitrogen reduction.  Total reductions of nitrogen and
phosphorous for the year are shown in Table 3 with average influent and
effluent concentrations.  Nitrogen and phosphorous removal during the
test are shown in Figure 5.  Removal of nurients was much lower than
reported by other investigators, at least partly attributable to much
shorter detention time.

The plant roots extended downward only about 2 inches into the 54-inch
deep pond, exposing the roots to only 4% of the water.  The nutrients
must be carried to the roots by turbulence, of which there was little,
and diffusion.lnA shallower pool, such as the 18 inches recommended by
Miner, et. al.   would probably be more satisfactory for this applica-
tion.  During most of the test period the plants were crowded and not
growing vigorously, so managed harvesting might have improved perform-
ance.

Nitrogen and phosphorous balances for the first ten months of operation
of the pond are given in Table 4.  Stand density, based on weight and
area of plants removed for ensiling in August, was 111 tons per acre.
Nitrogen and phosphorous in the plants are based on stand density, area,
known moisture and nitrogen contents and estimated phosphorous content.
Only about 10% of the two nutrients was removed, and only about 10% of
the removed nutrients can be accounted for in the plant tissues.  Some
of the missing nutrients may be in the detritus on the bottom of the
pond.  With a mass growth rate of 50% every week, 1/3 of  the plants
could be removed each week, for a total of 218 tons in the ten months
of testing.  This management scheme would have removed 476 pounds of
nitrogen and 127 pounds of phosphorous,  instead of the 24 and 6.5
pounds, respectively, shown in the table.

Effect of short detention times on nitrogen and phosphate removal is
shown in Figure 6.  Removal is generally greater at greater detention
times but data are very irregular, possibly due to weather and transient
effects, and inconclusive.

Harvesting - Processing

Harvest rates of over 4 tons per  hour per foot,of width  have been ob-
served for flat-wire-belt conveyors with reels   .  A  conveyor uses  less
than 0.33 horsepower-hours per  ton.  Cost of  harvesting was $2.13 per
wet ton; costs at a fixed site with reliable  machinery could be as
little as 25% of that figure.

A forage chopping cylinder reduces water hyacinth satisfactorily, but
feed  and discharge  systems of most  choppers must  be modified to give a
satisfactory capacity   .  A 16-inch wide cylinder chops  almost  2  tons
 per hour per  inch of cylinder width and required  a minimum  of 0.18
 horsepower-hours  per ton.

                                    725

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Table 3.  Nutrient Removal for the 12-Month Study Period
Average Average % Removal
Influent Effluent
ppm ppm
Total Nitrogen
Kjeldahl Nitrogen
Ammonia Nitrogen
Nitrate Nitrogen
Total Phosphates
Orthophosphates
Table 4. Nutrient Balance in

Entering pond in 10 months
Leaving pond in 10 months
Removal
Removed in Plants, August
Inventory in Plants, February
Missinq
12.11 10.98 9.3
5.57 4.80 13.8
3.88 3.31 14.6
6.54 6.18 5.3
4.19 3.81 8.0
3.18 3.03 4.8
Water Hyacinth - Covered Oxidation Pond
Total Total
Nitrogen Phosphorous
Ib % ID %
6670 2310
6040 2100
630 9.4 210 9.1
24 0.4 6.5 0.3
41 0.7 11 0.5
565 8.4 193 8.2
                                  729

-------
30-
<

O

LU
OC
20-
 104
 O-
                                   TOTAL  NITROGEN
                                    ' TOTAL PHOSPHATES
     15-HOUR DETENTION
    APR  ' MAY  ' JUN ' JUL  ' AUG '  SEP ' OCT ' NOV ' DEC '  JAN

                           MONTH
   Figure 5.  Nutrient Removal in Water Hyacinth - Covered
               Oxidation Pond
                          130

-------
  2CH
                                   TOTAL  NITROGEN
_J
  10-
O
2
LJ
o:
   o-
                                TOTAL PHOSPHATES
    O
                5          10          15          2O

                 DETENTION  TIME ,  HOURS
25
     Figure 6.  Effect of Detention Time on Nutrient Removal
               in a Water Hyacinth -  Covered Oxidation Pond
                             737

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A light-weight, decreasing-volume screw press can readily remove over
75% of the water from ¥iter hyacinth and is reasonably tolerant of un-
even loading and trash  .   A 12-inch press can easily process 12 tons
per hour and a 16-inch press should be able to process 28 tons per hour.
Estimated energy requirement is 2.5 horsepower-hours per wet ton or 4
horsepower-hours per ton of water expressed.

Relatively sophisticated combinations of chemical, thermal and mechani-
cal juice solids recovery can be used on a fixed site with enough space.
Recoveries of up to 63% were achieved in the laboratory   and could be
exceeded with improvement in equipment and technique.

Good quality silage can be made from pressed water hyacinth by adding
about 4% cracked corn or dried citrus pulp and storing anaerobically  .
If moisture content of the pressed water hyacinth is below 85%, drain-
age will be negligible.  Excellent silage was made in 21 days and 60-
day silage was very acceptable.

Water hyacinth must be dried thoroughly and quickly to make dry feed.
Whole plants in static beds dry slowly and with only 13% efficiency.
Pressed plants dry only 10% faster than whole plants and 60% more ef-
ficiently, but the principal benefit of pressing is the large, rapid
water reduction with low energy input and the small gain in drying per-
formance is a secondary benefit.  Without agglomeration of fines, per-
formance would have been much better.  A 6000-pound-per-hour rotary de-
hydrator dried 6800 pounds of pressed water hyacinth per hour from 88
to 22% moisture, using 1500 BTU per pound of water evaporated; the high-
er efficiency is partially attributable to fluffing of the product be-
fore and during drying.

Pure dried water hyacinth flows poorly and is very frictional and abra-
sive, causing very low pelleting rates and very high energy requirement.
Mixed feeds containing water hyacinth, corn and soybean meal can be pel-
leted at high rates and reasonable power requirements but pellet den-
sity and durability are lower.

Material balance for the complete processing system producing 100
pounds of dried water hyacinth is shown in Figure 7.  Larger or smaller
systems can be scaled proportionally.

Estimated processing costs per dry ton of product are $12.12 for har-
vesting, $1.26 for chopping, $5.25 for pressing, and $26.60 for drying.
Resulting product costs on a bone dry ton basis are $13.38 for compost
at 78% moisture, $33.03 for silage at 80% moisture and $45.23 for dry
feed at 10% moisture.  Bases for these costs are a 100-wet-ton-per-hour
interim'ttant operation, 20% solids loss between harvest and product,
$3.00 labor, $0.25 diesel fuel, $0.20 bunker C oil for drying, and typ-
ical machine costs and efficiencies.  Neglected were costs of buildings
and transfer and storage equipment.  The mechanical operations are cap-
ital-intensive, while drying is energy intensive.  Capital and labor
costs per unit of production would be smaller for larger and continuous

                                   132

-------
2381     chopped hyacinth
         2262  water
          119  solids
               17  protein
               20  ash
                                    9,700 BTU
         pressed hyacinth
          360  water
           90  solids
               10  protein
               10  ash
1931
                                  525,000 BTU
                             350  water
         dried hyacinth
           10  water
           90  solids
               10  protein
               10  ash
juice
1902  water
  29  solids
       7 protein
      10 ash
1715
 192
1






I


solution
1708 water
7 solids
1 protein
5 ash


water
>
216




288,000 BTU\

24
         residue
          194  water
           22  solids
                6   protein
                5   ash
         dried  residue
            2  water
           22  solids
                6  protein
                5  ash
     Figure 7.   Process Material  Balance for 100 Pounds  of  Dried
                Water Hyacinth
                                 733

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operations.

Composition

Analyses of the chopped and pressed water hyacinth from the oxidation
pond and Lake Alice are given in Table 5.  Moisture and protein contents
of the oxidation pond water hyacinth are higher than those of the lake
water hyacinth, in response to fertilization.  Ash content is high in
both and should be considered in diet design, both with regard to quan-
tity and balance.

Compost

Composted water hyacinth holds water unusually well.  Plants grow poor-
ly in pure compost, but perform satisfactorily in sandrcompost ratios
around 3:1.  Estimated market value of the compost is about $46 per ton.

Utilization by Ruminant Animals

Cattle and sheep voluntarily consumed diets  containing processed water
hyacinth.  When only  pressed dried water hyacinth was offered to steers,
consumption was less  than  1% of body weight; consumption was Increased
to 1% by adding 30% molasses and  to 1.556 by  pelleting  .  Steers con-
sumed 2.1% of  their body weight of a pelleted diet containing dried
water hyacinth as  33% of the organic matter; consumption was not gjg-
nlflcantly lower than that of a similar  coastal Bermudagrass diet  .
Cattle accepted most  water hyacinth silages  Immediately, but consis-
tently refused silages with no dry carbohydrate additive.  The most
acceptable silages had the highest additive  (dried2§itrus pulp or corn)
levels, lowest pH, lowest  ash, and highest protein   .  Oxidation pond
water hyacinth silage was  accepted better than Lake  Alice water hya-
cinth silage.

Digestibility  by cattle and sheep of diets containing  processed water
hyacinth varied with  level of water hyacinth in the  diet,  source of
water hyacinth and method  of processing. Dry matter digestibility in
dried water  hyacinth  was half that  in Bernudagrass  hay, apparently re-
flecting nutrient  loss 1n  the press juice   ; however,  there  were  no
differences  between  the two d1etsdfor net retention  and apparent  ab-
 sorption of  ten  mineral elements".   Digestibility  of  organic  matter
 and  crude  protein  was higher  in  oxidation  pond  than  in lake  water hya-
 cinth  silage,  as shown in  Table  6,  but  was  highest  in  pangolagrass
 silage   .

 Animal  performance was best when level  of  water hyacinth,  on a dry or-
 ganic matter basis,  was  less  than 25% of complete diets  for  cattle and
 sheep.   All  animals remained in  good  health throughout all  experiments.
 Levels  of  potential  toxicants,  namely oxalates,  nitrates,  tanmns,
 cyanides and dicouawrin apparently were within safe ranges as no tox-
 idty was  observed   .
                                   734

-------
Table 5.  Composition of Processed Water Hyacinth
Oxidation Pond
Chopped Pressed
Dry Matter, % wet basis 4.6 11.7
Crude Protein, % dry basis 14. 8 12.7
Ash, % dry basis 22.5 13.0
Calcium, % dry basis
Phosphorous, % dry basis
Potassium, % dry basis
Sand-s-lica, % dry basis
Crude fiber, % dry basis
Cellulose, % dry basis
Ether Extract, % dry matter
Gross energy, local per gram
Lake
Chopped Pressed
5.0 9.6
11.3 10.6
23.0 17.3
2.28
0.4*
2.0*
5.9*
—
26.1**
	
	

Silage***
18.6
9.1
11.2
2.4
0.4
4.7
—
21.7
	
1.9
4.0
*from Stephens
                22
**from  Salveson
***from Kiflewahid25
 Table  6.   Digestibility of  Nutrients  in  Water  Hyacinth Silage by Sheep

   ~Organic MatterCrude  Protein
 	Digestibility %     Digestibility %
 Oxidation Pond Water Hyacinth
    + DCPb                         48                51
 Lake Alice Water Hyacinth + DCP   40                53
 Pangolagrass + DCP                54                76

 afrom  Baldwin23
 bDried citrus pulp added at ensiling  time
                                   735

-------
     In feedlot and digestion trials, dried water hyacinths satis-
factorily replaced the conventional roughages, cottonseed hulls and
pelleted sugarcane^bagasse, at levels from 10 to 20% of complete
high energy diets.25
     All criteria measured showed processed water hyacinth has a re-
placement value of at least equal to cottonseed hulls and sugarcane
bagasse pellets.  Current market price of these products is $40 to
$45 per ton.
Fiber
     After attrition and washing, 18 to 20% of the plant material was
recovered as clean fiber.26  In the sulfite process and sulfite-
bisulfite process, pulp yield was as high as 80 to 88% of the fiber
placed  in the digester; the highly alkaline hydroxide and kraft pro-
cesses yielded only 40 to 50%.  Paper strengths were low when yield
was above 65 to 75%.  Cooked fiber bundles had to be beaten to
separate them into individual fibers.
     Mechanical characteristics of the papers are shown in Table 7.
Freeness of the water hyacinth pulp  is much lower than that of pine
kraft  pulp and reduces the freeness  of blends.  Hyacinth pulp drained
to paper on a screen  in 5 to  10 minutes, while pine kraft pulp re-
quires  only about 30  seconds; a given plant would have only 6% the
capacity using water  hyacinth pulp as it would have with pine  kraft
pulp.   Slow drainage  also produces a paper of non-uniform  strength
and texture.  Water hyacinth  paper had usually high shrinkage  and
was dark and dirty-appearing.

Tear factor decreases and breaking length  increases with increasing
water  hyacinth  content in blends  and were  highest for  the  alkaline
processes and  lowest  for the  sulfite-bisulfite process.  Burst factor
increased with  increasing water  hyacinth content, was  highest  for
kraft  process  and lowest for  the  sulfite-bisulfite  process.  Use
of  petioles only  instead of  the whole plants  slightly  improved
breaking length and tear factor  but  decreased burst factor and
freeness.   Breaking  length and burst factor  are  higher than those
of  pine kraft,  but low tear  factor would make the  paper unacceptable
for packaging  and low freeness and yield would make it uneconomical
to  produce,

 CONCLUSIONS AND RECOMMENDATIONS
 A water hyacinth covered pond with a detention time of over 10 hours
 removed 9% of the nitrogen and 8% of the phosphorous from secondary
 treated sewage effluent.   Removal is increased to 80% and 60%, re-
 spectively by increasing detention time to 5 days.   Low plant vigor,
 caused by crowding and low temperature, and excessively deep water
 reduce removal.  Only 10% of the removed nutrients were found in
 the plant tissue.  Plants should be harvested regularly at the rate
 of 25 to 33% per week to maintain maximum growth and nutrient uptake.


                                   136

-------
Table 7.  Mechanical Characteristics of Water Hyacinth Pulps and

           And Papers at 10-Minute Beating Time
                                   Freeness   Tear   Breaking  Burst
                                            Factor  Length    Factor
                                      ml            m
Pine kraft
65% pine kraft, 35% water hyacinth
50% pine kraft, 50% water hyacinth
Sulfite, whole water hyacinth
Sulfite, water hyacinth peticles
Sulfite-bisulfite, water hyacinth
Hydroxide, water hyacinth
Kraft, water hyacinth
720
160
90
40
20
45
40
30
205
130
95
28
30
15
32
35
4300
9600
10000
7200
7900
6600
8700
>nooo
26
52
54
53
42
40
52
>75
afrom Nolan26
                                   737

-------
Compost can be produced from water hyacinth at a cost of about $3.00
per ton and sold for about $46 per ton.  A readily developable mar-
ket to nurserymen and home gardeners is available.

Cattle and sheep readily consumed water hyacinth silage and will con-
sume dried water hyacinth up to 25% of a complete diet.  Processed
water hyacinths had nutritional value in cattle and sheep diets
primarily as a source of energy, mineral elements and roughage.
Animal performance and health on diets containing processed water
hyacinths were satisfactory.  Estimated production cost of dry feed
is $40.70 per ton and silage is $6.62 per ton.  Estimated value as
dry feed is $40 to $45 per ton.  Market development depends on con-
sistency of quantity and quality of supply and price.

Paper can be made from water hyacinth, but yield  and freeness are
low, making production uneconomical.  The paper has good breking
and burst strength but low tear strength.  Because of  low pro-
duction rate, production cost would be too high to be  competetive.

Compost appears to be the most feasible product,  followed by cattle
roug  hage.
                                   138

-------
                              REFERENCES

1.  Holm, L. G., L. W. Weldon and R. D. Blackburn.  Aquatic Weeds.
      Science.  166:699-709, 1969.

2.  Penfound, W. T. and T. T. Earle.  The Biology of the Water
      Hyacinth.  Ecological Monographs.  18(4):447-472, 1948.

3.  Bock, J. H.  An Ecological Study of Eichhornia Crassipes with
      Special Emphasis on  its Reproductive Biology.  Berkeley,
      University of California, Thesis, 1960.

4.  Knipling, E. B.,  S. H. West and W. T. Haller.  Growth Character-
      istics, Yield Potential, and Nutritive Content of Water
      Hyacinths.   Proceedings: Soil and Crop Science Society of
      Florida.  30:51-63,  1970.

5.  Haller, W.  T.  and D.  L. Sutton.  Effect of pH and High Phos-
      phorous Concentrations on Growth of Water  hyacinth.  Hyacinth
      Control Journal.  Vh59-61, 1973.

6.  Haller, W.  T., E. B.  Knipling and  S. H. West.  Phosphorous
      Uptake  and Distribution  in Water Hyacinth.  Proceedings:
      Soil  and  Crop Science Society of Florida.   30:64-68, 1970.

7.  Sheffield,  C.  W.  Water Hyacinth   for Nutrient Removal.  Hyacinth
      Control Journal.  6:27-30,  1967.

8.  Clock,  R.M. Nitrogen and  Phosphorous Removal from  a Seconday
      Sewage  Treatment  Effluent.  Gainesville, University  of  Florida,
      Doctorial Dissertation,  1968.

9.  Steward,  K. K.  Nutrient Removal  Potentials  of Various Aquatic
       Plants.  Hyacinth Control  Journal  8_: 34-35, 1970.

10.  Miner,  R. J.,  J.  W. Wooten and  J.  D.  Dodd.   Water  Hyacinths  to
       Further Treat Anaerobic Lagoon  Effluent.   In:  Livestock Waste
       Management and Pollution Abatement.   St. Joseph,  Michigan,
       American Society of Agricultural Engineers, 1971, 170-173.

11   Boyd, C.  £.  The Nutritive Value of Three Species  of Water Weeds.
       Economic Botany.   23(2):123-127, 1969.

12   Hentges, J. F.   Processed Aquatic Plants for Animal Nutrition.
       Proceedings: Aquatic Plant Research Conference.   Gainesville,
       Governors Aquatic Research and Development Committee,  1970,
       62-67.
                                   139

-------
13.   Casselman, T.  W., V.  E.  Green,  Jr.,  R.  J.  Allen,  Jr.,  and  F. H.
       Thomas.  Mechanical  Dewatering of  Forage Crops.   Gainesville,
       University of Florida, 1965.   AES  Technical  Bulletin 694.

14.   Pirie, N. W.  Leaf Protein: Its Agronomy,  Preparation, Quality
       and Use.  Oxford, Blackwell  Scientific Publications, 1971.

15.   Bagnall, L. 0.  A Simple Screw Press for Water Hyacinth.   Procee-
       dings:  Association of Southern Agricultural Workers. 70_:28,
       1973.

16.   Phillipy, C. L. and J. M. Perryman.   Mechanical Harvesting of
       Water Hyacinth (Eichhernia crassipes) in Gant Lake Canal,
       Sumter County, Florida.Tallahassee, Florida Game and Fresh
       Water Fish Commission, 1972.

17.  Stewart, J. S.,  III.  Energy and Flow Requirements for Chopping
       Water Hyacinths.  Gainesville, University of Florida, ME
       Thesis,  1972.

18.  Bagnall,  L. 0.,  R. L. Shirley  and J. F. Hentges.  Processing,
       Chemical  Composition  and Nutritive Value of Aquatic Weeds.
       Gainesville, University  of Florida Water Resources Center, 1973.

19.  Bagnall,  L. 0.   Mechanical REcovery of Water  Hyacinth  Press Liquor
       Solids.   St. Joseph,  Michigan, American Society of Agricultural
       Engineers,  1973, Paper 73-562.

20   Bagnall,  L. 0.,  J. A. Baldwin  and J. F. Hentges.  Processing and
       Storage of  Water Hyacinth Silage.  Hyacinth Control  Journal.
       12:{in press), 1974.

21   Byron, H.  T.,  Jr.  Nutrient Value of Water Hyacinth Silage for
       Cattle and  Sheep.   Gainesville, University  of  Florida,  MSA
       Thesis, 1973.

 22   Salveson, R.  E.   Utilization  of Aquatic Plants in  Steer Diets:
       Voluntary Intake and  Digestibility.   Gainesville, University
       of Florida, MSA Thesis,  1971.

 23.  Baldwin, J. A.  Utilization  of Ensiled Water  Hyacinths in
        Ruminant Diets.   Gainesville, University of Florida, MSA
       Thesis, 1973.

 24.  Stepehens, E. L.  Digestibility Trials on Ten Elements and Three
        Toxicants in Aquatic Plant Diets  Fed Steers.  Gainesville,
        University of Florida, MSA Thesis,  1972.

-------
25.  Kiflewahid, B.  Composition and Digestibility of Processed Water
       Hyacinths.  Gainesville, University of Florida, Doctoral
       Dissertation, 1974.

26.  Nolan, W. J.  and D. W. Kirmse.  The Papermaking Properties
       of Water Hyacinths.  Hyacinth Control Journal. 12:(in press),
       1974.
                                   747

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     THE AVAILABILITY OF DAPHNIA FOR WATER QUALITY IMPROVEMENT
                   AND AS AN ANIMAL FOOD SOURCE

                          By Ray Dinges*

INTRODUCTION

Modern aerobic biological wastewater treatment facilities are
capable of reducing biochemical oxygen demand of their influ-
ents by some 85 to 90 per cent.  The biological activity occur-
ring in such plants has been described by Dinges^- as the fermen-
tation phase of biological treatment and is characterized by ex-
ternal digestion facilitated primarily by solubilization of
organic material by bacterial enzymes.  See Figure 1.  Secondary
to the fermentation phase is the consumption phase which is
characterized by internal digestion.  The predaceous consumption
phase essentially occurs in receiving waters.  It should be
understood that the three phases outlined signify dominant fea-
tures and are not separate and apart but overlap to different
degrees dependent upon water quality and physical conditions of
the environment.  Insects, for example, may be present in all
three phases.

This paper deals with what has been termed the consumption
phase, and specifically, a Daphnia dominated community.  In
recent years there has been considerable interest developed
regarding the possibilities of expanding conventional processes
of biological wastewater treatment for reduction of remaining
organic fractions by utilizing higher life forms.  Uhlmann24
Loedloff3, Erlich^, Scheithauer and BickS, and DeWitt and
Candland^ have all investigated the potential of utilizing
Daphnia to attain improvement of wastewater effluent.
Kryutchkova? discussed energy flows in wastewater stabilization
ponds as related to consumption of particulates by zooplankton.
Greer and ZiebellS utilized asiatic clams Corbicula for reducing
the content of phosphorus from stabilization pond effluent
through their consumption and deposition of algae and other
suspended matter.  Development of a wastewater treatment pro-
cess making marine filtering organisms available to consume
particulates from treated effluent is continuing at the Woods
Hole Oceanographic Institution in Massachusetts.

                      DESCRIPTION OF DAPHNIA

As reported by Ward and Whipple^, Daphnia habitats include tem-
porary pools, small ponds and lakes.  Daphnia are common aquatic

* Division of Wastewater Technology and Surveillance, Texas
  State Department of Health, 1100 West 49th Street, Austin,
  Texas, 78756.

-------
                    Figure 1
                  BIOLOGICAL
           ORGANIC  STABILIZATION
                  NON  VIABLE
                ORGANIC WASTE
             FERMENTATION  PHASE
BACTERIA
FUNGI
PROTOZOA
ALGAE
 ETC.
External digestion
                   Ciliotes
                   Stalked Ciliates
                   Rotifers
                   Etc.
                              SOLUBLE ORGANICS
                              COLLOIDS
                              BIOMASS
                              DETRITUS
             CONSUMPTION  PHASE
OAPHNIA
CILIATES
 STALKED CILIATES
 ROTIFERS
 ETC.
Internal digestion
                   Bacteria
                   Fungi
                   Protozoa
                   Algae
                   Etc.
                               SOLUBLE ORGANICS
                               COLLOIDS
                               BIOMASS
                               DETRITUS
         PREDACEOUS CONSUMPTION
         FISH, INSECTS, ETC.
                      743

-------
crustaceans about 1/8 inch in length and feed upon algae, bac-
teria, protozoa and debris.  Food particles of a proper size
are apparently filtered from water and consumed in a rather
indiscriminate manner.

Daphnia exhibit both sexual and parthenogenic reproduction.  A
population will consist almost entirely of parthenogenic females
when environmental conditions are satisfactory.  A parthenogenic
female may produce 30-40 new Daphnia every two days.  Male
Daphnia appear when environmental conditions become less than
optimal and sexual eggs are produced.  Two eggs are enclosed in
a dense, durable structure called the ephippium.     Sexual egg
production serves to assure survival of Daphnia when habitat
conditions again become favorable.  The life span of Daphnia is
about one month.  Two species found in Texas wastewater stabili-
zation ponds were Daphnia  pulex and Daphnia similis.  Daphnia
similis is shown in Figure 2.

                       ENVIRONMENTAL FACTORS

Stabilization  ponds supporting Daphnia populations were noted to
have effluents that were macroscopically clear and stable.  With
cooperation of field  staffs of the Department and the Texas
Water Quality  Board,  as well as municipal employees and the
chemists of our Central Laboratories, a concerted effort was
made during 1970 to 1973 to identify ponds supporting Daphnia
populations.  Occurrence of Daphnia was found to be quite  uncom-
mon, as they were present  in only 29 pond systems out of the
470 domestic wastewater treatment facilities in Texas utilizing
ponds.  Field  surveys were conducted to evaluate environmental
factors affecting Daphnia  with the intent of establishing,  if
possible, a continuous culture for effluent  improvement.
See Figure 3.

LIGHT

Observations made during the first year of the  field studies
revealed quite clearly that Daphnia  population  pulses  in stabi-
lization ponds were seasonal phenomenon.  Daphnia populations
appeared during the early  winter months and  disappeared  in late
spring.  Figure 4 represents available  sunshine and air  tempera-
ture  data  on a monthly average basis as indicated by weather
records for Austin, Texas.  It was postulated  that  a Daphnia
population pulse was  either  dictated by temperature, or  photo-
period.

After having made a review of the  literature and conducting
 further studies,  it was concluded  that, in Texas, photoperiod
 is the dominant  environmental factor affecting Daphnia  in
 stabilization  ponds.  The  consequence  of  long periods  of bright


                               1HH

-------
              Figure 2
Daphnia slmllls

-------
          Figure  3
DAPH NlA    PONDS
     A  —  pulex
         —  si mi I is
                 746

-------
              Figure
     AVAILABLE   SUNSHINE
JAS  ONDJ  FM  AMJJ
                 747

-------
sunlight is an excessive production of phytoplanktonic algae
in ponds which results in high pH caused by algal uptake of
available free carbon dioxide and the subsequent shift in the
carbonate equilibrium at the expense of bicarbonates.  Ammonia
is almost always present in Texas stabilization ponds.  As
early as 1934, ChipmanlO, demonstrated toxicity of ammonia to
Daphnia at elevated pH levels.

TEMPERATURE

Ammonia toxicity results from dissociation of ammonia, which
is a function of both pH and temperature, with ammonia becoming
more toxic with an increase in temperature.  Stahl and Mayll,
and others have shown that microstratification occurs in shallow
stabilization ponds as a result of high temperature  and lack of
wind induced mixing.  Stagnant lower layers in a stratified
pond often are anaerobic and provide suitable environments for
sulphate reducing bacteria.  Warm water temperature  enhances
growth  rate of the bacteria and sulphides produced are quite
toxic.   Scheithauer and  BickS have stated that Daphnia can tol-
erate up to 3.0 ppm of hydrogen sulphide, but prefer a level
less than  0.4 ppm.

Several observers have attributed demise of Daphnia  populations
to water temperature  increase.  Algae  free Daphnia cultures
were maintained in shallow,  outdoor  concrete containers exposed
to full sunlight during  the  past three  summers.  Water tempera-
tures recorded daily  in  mid-afternoon  during the summer of  1970
in an exposed culture container 14 inches  in depth were typi-
cally in the  upper 80Ts  F°.   Monthly mean, maximum and minimum
surface water temperatures  of Texas  ponds  during the period
from 1967  to  1973 are depicted  in Figure 5.  It appears logical
to assume  that endemic strains  of pulex and similis  are able  to
withstand  normal water temperatures  in Texas stabilization  ponds.

DEPTH

The  physical  characteristic most common to ponds with Daphnia
populations was that  of  water depth.   The  usual depth of  Texas
stabilization ponds  is  3 feet.  Almost all ponds  supporting
Daphnia populations  had  depths  varying from 5  to  15  feet.   It is
believed that lower  layers of a deep pond  have  reduced pH^levels
suitable as  an  environment for  Daphnia provided that sufficient
dissolved  oxygen  for respiratory  needs is  present.   Abnormal
pond depth usually  resulted from:

   1.   Deep ponds  provided to serve  as irrigation  water
       holding basins.
   2.   Terrain considerations -  Pond systems constructed
       on slopes  or  in uneven areas  that made  it uneconomic
       to level  bottom.

                                148

-------
I
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                                    Figure 5

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-------
  3.  Air-Aqua - Standard depth of 10 feet.

Several pond systems with Daphnia populations reported in the
literature also were more than 3 feet in depth.  Shallow Daphnia
ponds mentioned were mixed by recirculation, or aeration.

MINERAL

Taub and Dollar^ t ±n devising a compatible media for concurrent
culture of Daphnia pulex and Chlorella. reported that certain
ionic relationships resulted in Daphnia mortality.  They conclu-
ded that chloride should be the dominant anion and that a sodium
to potassium ratio of less than 10:1 could result in toxic
conditions.  In addition, nitrate toxicity was variable, but
decreased with an increase in chloride.  They further concluded
that chloride content should always exceed that of nitrate and
nitrate level be kept as low as consistent to algae growth.

Mineral content and ionic relationships in Daphnia pond waters,
therefore, assumed importance, as a serious handicap would
exist in using these animals for effluent  clarification  if
certain types of waters  should preclude their production.  Water
samples were collected for mineral analyses from all ponds sup-
porting Daphnia.  On an  average basis, the dominant anion found
in  Daphnia pulex pond waters was bicarbonate, but at times
chloride was dominant.   In waters of Daphnia similis ponds,
chloride was usually the dominant anion, but on occasions
bicarbonate was dominant.  The lowest  sodium-potassium ratio
found in Daphnia pulex pond waters was 6.5:1, with a low of
4.7:1 in Daphnia similis pond waters.  In  most instances, there
was a great difference between chloride and nitrate levels,
with the chloride ion being dominant.  However, on two occasions,
nitrate levels in excess of chloride levels were found in waters
of  a pond supporting Daphnia pulex.  The highest nitrate level
found in Daphnia pulex pond water was  82 ppm and 55 ppm  in
Daphnia similis pond water.  Results of field  investigations
indicate that mineral composition of waters in Texas wastewater
stabilization ponds is suitable for Daphnia production.  Mineral
content of pond waters was found to determine  dominance  between
Daphnia pulex and Daphnia similis. with pulex  being restricted
to  relative soft, iron bearing waters  of East  Texas, whereas
similis existed in hard, mineralized waters found in West Texas.
Table 1 presents observed mean and extreme mineral levels in
pulex and similis ponds.  An overlap in range  occurs on  any
individual mineral parameter between similis and pulex pond
waters, but means and maximums exhibit significant disparity.
                                750

-------
                             Table 1

         Mineral Levels in 19 Daphnia similis Ponds N=70
       ppm

Calcium
Magnesium
Sodium
Potassium
Bicarbonate
Sulphate
Chloride
Dissolved Solids
Hardness
Total Alkalinity
Minimum

   13
    3
   59
    3
  133
   28
   53
  437
   80
  170
Maximum

   186
    70
   630
    33
   620
   580
   790
 2,490
   660
   510
 Mean

   61
   23
  291
   15
  329
  218
  276
1,254
  249
  276
          Mineral Levels in 10 Daphnia pulex Ponds N=21
       ppm
Minimum          Maximum
               Mean

                 18
                  5
                111
                  7
                183
                 48
                 80
                496
                 76
                155
Calcium                    6                56
Magnesium                  2                11
Sodium                    55               225
Potassium*                 2                12
Bicarbonate               31               400
Sulphate                  16                85
Chloride                  40               160
Dissolved Solids         308               800
Hardness                  27               172
Total Alkalinity          25               328

      * N=16

BIOLOGICAL

Fish probably would represent the most significant predator on
Daphnia. as Daphnia are recognized as excellent fish food or-
ganisms.  In Texas, Dingesl3 found that five fish species are
potential inhabitants of wastewater stabilization ponds, or in
otherwise dry stream courses receiving flow of stabilization
pond effluent.

  1.  Mosquito Fish - Gambusia  spp.
  2.  Warmouth - Chaenobryttus  coronarius
  3.  Golden Shiner Minnow - Notemigonus crvsoleucas
  4.  Southern Black Bullhead - Ictaluras melas
  5.  Carp - Cyprinus carpio

Of the  fishes listed above, only mosquito fish were ever noted
to occur in ponds  supporting Daphnia populations, and then only
                                757

-------
in small numbers.  Few ponds in Texas are known to support fish
populations other than Gambusia, although almost all systems
have been reported to have had fish introduced into the ponds.
Several insects would be potential Daphnia predators.  Of these,
probably diving beetles Dytiscidae would be most important.
Both the larvae and adults of this insect are very efficient
predators in the aquatic environment.  Diving beetles could
possibly retard initial development of a Daphnia population,
especially if they were present in large numbers, but Daphnia
productive capacity is so great that a population would soon
expand beyond the point where predation would make much of an
impression.

Daphnia are not alone in clarification of water.  The rotifers
Brachionus and the stalked ciliates Vorticella were often noted
as bionts on Daphnia and both feed upon suspended organic mat-
ter.  Among other metazoa recorded were Ostracoda, Amphipoda,
Mollusca, Acari, Insecta, Moina and other varieties of Branchi-
poda all contributing to the stabilization of organic material.

                         DAPHNIA CULTURE

Waters in a Daphnia culture unit are maintained in a clear,
clean condition.  Uhlmann^ termed this condition as a
klarewasserstadium, or clear water stage.  In a klarewasser-
stadium, the population is in a state of semi-starvation with
males being present and sex eggs evident.  Daphnia populations
tend to become balanced with available food supply and popula-
tion growth rate is restricted by presence of sexual animals
and a reduction in parthenogenic birth rates.  A klarewasser-
stadium would be maintained if the objective of culture is
production of high quality effluent.  The population would be
fed at a rate in excess of their dietary requirements in order
to maintain continual parthenogenic reproduction at an advanced
level if biomass production for commercial purposes is intended.

CULTURE UNIT

A small pond was constructed by the City of Giddings, Texas, to
serve as a Daphnia culture unit at their Northside wastewater
treatment plant, which consists of an Imhoff Tank and three
stabilization ponds in series.  A portion of effluent from the
final pond was diverted to the culture unit.  The outlet from
the pond was provided with a concrete, broad crested, rectangu-
lar weir eight feet in length and with a crest width of six
inches to reduce outflow velocity and washout of Daphnia.
(Dingesl1* has prepared a paper on biological considerations
of stabilization pond design for enhancement of zooplankton
production, which is too lengthy to discuss here).
                               752

-------
The first study phase was an investigation of the effectiveness
of pH control by the regulation of pond exposure to sunlight and
the resulting reduction of algae growth.  The second phase eval-
uated both pH control by chemical addition and sulphide reduc-
tion through the mixing of pond waters by diffused air.

EFFLUENT QUALITY

Effluent quality results of samples collected weekly during the
first run are presented in Table 2 and results of samples col-
lected weekly during the second run are presented in Table 3.

                             Table 2

          Mean Effluent Quality of Daphnia Culture Unit
                        1-6-72 to 3-23-72

   Detention - 11 days  N=10  Loading  - 35 Ibs. BOD5/acre/day

                      Influent      Effluent     % Reduction
     , ppm
COD, ppm
VSS, ppm
Total coliforms
Fecal coliforms
                          45.4
                         271.1
                          77.2
                      32,860.
                       6,880.
   14.2
   90.6
   10.8
9,160.
   84.
68.7
66.6
86.0
72.1
98.7
                              Table  3
      Controlled  Operational  Period of  Daphnia  Culture  Unit
                        2-27-73  to  4-9-73

     Detention  Time  - 11 days  Loading - 43.6  Ibs.  BOD5/acre/day
     Date
 February  27
 March      6
           8
          13
          16
          19
          21
          27
           3
           9
        Mean
April
Influent, ppm
BOD5
90
80
105
25
30
30
40
40
85
50
57.5
COD
245
310
320
75
115
45
110
110
160
95
158.5
VSS
179
121
142
27
29
33
56
58
63
76
78.4
                                             Effluent, ppm
                                           BOD5   COD    VSS
                                           11.8
              60
              60
              90
              60
              65
              90
              25
              70
              95
              85.
              70
       8
      11
       8
       8
       9
       3
       5
      41*
      19
      18.
      13
        Percent Reductions - BOD& - 79, COD - 55, VSS - 83

      * Rotifer bloom in plant pond and passed through unit.

                                753

-------
The BOD^ remaining in the effluent from a Daphnia culture unit
could be expected to approach the level of soluble BOD5 of the
influent.  Soluble nutrient minerals were not removed in passage
through the culture unit.  Phosphate levels remained about the
same and nitrates and organic bound iron increased.
See Figure  6  and Figure 7.

LOADING

Maximum permissible hydraulic and organic loading and minimum
detention time on the culture unit were not determined.  It is
believed that a unit with a 5-day detention time would be sat-
isfactory and that a 10-day detention time would be conserva-
tive, but could serve as a working basis for unit design until
the process is further evaluated.

Organic particulates entering a unit are mostly consumed in the
immediate vicinity of inflow where masses of Daphnia congregate.
Organic loading, measured by the BOD5 test, would not actually
be relative as the test  is based primarily upon respiratory
requirements of fermentative organisms.   The significant por-
tion of the oxygen demand in a culture unit would probably be
exerted by the Daphnia dominated zooplankton community.

Daphnia survive quite well when only a trace of dissolved
oxygen exists.  Mixing of nominally clean waters of unit for
soluble sulphide suppression would take precedence over aera-
tion per se, although mixing of contents entails aeration.
Mixing-aeration should be minimal to needs as Daphnia are weak
swimmers unable to resist even slight currents.  The single
point, fixed feature of  mixing-aeration utilized in the Giddings
culture unit was excessive, and dissolved oxygen levels fluc-
tuated around 5 ppm.

                PRODUCTION - HARVEST - MARKETING

DeWitt and Candland^ relate that commercial Daphnia harvest in
California stabilization ponds commenced in 1968.  The amount
harvested was HO tons, with one pond yielding 25 tons at a rate
of 1.5 tons per acre per month.  Bogatova and Askerovl^, mass
culturing Daphnia in concrete tanks on media including yeast
and fertilizer, achieved a sustained yield of 76.7 Ibs. per
acre foot per day.  One  Texas pond system, consisting of four
35 acre ponds 5 ft. in depth, produces Daphnia  4-5 months each
year.  Even at a somewhat  lower production rate than those
above, the harvestable tonnage of Daphnia would be considera-
ble.  Daphnia in California were harvested by hand nets and a
pump equipped with screening device.  A man with a dip net was
said to be able to harvest up to 100 Ibs. per hour when standing
crop was high.   A 100 gpm pump was used to pump pond water


                               754

-------
             Figure 6

NITRATE  ACCUMULATION  IN
          CULTURE  UNIT
               1973
                          	Influent
                             Effluent
         MAR.
APR.
MAY
                 755

-------
               Figure 7
     {RON  ACCUMULATION IN
         CULTURE  UNIT
               1972
0.40r-
0.30 -
0.20 -
0.10-
       JAN.
FEB.
MAR
                   756

-------
onto a screen on one occasion and rate of Daphnia harvest
attained was 2 Ibs. (wet-drained) per minute.

Various types of manufactured screening equipment suitable for
Daphnia harvest are available as well as low speed separators.
Screen mesh sizes of 40-60 would serve to remove most Daphnia
from water.  A proposed approach to Daphnia harvest would be to
pump culture unit water through removal device with discharge
back to unit.  Baby Daphnia and food material would return to
unit and aid in sustaining production.  A movable, lightweight,
perforated boom supported by floats could serve as a collector
for pump input.  Daphnia tend to swarm and form dense localized
concentrations.  The boom could be moved to  such areas as re-
quired.

There is little need to expound about an ever increasing need
for protein as animal food additives.   Wastewater produced
Daphnia may represent a significant unexploited potential source
of animal protein for this purpose.

Analysis of a sample of Daphnia pulex collected from the
Giddings culture unit indicated a content of 65.3 per cent
protein on a dry weight basis.  The potential market for
Daphnia is probably considerable.  The existing market is quite
limited.  DeWitt and Candland6 stated that the 40 ton harvest
of 1968 in California was sold to wholesalers to be used as
aquarium fish food.  They report that frozen Daphnia brought
15 to 25 cents per pound when sold to wholesalers, depending
on market need and size of order.  They further pointed out
that the aquarium fish food market would soon be flooded should
Daphnia production become commonplace.

It is believed that one excellent use for Daphnia would be as
protein supplement in fish food  pellets used by hatcheries and
commercial fish farmers.  Ghadially16 has pointed out that
Daphnia are  first class fish food, but that  an exclusive diet
of Daphnia provides excessive roughage causing a  laxative effect
resulting  in poor growth.  Production of bait fish, such as
golden  shiner minnows, in a  nearby separate  pond  and fed on
harvested  Daphnia could prove to be  feasible.  Daphnia could
also be employed as protein  additives in pet and  livestock
foods.

                           DISCUSSION

Several thousand stabilization ponds are currently  in use in  the
United  States.   Some  would favor outright  abandonment of  ponds
for wastewater  treatment  considering that  their  ultimate  effi-
ciency  has been  attained  and found wanting  due  to excessive pro-
duction of volatile  suspended solids which  are  largely composed


                                757

-------
of algae cells.  Others feel that this economic, dependable
process can be further improved through objective research with
close cooperation between engineers and biologists.  Figure 8
reveals relationship of light-temperature to volatile solids
levels in Texas stabilization ponds as determined from results
of analysis of 2,02M- effluent samples collected from 1967-1973.
Minimum average indicated production is about 40 ppm, with
volatile solids level in summer being around 80 ppm.   May-
September anomalies denoted by arrows could reflect seasonal
algae species change.  The May anomaly had been noted pre-
viously by Dingesl'.

An organism that may have potential value for improving waste-
water treatment plant effluent either alone, or in conjunction
with Daphnia culture is the introduced asiatic fresh water clam
Corbicula.  These small clams have a very high reproductive
potential and are able to clarify water rapidly.  They have
been spreading throughout the United States since the 1930fs
and Dinges^-8 has reported that they now infest three Texas river
basins.  During this study, clams were placed in wire baskets
and suspended near the outfall of the Giddings stabilization
pond.  The animals promptly died.  There is a need to define
their basic environmental requirements.  It may well be that
like Daphnia. they are sensitive to ammonia and soluble sulphide
toxicity.

                           CONCLUSIONS

1.  Incorporation of advanced biological methods into wastewater
treatment systems to make use of fertile effluents for cultiva-
tion of beneficial organisms appears feasible.
2.  Daphnia culture in wastewater effluent requires pH control
within a range of 7.0-7.5 and sufficient mixing for suppression
of soluble sulphides.
3.  Equipment exists for harvest of Daphnia. but the market is
now limited to aquarium fish food.  It is believed that a poten-
tial market for Daphnia as a protein source for animal foods
exists.
4.  Advanced biological wastewater treatment offers the possi-
bility of salable products to offset treatment costs, or perhaps
to turn a profit.  The resulting clarified effluent is condi-
tioned for biological, or chemical-physical methods for further
nutrient removal and is still available for irrigation or indus-
trial use.
                               755

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                       Figure 8

     TEMPERATURE —  LIGHT 	V.S.S.
     IN  TEXAS  STABILIZATION  PONDS
                 1967 - 1973
  700
 600
 -
.- 5 00
c
3
*»400
«
r
35
Q.
M
1
1

5
               VolotJie
              - Available
Suspended! Solid
 Sunshlnt
            	Temperature
            N » 2024
              1   i
                                            90
                                            •
                                              u
                                            TO
                                            60
                                            50
                         o
                         £
                         a.
                         _
                         .
           SONDJFMAM
             MONTH  OF THE YEAR
                         759

-------
                         ACKNOWLEDGEMENT S

The  support of Henry  L.  Dabney,  Division Director, and assis-
tance  from my  fellow  employees and many other people involved
in the studies reported  upon are appreciated.

                            REFERENCES

1.   Dinges, R.  Ecology  of Daphnia in Stabilization Ponds.
       Austin,  Texas State Department of Health, 1973, 155p.
2.   Uhlmann, D.  Influence of Dilution, Sinking and Grazing
       Rate on Phytoplankton Populations of Hyperfertilized
       Ponds and Micro-ecosystems.  International Association
       of Theoretical  and Applied Limnology. 19:100-124.
       November 1971.
 3.   Loedloff, C. J.  The Function of Cladocera in Oxidation
       Ponds.  Proceedings of the Second International Water
       Pollution Research Conference, Tokyo.  p. 307-325, 1964.
 4.   Erlich, S.  Two Experiments in the Biological Clarification
       of Stabilization Pond Effluents.  Hydrobiologia.
       27_:70-80, 1966.
 5.   Scheithauer, E.,  and H. Bick. Okologisch Untersuchungen
       an Daphnia magna und Daphnia pulex im Freiland und im
       Laboratorium.   Scientific Papers from Institute of
       Chemical Technology, Prague.  8_:439-481, January 1964.
 6.   DeWitt, J. W., and W. Candland. The Water Flea.  The
       American Fish Farmer,  p.  8-10, January 1971.
 7.   Kryutchkova, N. M.  The Role of Zooplankton on the
       Self-Purification in Water Bodies.  Hydrobiologia
       31_:585-595, 1968.
 8.   Greer, D. E., and C. D. Ziebell. Biological Removal of
       Phosphates From Water.  Journal of the Water Pollution
       Control Federation.  44_:2342-2348, December 1972.
 9.   Ward, H. B., and G.  C. Whipple. Freshwater Biology.
       New York, John Wiley and Sons, Inc., 1959, 1248p.
10.   Chipman, W. A., Jr.   The Role of pH in Determining
       the Toxicity of Ammonium Compounds (Doctoral Thesis)
       University of Missouri.  1934, 155p.
11.   Stahl, J. B., and D. S. May. Microstratification in
       Wastetreatment Ponds.  Journal of the Water Pollution
       Control Federation.  jJ9_:72-88, January 1967.
12.   Taub, F. B., and A.  M. Dollar. A Chlorella-Daphnia
       Food-Chain Study:  The Design of a Compatible
       Chemically Defined Medium.  Limnology and Oceanography.
       9_:61-74,  1964.
13.   Dinges, R.  The Ennis Study  - Experimental Chlorination
       of Stabilization Pond Effluent.  Austin, Texas State
       Department of Health, 1970, 117p.
                                160

-------
1M-.  Binges, R.  Biological Considerations in Stabilization
       Pond Design.  Paper Presented to 56th Texas Water
       Utilities Short School, Texas A & M University,
       College Station.   (Unpublished) March 1974.
15.  Bogatova, I. B., and M. K. Askerov. Experience  in Large
       Scale Breeding of Water Flea Daphnia magna.   Rybn Khoz.
       12_:21-26, 1958  (Biological Abstract No.  227M-2, 1965).
16.  Ghadially, F. N.  Advanced Aquarist Guide.   London,
       The Pet Library Ltd.  1969, 282p.
17.  Dinges, R.  Cyclical Chemical Changes in a Eutrophic
       Reservoir.  Report - Austin, Texas State Department
       of Health.   (Unpublished) 1970, 19p.
18.  Dinges, R.  Asiatics Invade Texas.  Water-Southwest
       Water Works Journal,  p. 7-8, June 1973.
                                 161

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           REPORT ON PILOT AQJJACULTURE SYSTEM USING DOMESTIC

             WASTEWATERS FOR REARING PACIFIC SALMON SMOLTS


                   George H. Allen and Larry Dennis—


                          BACKGROUND TO PROJECT

     The utilization of animal wastes, and particularly human wastes,
to fertilize waters for improving the growth of aquatic organisms has
an ancient past  (Allen'' 2;  Mortimer and Hickling*).  Most reclaimed
wastewaters used for food and fiber production  in the United States  is
for increased plant growth on land  (Law^; Wilson and Beckett5).  "Waste
treatment lagoons" have grown rapidly in treating a wide variety of
wastes, especially treatment of domestic wastes by smaller communities
(McKinney, Dornbush and Vennes6).   Such lagoons are now attracting at-
tention because  of their potential  for management as highly fertilized
fish ponds (Ryther, Dunstan, Tenore and Huguenin7).  Catfish (Muggins
and Backman°; Hallock and Zeibell^), bait fish  (Trimberger'0) and sal-
mon ids  (Anon.  ) have been  reared  in these  lagoons  in the  United States.

     In California about one percent of the  state's water  demand is  sup-
plied by  re-use  of wastewaters  (Deaner'*).   About 85% of the use
California is for agriculture,  thus wastewater  re-use for  production
food and  fiber in California  is primarily in agriculture  (Deaner1-*).
Of the  1^0 reclamation sites  listed for 1971, about  100 included some
type of lagoon in the system, but  no uses involving  fish  rearing were
listed.   Reclaimed water was  reported in use in four recreational lakes
and in  four other lakes for miscellaneous purposes.  Studies of these
systems have been primarily on  their reliability of  operation  in order
to protect public health (Jopling,  Deaner and Ongerth"'*).  The poten-
tial uses of algae grown in wastewater ponds,in California has had a
long history of  investigation in California.

     In 1955, the City of Arcata constructed a  55-acre oxidation pond
on the  intertidal flats of the  north arm of  Humboldt Bay  (Figure 1).
This was  the first stage in protecting commercial oyster beds, and other
aquatic resources, from human fecal contamination.   Fisheries personnel
early became interested in the  potential of  this oxidation pond for  aqua-
culture,  and three studies were directed, either entirely  or in part,
toward  this aim  (DeWitt^; Hansen'6; Hazel'7).  Subsequently the City
has installed a  primary treatment  plant, a facultative aeration pond,
and a chlorination unit at the  oxidation pond outlet (Allen, Conver-
sano and
ion  of
M Fisheries, School of Natural Resources
~  Humboldt State University, Arcata, California, 95521.

                                   752

-------
     Initial formal proposals for aquaculture studies in the Arcata
system were made in 1963, with financial support available from the
Humboldt County Board of Supervisors.  A pond system was approved for
funding as a demonstration project by the U.S. Public Health Service
but never received sufficiently high priority for appropriations.  Pre-
liminary studies of the pond water through 2-week standing water bio-
assay using chinook salmon fry indicated that the undiluted water of
the oxidation pond under oxygen saturation was non-toxic during winter
months (Allen and O'Brien'9).  When the ratio of sewage to diluting
water from  infiltration  in to the sewer collecting system increased
with cessation of winter rains, the oxidation pond effluent became tox-
ic to fingerling salmon by June.  These studies indicated the feasibil-
ity of a pilot project, which was subsequently funded by the California
Department of Fish and Game, Wildlife Conservation Board.  Two ponds,
each about  1/3 acre (0.15 hectare) in surface area, were placed  into
operation in July  197K  Fryer^0 recently reported the need to demon-
strate in pilot projects preliminary results on vibriosis control, de-
veloped  in  laboratory experiments.

     Modern methods for the  culture of  salmon and trout  (Qncorhynchus
and Sal mo) employ  highly mechanized operations (Leitr \tz2TJ~.  This has
resulted because of historically high labor costs in comparison  to
energy costs.  Mechanization has  included automatic feeding machines
using pellets made in part from high-quality animal protein, particu-
larly marine fish.  The cost of both plant and animal protein used in
fish foods, along with the cost of energy to run machines, has been
sharply  rising within the last few years.  Thus any simple aquaculture
system for  rearing trout or  salmon utilizing natural food chains de-
veloped  by  low-cost fertilization, or by feeding low-cost raw materials,
will have immediate practical application in salmonid culture.

                      GENERAL OBJECTIVES OF PROJECT

     The  initial objective of our pilot project was to test by empiri-
cal means the capacity of brackish and  saltwater fertilized with treat-
ed domestic wastewaters to produce food for juvenile salmon.  Our  in-
vestigations have  the ultimate practical objective to ascertain  if this
cultural technique can rear  young salmon to migratory size (smolts) as
effectively and economically as salmon  cultural methods  now employed.

     Once data have been gathered to meet our primary objectives,  the
system lends itself to experiments with a wide range of  local species
of fish  and shellfish, both  singly, or  in various selected combinations
(polyculture).

                      OBJECTIVES OF THIS REPORT

     This paper reports  the  results of  six salmon rearing experiments
completed from July 1971 through December 1973.  Four experiments  in-
volved short-term  rearing of fingerling coho salmon  (0.  kisutch)

                                  763

-------
conducted during summer through late-fall periods.  Two experiments in-
volved J>-k month rearing of chinook salmon fry (0. tshawytscha) during
winter-spring growing periods.  As previously mentioned, for both coho
and chinook salmon experiments, the objective was to produce smolts.
Smolts are juvenile salmon that lose characteristic lateral marks (parr
marks) as freshwater feeding fish, become silvery in color, and physio-
logically become adjusted for salt-water osmoregulation.  Results of
these experiments are reported in terms of survival and growth.  Factors
causing mortalities are discussed, along with evaluation of the results
and future plans.

                          ACKNOWLEDGEMENTS

     Funds for the capital cost were provided by  the Wildlife Conserva-
tion Board, California Department of Fish.  Operating funds have been
provided through the California State gniversity  Coherent Area Sea Grant
Program.  The continued interest and encouragement of both the adminis-
trative and elected officials of the City of Arcata have been a great
source of satisfaction as  it  involves considerable cooperation and good-
will by all parties  involved.  Mr. Al Merritt, in charge of the recir-
culating fish hatchery at  the University campus,  reared most experiment-
al salmon used  in the system.  He also conducted  bioassay  in our hatch-
ery of culture waters used  in pond experiments.   Many undergraduate and
graduate students have conducted studies  in the system and provided much
voluntary help.  Many faculty have directed student projects involving
aspects of the pond system.  To these many people we wish to extend ac-
knowledgement of their aid, and express our sincerest appreciation.

                 DESCRIPTION AND OPERATION OF SYSTEM

PHYSICAL FEATURES

     Fish ponds are located on the west side of the oxidation pond to
allow access to a small creek channel located between the oxidation
pond and a landfill dump  (Figure 1).  Saltwater taken from this ditch
is introduced into the ponds through a 3'-diameter tar-coated pipe fit-
ted on the pond-side with  an Armco cast-iron valve (Headgate, Figure 2).
Wastewater is introduced  into North Pond by a 1'-diameter pipe located
in the northeast corner of North Pond which connects with the oxidation
pond.  There is no pipe connecting the oxidation  pond with South Pond,
but fertilization can be  accomplished by use of a Barnes trash pump ca-
pable of pumping at about  350 gallons per minute.  A fish-collecting
basin located immediately  in front of each headgate is the deepest part
of each pond (about 2.0 meters).  Each basin also contains a small sump
allowing complete dewatering of each pond by use  of the trash pump.
Headgates are fitted with  stainless-steel screens of 1/8-inch diameter
circular openings.

     Three different types of material were used  in construction of the
ponds.  A yellow sandy loam  (Hookton Series) was  used for pond banks.


                                  764

-------
        HUMBOLDT
        BAY ARE.A
                            ARCATA
                             (NORTH)
                              BAY
           SOUTH
            HUMBOLDT
             BAY
         ARCATA
         STUDY
         AREA
SANITARY  LAND FILL


•ARCATA WASTE WATER
TREATMENT  PLANT
         POTENTIAL AREAS
         FOR  MAR| CULTURE.

         OXIDATION  POND
                                             JACOBY
                                              CREEK*
higure 1.  Location of study areas mentioned  in text,
                         765

-------
                                                      SLOPE
                                                       HINS6
                                                       POJNT
                                                      "-32'
                                                    (APPRO*.)
ure 2.   Physical features of 'orth  (top) and South (bottom)  fish ponds.

                              766

-------
River-run gravel was cast around each headgate to allow light machinery
access to the area for excavating of fish-collecting basins.  A third
material, oyster shell, was specifically employed to provide artificial
substrate for production of food organisms.  This material was placed
in several low spots in the pond bottoms and along portions of the pond
banks.  A fourth material (mud) was the original intertidal sediment
plus overlying organic matter deposited during operation of the oxida-
tion pond.  Mud covered a majority of the pond bottoms.  Pens two-meters
square of nylon bobinetting were constructed in each pond over a sample
of each of the four substrates.  These pens (Exclusion Pens, Figure 2)
were originally used to exclude fish from these substrates, but now
serve to rear known numbers of fish over each bottom type.  The ponds
have been operated as static systems to conform to directions from the
North Coast California Regional Water Quality Control Board (no waters
containing unchlorinated effluents may be discharged into Humboldt Bay
without at least two weeks retention time).  In practice, we have rarely
discharged unchlorinated wastewater that has not had about a month re-
tention  in the system.  Ponds could be operated as a tidal-flushing sys-
tem but due to the expense and complexity of having to chlorinate these
flows, we have not as yet operated with tidally-flushed ponds receiving
a continuous addition of wastewaters.

                              B10ASSAYS

     In order to assist in identifying periods of mortality, or to iden-
tify causes of mortality, lots of salmon taken from those planted into
the ponds were reared in aquaria in the hatchery (Hatchery Bioassay) or
in floating pens in the fish ponds (Live-car Bioassay).

HATCHERY BIOASSAY

     Standing-water bioassays were conducted in 20-gallon aquaria kept
at hatchery water  temperatures by placing the aquaria  in hatchery
troughs.  The aquaria were serviced with compressed air introduced
through air stones to maintain oxygen saturation in the water.  There
was no feeding of  lots of salmon being tested in these bioassay experi-
ments.

     Hatchery bioassay was undertaken to measure mortalities due to
toxicity of water  used in salmon culture.  Various combinations of
oxidation pond water, saltwater from Humboldt Bay or from the open
ocean at Trinidad  Head, as well as the mixtures of oxidation pond wa-
ters and Humboldt  Bay water employed in North and South ponds, were
tested.  Although  not designed for the purpose of measuring improve-
ment of water quality from operation of the system, data from such
hatchery bioassay  can be used to estimate how well Arcata plant efflu-
ent meets recently adopted state standards on toxicity (Calif. State
Water Resources Control Board22),  Only a small part of hatchery bio-
assay data are reported here.

LIVE-CAR BIOASSAY

                                  767

-------
     Floating pens (live-cars) were employed to obtain daily counts of
mortality of salmon.  Two types of cages were employed (Table 1).  One
type (cage) was made from 2" x *+" wooden frames fitted with nylon bobf-
netting.  A large cage 4' x V x 2' employed mesh of hexagonal openings
7 mm in longest and 3 mm in shortest diameter.  A small cage 21 x 2' x 2'
employed circular mesh of 1 mm diameter.  A second type of live-car used
was called an "insert" because of  its mode of construction.  A 2' square
wooden frame was fitted with plastic floats and a hinged lid.  An inside
lip was made to hold a frame which was used to support a collapsible
hanging net of mesh 7 mro by 3 rom hexagonal openings.  The  lower port-ion
of the net was kept vertical and square by sewing into the lower peri-
meter a stainless steel square frame.

     Accurate daily mortality counts using cages were not  available.
Due to the size and weight of the  cages it was difficult for a single
man to entirely remove the cage from the pond.  Dead salmon could remain
undetected on the bottom of the cage where a carcass could be completely
consumed by amphipods  in less than  12 hours when high numbers of amphi-
pods were present in the ponds.  The insert live-car could be easily re-
moved from the pond by one man, thus greatly  increasing reliability of
daily mortality measures with this  type of  live-car.

AERATION AND MIXING

Upwe11i ng

     Initially we used a low-cost  system producing "upwelled" water (Fig-
ure 3).  Water was  taken from the  surface by a small electrically-driven
water pump and discharged  through  a simple manifold at the bottom of each
fish-catching basin.  The  intake jet was kept on the surface by  a styro-
foam plastic float.  The systems created an "upwelling"  immediately ad-
jacent  to each headgate.  Whenever aquatic  higher plants occurred  in the
system, constant plugging of  intake and impellers  in the pumps developed.
The pumps were not marine models and deteriorated rapidly  in the waste-
water-saltwater solution.
     A  spray  system was  the  second method  of  aeration  employed  (AIR-0-
 LATOR,  Roycraft  Industries).   The  instruemnt  drives  surface water  into
 the  air by  use of a small  impeller rotating at  very  high  speeds  (Figure
 k).  As originally mounted by  the manufacturer,  the  instrument  did  not
 provide protection for salmon  against  the  impeller blades.  We  mounted
 the  instrument on a styrofoam  block, fitted  into the end  of a 55-gallon
 oil  drum.   The  lower  end of  the  drum was covered with  nylon-mesh to ex-
 clude  fish.   The water enters  the drum near the bottom of the pond,
 moves  vertically, to  be  sprayed  into the air.   These units have been
 located near  the fish collecting basins.   AIR-0-LATOR  parts used by us
 were primarily fashioned for freshwater operations,  thus  galvanic  ac-
 tions  due  to  saltwater caused  complete disintegration  of  some parts of


                                   768

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Table 1.  Description of "live-cars" used in monitoring water
          quality of pond waters for juvenile salmon and for daily
          measure of mortality
 Live-Car     	Descr ipt ion	
                  Frame                                Mesh
 Large Cage   4' x V x 2'      Nylon bobinetting; hexagonal mesh;
                                7 mm long and 3 mm shortest dia-
                                meter.

 Small Cage   2' x 2' x 2'      Nylon bobinetting; circular mesh;
                                1 mm in diameter.

 Insert       21 x 2' with      As  in large  live-car; mesh on re-
              plastic floats    movable frame hung 3' deep with
                                stainless steel frame at bottom
                                to maintain  shape.

 Fry          11" x  I1*" x 20"   Wire mesh of 1/8" square openings.
                                  765

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Figure 3.  Upwelling mixing and aeration unit, South Pond
           headgate.  Pen  (SC) over gravel shown right
           corner; crab pen middle background.
Figure k.  AIR-0-LATOR spray system, South Pond, with
           Pen (SC) over gravel.  Fish barn in back-
           ground to house recirculating system for
           holding and marking salmon.
                       170

-------
the unit.  The electric motor unit (Franklin), however, appears quite
rugged and watertight, and has performed adequately.  Plastic parts
have now become available and are to be tested in the system.

Air

     North Pond was fitted with a forced-air aeration system during the
summer of 1973 (Figure 5).  Air lines with associated jets are located
immediately off the portion of the North Pond bank not associated with
an oyster shell covering.  A vertical 2-foot wall exists in this portion
of the pond, and the air jets breaking the water surface plus the verti-
cal bank serve to discourage predation by fish-eating birds (herons,
egrets, night herons).  A second pipe leads directly to the fish catch-
ing basin to a U-shaped manifold insuring complete mixing of all pond
waters.

                       POND WATER PROPERTIES

     Daily observations of water temperature, turbidity, hydrogen  ion
concentration, dissolved oxygen, and salinity were made from surface
samples obtained at the headgate of each pond during morning hours  (8-
10 AM).  Additional water properties were studied only  intermittently.

     Considerable day-to-day fluctuations in some of the properties oc-
curred (pH, turbidity, oxygen) (Allen23).  |t was possible to fit curves
to the raw data by eye showing major trends during each experiment
(Figures 6-9). Time of each experiment  is  listed  in  Table 2.

     Water temperature was primarily controlled by air temperature.
Local weather conditions (storms in winter, cloud or fog cover in sum-
mer) cause yearly variations.  During summer months, the ponds ranged
near 20 C (Figure 6).  Such temperatures are found  in estuaries and  la-
goons  in northern California and Oregon at this time of the year (jo-
seph2**, Hayes 5, Reimers2").  Ice covered our ponds  once during the two
winters of operation to date and then only for a day.  Diurnal changes
in temperature, as determined from cursory analysis  of thermograph
charts obtained from an  instrument operated about a  foot under the wa-
ter surface, are relatively small (2-3 degrees at most).

     Turbidity, primarily a function of phytoplankton populations,
showed high variability during coho rearing periods  (fall and early win-
ter) (Experiments I,  III, IV, VI) (Figure 7).  High  turbidity from phy-
toplankton occurred during both chinook rearing periods (winter-spring)
(Experiments  II, V).

     Hydrogen  ion, primarily a function of photosynthesis, was always
quite  high, generally between values of 8 to 9.5, with a few cases of
values near 10 (Figure 8).  No similarity in trends  could be discerned
during winter periods (Experiments I, III, IV, VI) although the average
value was about pH-8.  In contrast, the winter-spring conditions pro-
duced  pH's tending toward values in late spring of 9-9-5.

                                  777

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• I
' .
                                                                                  <
               Figure 5.  North Pond during pond modification, summer  1973t showing

                         retaining wall and air line at base.

-------
o
LU
or
Of.
Id
a
I
UJ
     15-
10
          j
                              M     J     J     A

                                    MONTH
N
         Figure 6.  Trends  in  pond  water  temperatures,  North Pond, salmon rearing

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UJ
o
a.
a.
U.
o
 Q.

 Ld
no



IOO



 90



 80



 70
  50



  40



  30



  20



   10
LL



 I
           JFMAMJJASONDJ


                                        MONTH


          Figure 7.  Trends in water clarity, North  Pond, salmon rearing experiments

                    I-VI.

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   10
    a
X
 d.
                      M
M    J     J    A

        MONTH
N
D
       Figure 8.  Trends  in hydrogen  ion concentrations, North Pond, salmon rearing
                  experiments  I-VI.

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     Dissolved oxygen was always high, and only on a few occasions fell
below 5 mg/1 (Figure 9).  In those instances, salmon mortalities could
be correlated with these low values,  especially during periods when wa-
ter temperatures were high.   No pattern was discerned in oxygen trends
during the fall-winter experiments (Experiment I, Ml, IV), except for
Experiment VI which under Air aeration maintained a constantly high oxy-
gen level.  Winter-spring patterns were somewhat similar with high ini-
tial levels followed by a slowly dropping trend, and a sharp drop at the
latter portions of the experiment (Experiment I I and V).  These low
points are associated with periods of intense overcast from local heavy
fogs that have followed a period of bright sunshine.

     Salinities have been the most stable property, with any sharp chan-
ges occurring toward lower salinity values as associated with periods
of heavy rainfall.  Adjustments in pond salinities were made to experi-
mental values by small discharges of pond water to the bay and addition
of higher-salinity water in return.  During spring periods, salinities
have slowly tended to increase, probably due to evaporation.  Salinity
ranges have not been graphed but are listed with tabular  data as needed.

           DESCRIPTION OF EXPERIMENTS AND SALMON SURVIVAL

     Salmon rearing has been grouped  into six periods (experiments)(Ta-
ble 2).  Eleven lots of salmon were planted as part of these experiments.
To date, coho salmon have been planted only as fingerlings (juvenile
salmon 6-9 months old).  Coho  rearing has been conducted during the sum-
mer (Experiment IA; Experiment III), or during late fall,  (Experiment
IVA; Experiment IB, IVB, and VI).  Chinook salmon experiments employed
fry (young salmon that have absorbed their yolk-sac and have been active-
ly feeding from two-four weeks).  Chinook rearing has taken place only
in winter (Experiment II and V).

     Salmon utilized in our experiments were obtained from the Califor-
nia Department of Fish and Game hatcheries located on Mad River, Hum-
boldt County, California, and on the upper Klamath River (Iron Gate
fish hatchery).  Salmon planted into the ponds were mainly reared at
the Humboldt State University  recirculating fish hatchery although some
were reared at the Mad River hatchery or a Humboldt County hatchery lo-
cated on Prairie Creek.

EXPERIMENT  IA

     Water was initially introduced into North Pond on July 21,  1971
and it was allowed to mix until July 23 when initial plants of coho sal-
mon were attempted.  From July 23-28, 5,000 fingerling coho salmon were
introduced  into each pond.  Water quality in North Pond was poor and
salmon with darkened coloration and listless behavior were observed on
the surface after about three days residence.   In South Pond, coho plant-
ed had been acclimatized for U8 hours at 15 o/oo salinity at 16-17°C
temperature, but this was unsuccessful as acute mortalities occurred al-
most  immediately  in salinities of 32-32.5 o/oo  (parts per thousand).

                                   776

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    15--
HI
x
O

a
uJ
~
O
t/1
01

5
     10
                      n
A    M
J     J     A


  nONTH
N
        Figure 9.  Trends  in  oxygen  concentration,  N'orth Fond, salmon rearing

                   experiments  I-VI.

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Table 2.  Summary of rearing experiments in North Pond with juvenile Pacific salmon using Humboldt
          Bay seawater mixed with wastewater from City of Arcata oxidation pond.

Expert- Rearing
ment No. Period
IA Jul -
IB 7 Dec
II 1 1 Feb
17 Apr
III 7 Aug
IVA Oct 72
IVB 3 Nov
V 26 Jan
16 Mar
26 Apr
VI 2 Nov
Oct 71
71 - 22 Jan 72
- 2 Jun 72
- 2 Jun 72
- 22 Sept 72

- 17 Nov 72
- 26 May 72
- 26 May 73
- 26 May 73
- 15/16 Dec 73
Species
Coho
Coho
Chinook
Ch inook
Coho
Coho
Coho
Ch inook
Chinook
Chinook
Coho
Size of
Fish
Fingerl ing
Fingerl ing
Fry
Fry
Fingerl ing
Fingerl ing
Fingerl ing
Fry
Fry
Fry
Fingerl ing
Days of
Rearing
-
45
111
45
45
-
14
119
70
52
43
Aeration and Mixing
System
Upwe 1 1 i ng
Upwel 1 ing
Upwel 1 ing and Spray
Upwel 1 ing and Spray
Spray
Spray
Spray
Spray
Spray
Spray
Ai r—
Sal inity
(parts per
thousand)
19-24
13-16
11-12
11-12
16-23
13-16
15-17
10-1 3"^
10-13-L7
10- 13!'
10-18
 ]/  South Pond;  18-20 and spray aeration.
 2/  South Pond;  Spray.

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 The number of salmon recovered from around the edges of the ponds was
 about seven times as great from South Pond as was recovered from North
 Pond.  North pond fish tended to sink to the bottom, while South Pond
 fish skittered on the surface and tended to float.  Feeding salmon were
 observed on South Pond surface but rarely on North pond.  Some coho sal-
 mon survived in South Pond until early September.  Additional  small num-
 bers of coho salmon were introduced into North Pond during August but
 these all  succumbed as indicated by total mortalities recorded in live-
 car bioassay.   Bioassay in live-car continued to show high mortalities
 through late September.

      During July-September 1971, salinities in North Pond ranged be-
 tween 19-24 o/oo.  Water temperatures ranged around 20°C.  Oxygen lev-
 els showed typical  wide diurnal  variations associated with highly eu-
 troplc conditions.   Salmon mortalities in live-car bioassay were corre-
 lated with dissolved oxygen level  of from 2-3 mg/1 .

 EXPERIMENT IB

      Beginning in mid-October,  1971,  coho salmon planted into  live-cars
 in  North Pond  showed about 70 percent survival  over a two-week period.
 During November,  survival  increased to about 80 percent  of the salmon
 retained in live-cars over a  month's  time.   Based on these results,
 17,000 coho salmon  averaging  10.5  grams  per fish, were introduced into
 North Pond,  between October 18 and 7  December 1973 (15,000 were planted
 7 December 1973).   On January 22,  1972,  9.500 salmon were recovered and
 released into  Humboldt  Bay.   The average weight of the surviving  salmon
 was 15.0 grams.   Survival  was 55 percent over the 45-day rearing  period
 for the  bulk of  this fish  planted  (7  December 1973).

 EXPERIMENT I I

      North Pond  remained empty from 22 January  until  February  9,  1972,
 when  effluent  and saltwater were mixed into the pond.   Some additional
 water  was  added on  February 10 and 11.   Salinities on  11  February varied
 from  11  o/oo at the  surface to 15.5 o/oo at 3-foot depth.   The  pond was
 initially  aerated by upwelling,  then  by  spray.   The  AIR-0-LATOR unit
 failed during  the experiment  and was  replaced by the upwelling  pump  re-
 fitted for aerial spray.   In  late  April  the AIR-0-LATOR was replaced
 with  a new unit,  and the pump refitted for  upwelling.  On  2 June  the
 pond was drained.

      Between February  11-14,  1972,  10,000 chinook fry were  planted  into
 North  Pond,  followed by 2,500 fry  marked  by excision of the  left  ventral
 fin  (LV).   On  2 June, 2.5 percent  and  4.0 percent of these  two  groups
 of  salmon  were recovered as smolts.

 EXPERIMENT  I I I

     On June 28,  1972 a mixture  of  saltwater and  oxidation  pond water
was  introduced into  North Pond to  form a  salinity of 16 o/oo.   Live-car

                                  779

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bioassay showed high mortalities of coho salmon through July but with
slowly improving survival rates throughout the month.  During live-car
rearing periods temperatures fluctuated around 20° + 2°c.  From July
28 to August 11, three introductions of finger ling cbho of about 1,000
fish each were made into water of about 18 o/oo salinity.  On 22 Sep-
tember the pond was drained and 3.9 percent of the 3,000 salmon recover-
ed.  In addition over 50,000 fingerling topsmelt and sticklebacks were
recovered.  These latter fish had developed entirely from eggs and lar-
vae  in the saltwater  introduced from Humboldt Bay at the initial filling
of the ponds.

EXPERIMENT  IVA

     North Pond was filled with seawater and effluent  in late Septem-
ber, 1972 and adjusted to produce a salinity of  15 o/oo.  Live-car bi-
oassay was begun on ^ October, with no mortality over  a  10-day period.
In order  to  repeat Experiment  I with a longer period of  rearing, 16,000
coho fingerlings were placed  into the pond on October  13.  A group of
50 salmon was placed  into the  live-car.  On October  13,  19 salmon died
in the  live-car  (38 percent), which correlated with  a  period of  low oxy-
gen  level  (£3 mg/1). On November 3, only  12 percent  of the live-car
fish remained.   High  numbers  of  distressed  fish  were being eaten by
birds  (Blankinship*7).   We  assumed that most  fish  in this experiment
had  died.

EXPERIMENT  IVB

     As  part of  the objective of  Experiment  IVA  was  to allow a  study  of
bird predation,  we  had  retained  5,000  fingerlings  in our hatchery as
 reserve,  and these were  placed  into  the pond  on  November 3.  On  17  No-
vember  the  pond  was drained and  4,220  fingerlings  were recovered (84
percent  survival  over 14 days of rearing).   No mortalities  occurred  in
 live-car bioassay during the  rearing period.

 EXPERIMENT  V

      Both North and South Ponds  were  filled with seawater during Decem-
 ber  1972 but had to be  emptied in early  January  1973 for some  pond  re-
 pair and maintenance  work.   Oxidation  pond effluent was introduced  in
mid-January to obtain two levels of  salinity (about 9  o/oo for North
 pond and 18 o/oo in South Pond).   Two  stocks of  chinook salmon were
 made available for this experiment.   One  stock was from early-run sal-
 mon returning to Mad  River  hatchery,  and  the second stock later-running
 salmon to Iron Gate fish hatchery on the  Klamath River.  A group of late-
 run salmon was retained in  the Humboldt State University hatchery for
 planting in late April   in order to allow the salmon to become large
 enough to be marked by fin excision (left ventral  fin  - LV).  South Pond
 was drained on 25 May and North Pond on 26 May 1973.  Surv.val of fish
 over the three rearing periods ranged from 0.1  to 6 percent (Table 3).
                                   750

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Table 3.  Survival of chinook salmon fry planted into North and South Ponds,  Experiment V,
North Pond
Stock
Mark
Date Salmon Planted
Number Planted
Days Reared
Percent Survival
Early Run
None
26 Jan
1 ,800
119
6
Late Run
None LV
16 Mar 26 Apr
7,500 2,500
70 30
1 1
South Pond
Early Run
None
26 Jan
1,800
no
2
Late
None
16 Mar
7,500
70
0.1
Run
LV
26 Apr
2,500
30
0.2

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EXPERIMENT VI
     No studies were conducted during the summer of 1973 due to major
modifications and repairs undertaken for the system.  A 2-foot vertical
wall of redwood planks was constructed along all banks of North Pond ex-
cept in those areas where the bank was not protected by an oyster shell
layer (Figure 5).  A forced-air aeration system was installed during
this period as previously described.  Water from Humbojdt Bay was intro-
duced into both ponds during late August.  After draining North Pond
several times to adjust the aeration system, water from the oxidation
pond was added to both ponds to develop a salinity of about 25 o/oo.
This was to test Power's^0 hypothesis that amphipod growth and survival
would be maximized at this salinity level.  In early October, salinities
were reduced to about 15 o/oo.  On October k, live-car bioassays were
started, with no mortalities recorded through October.  On 2 November
1973, 5,000 coho fingerlings were introduced into both ponds.  After k$
days rearing, over 80% of the fingerlings were recovered for release in-
to Humboldt Bay.  Very few of the salmon had become smolts.  Because
there were no residual stocks of amphipods left within the pond follow-
ing summer construction, nor were there amphipods occurring naturally
in Humboldt Bay during introduction of saltwater, very few of these
forms were available as food for the salmon.  Consequently, larger sal-
mon actually showed a slight reduction in weight although smaller fish
retained or even slightly  improved their condition during the rearing
period.
                         SALMON PRODUCTION

POND REARED FISH
     The production of salmon from the system varied widely (Table 4).
For coho fingerling planted  in late summer and fall periods (Experiment
IB, IVB, VI) the trend has been from moderate to high survivals, coupled
with an inverse trend in actual fish production  (high to low).  The
single experiment with coho  fingerlings  reared during a summer period
(Experiment  III) showed a  low survival and low production of salmon but
a high survival and high production of other species of fish.  Both sur-
vival and production of chinook salmon from fry plantings were low, al-
though the chinook grew fast and smolted  (Experiment  II and V).

PEN AND LIVE-CAR REARED FISH
     The survival and growth of salmon reared  in pens and  live-cars was
often markedly different, usually greater, than salmon  living unre-
stricted  in the ponds, indicating a potential growth and survival great-
er than actually found for the ponds to date.

Pen

     Pens constructed over pond substrates were designed to exclude
fish over these substrates.  During Experiment  IB, on October 20, 1971,
in error 50 coho salmon were placed  into  Exclusion Pen NC  (gravel sub-
strate, Figure 2).  Electric shocking, and seining, recovered 35 salmon.
When the pond was drained on 22 January  1972,  17 fish were recovered.
                                  182

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So
Uj
    Table k.
       IB
  Summary of production—  in fish-rearing experiments,  June 1971  - December 1973,  in
  pilot-fish pond system using saltwater fertilized with advanced secondary treated
  effluent from City  of Arcata oxidation pond.
Exp. pond
No.


Species,
Size of Fish


Days
Reared


Percent
Survival


product ion—
as kilograms
per hectare
per day
Remarks on
experimental



result


       IA    North    Coho fingerling
            South    Coho fingerling
                                             0
North    Coho fingerling
      I I      North    Chinook fry
     III      North     Coho  fingerling
55


 3

 k
             Unstable water and bottom
             conditions; temperatures
             consistently over 20 C

             Salinities over 32 o/oo
             and high temperatures;
             high density of topsmelt
             and stickleback

    6.8      High density of planting
             induced intensive preda-
             tion by bi rds

0.05-0.07    Mortality possibly due to
             over-rearing.

    0.4      Nature of mortality unde-
             termined.   Food primarily
             consumed by other species

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Table *».  (Continued)
 Exp.   Pond
 No.
Species
Size of Fish
Days      Percent
Reared    Survival
Production—
as kilograms
per hectare
per day
      Remarks on
      experimental result
        North   Topsmelt, stickle-
                back, sculpins
  IVA    North   Coho finger ling
  IVB    North   Coho fingerling
  V     North   Chinook fry
        South   Chinook fry
 VI     North   Coho fingerling
        South   Coho fingerling
                     86
                   30-119

                   30-119
            high
             Bk



            1-6


           0,1-2

             96



             78
                                             3,7
  trace
  trace
                                           loss
                                           loss
Production entirely from sur-
vival of eggs and larvae in-
troduced with saltwater from
Humboldt Bay
Mortalities correlated with
period of low dissolved
oxygen
Experiment primarily to
assist in bird predation
study

Mortality possibly from
over-rearing

Mortality possibly from
over-rear!ng
Sufficient amphipod popula-
tion did not develop in
system
 I/  Production defined as the change in weight of those fish surviving from planting into ponds
 ~   until  recovered during pond draining.

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Table 5.  Comparison of survival of chinook fry planted into ponds
          and reared in exclusion pens (Experiment V).




Number planted
Number Recovered
Percentage
Surface Area
(Square meters)
North pond
Exclusion Pond
Pen Over
Gravel
77 7,500
40 85
51 1

4 1,500
South Pond
Exclusion Pond
Pen Over
Gravel
77 7,500
Ik 12
18 0.1

4 1,500
 Table 6.  Comparison of survival of coho fingerling salmon planted
           into ponds and reared in exclusion pens (Experiment VI).
North Pond
Exclusion Pond
Pens
Number planted!7 56 5,000
Number Recovered 46- 4,800
Percentage 82 9«
South
Exclusion
Pens
56
56
100
Pond
Pond

5,000
3,900
78
  M  7  unmarked  and  7  LV marked  coho  fingerlings  planted  into each
  ~~   of four  pens.

  2/  8  of  10  mortalities from pen  NB  (Hookton Soil).
                                 185

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This represented virtually no mortality during a 92-day rearing period.
At the same time,  live-car reared fish showed 20-30 percent mortalities,
compared to a 45 percent mortality suffered by the salmon released into
the pond.

     Based on this experience, we used the pens to study the effect of
having different densities of salmon feeding over the substrates in Ex-
periment V.  Our examination of the pen bottoms failed to disclose small
escape route for small fish under the corners of the pen frames except
for the pens over gravel.  Consequently salmon escaped through these
holes during pond draining, and an absolute count of salmon surviving
was made from the pens over gravel in each pond (Table 5).  In both
ponds, survival of Chinook fry in the pens was strikingly higher than
the generally low survival of the same lot of fish planted into the
ponds.

     During the summer of 1973, each pen bottom was made secure.  At
this time pen NB was  relocated over fresh Hookton soil.  We then planted
each pen at the same  density of salmon per unit area as we did in the
main ponds (Experiment VI).  All salmon survived in South pond pens,
while mortalities  in  North Pond pens were mainly associated with fish
grown over raw Hookton Soil (Table 6).  High survival in general made
it impossible to draw any conclusions on  substrate effect.  The new
Hookton soil probably did not provide sufficient food for the salmon.

Live-Car

     For coho fingerling salmon, the confinement of salmon in pens sus-
pended away from the  pond bottom resulted in less growth than salmon
reared in cages located on pond substrates.   In Experiment VI, both LV
and unmarked lots of  salmon were consistently smaller when grown in
cages than when reared in pens over bottom substrates (Table 7).  This
effect was first noted in Experiment M when the average size of the sal-
mon in North pond was 12.4 grams in pen located over gravel in compari-
son to 15.0 grams for the salmon reared rn the pond.  |t was not possible
to discern whether one substrate was more beneficial over another in
Experiment VI  because individual fish were not measured for weight or
length before being placed into pens.

     fn the single experiment with chinook fry lots reared in cages and
pens (Experiment V),  the rate of growth was slower in cages than in the
pond but the actual production of wet weight of salmon was higher in the
cages (Table 8).  These production rates were in the range exhibited by
coho fingerling in experiments IB and IVB (Table 4).

                         CAUSES OF MORTALITY

OXYGEN DEPLETION

     The use of oxidation pond effluent to fertilize seawater produces

                                  786

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Table 7.  Comparison of average weight in grams of small and large-sized coho fingerling salmon
          reared in cages and pens (Experiment VI).
Pond Rearing Site
North Insert and
Culture Raft
Insert Live-Car
Shel 1 Pen
Hookton Soi 1 Pen
Gravel Pen
Mud Pen
South Insert and
Culture Raft
Insert Live-Car
Shel 1 Pen
Hookton Soi 1 Pen
Gravel Pen
Mud Pen
Weight by group of fingerling
Per
k Oct
2 Nov
2 Nov
2 Nov
2 Nov
2 Nov
k Oct
2 Nov
2 Nov
2 Nov
2 Nov
2 Nov
iod
- 15 Dec 73-7
- 15 Dec 73
- 15 Dec 73
- 15 Dec 73
- 15 Dec 73
- 15 Dec 73
- 16 Dec 73-
- 16 Dec lk
- 16 Dec lk
- 16 Dec Ik
- 16 Dec 7*4
- 16 Dec lk
Days
73
kk
kk
kk
kk
kk
lk
kS
k5
k$
kS
kS
Small
(LV)
6.3 (2k)-
5.3 (23)
7-6 (5)
9.0 (5)
8.0 (7)
7.5 (7)
k.2 (2k)
k.8 (30)
8.0 (7)
6.8 (7)
9-5 (7)
8.5 (7)
Large
(Unmarked)
_
17.6 (26)
27.1 (7)
1.0 (1)
18.0 (7)
18.6 (7)

13.8 (30)
17.2 (7)
15.9 (7)
19.1 (7)
19.6 (7)

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     Table 7.   (Continued)
do
00
Pond Rearing Site
Period Days
South Crab Rearing Pervi' 2 Nov - 16 Dec 7** **5
Weight
Sma 1 1
(LV)
6.6 (21
by group of fingerling
Large
(Unmarked)
1 18.0 (2)
I/  Small salmon without a mark were placed in Insert live-car on k October, reared until
~   2 November, then moved to hanging pen in Culture Raft.

2/  Number of salmon recovered at end of rearing period shown in parenthesis.  Twenty-five
""   salmon were placed in Insert live-car.  Seven salmon each of small and large-sizes were
    placed into pens.

3/  Salmon entered pen on their own and were recovered on draining pond.

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Table 8.  Comparison of rates of production of wet weight of salmon
          of unmarked chinook fry reared in pens and cages, North
          Pond, Experiment V.
                                   Stock of chinook salmon
                          ETarTy' RunLate Run
                          Large       Pen on     Large     Small
                          Live-Car    Gravel     Live-     Live-
                                      Bottom     Car       Car
A.  Experimental Factors

    Surface area of rear-
    ing site  (square
    meters)                  0.4       4.0        0.4       0.1

    Planting  density num-
    ber of fish per
    square meter)             65         20        250       250

    Length,of  rearing  (days)  49         70          70        70

B.  Percent survival         100         49          51        40

C.  production  rates (kilograms per  hectare  per day)

    I.  Based  on gain  in
        weight  of  surviving
        salmon                 11       3.4          10        10

    I|.  Based  on net gain
        in weight  of salmon  8.5       2.8        4.0       1.4
                                   759

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 a highly fertile solution.   Consequently,  the high oxygen demand  of the
 system can produce low oxygen values in a  very short time period  when
 cloud cover is heavy or aeration systems are inoperative.   Periods  of
 low oxygen level during mid-October 1972 (Figure 9)  caused heavy  mor-
 talities during Experiment  IV.   Other than this instance,  and  the ini-
 tial  unstable conditions during Experiment (A,  aeration  units  have  been
 able  to avoid stratification in the ponds  and to maintain  oxygen  levels
 sufficient for juvenile salmon.

 HEAVY METALS

      Data on levels  of metals (acid-soluble Cu, Cr,  Fe,  Na,  Ni  and  Zn)
 in the pond bottom were available from a study by GrossZS,   of  fifteen
 locations investigated in the local  marine areas,  ten were either in or
 near  the North and South fish ponds.   Two  sampling sites were  located
 in the fish ponds, one each  In  North and South ponds.  The sediments of
 the oxidation pond,  and the  fish ponds,  contained unnaturally  higher
 amounts of Zinc than all  other  locations tests (5 samples  ranged  from
 85 to 360 ppm, one sample 25 ppm as  opposed to all  other areas  which
 ranged from only 2-43 ppm).

      There has been  no study yet to ascertain if heavy metals  in  the
 pond  sediments,  which undoubtedly represent an area  of accumulation due
 to the operation of  the oxidation pond,  are entering the food chain of
 the salmon.   A major source  of  food  consumed by the  salmon  has  been two
 species of amphipod  (Corophium  sp.,  and  Anisogammarus confervicolus)
 but these have not been analyzed for  heavy metal  contendThe  higher
 survival  of  salmon reared in live-cars and exclusion pens,  suggest  this
 possibi1ity,

 BIRD  PREDATION

     Although methods  of  controlling  loss  of fish  to birds at rearing
 facilities has been  reported extensively in  the literature  (Blankin-
 sn'P27),  little  data  are  available on the  numbers  of fish  actually
 eaten.  Thus  the pilot  project  at Humboldt Bay  has provided an  opportu-
 nity  to study bird predation on  unprotected  ponds.   These data will al-
 low for comparison of  rate of predation  on modified  ponds, and eventu-
 ally provide  data  on  the  design  of production facilities.

      Bird  predation as  estimated by studies  undertaken by  Blankinship
 on  unmodified  ponds showed a variation of  from  2-24  percent for coho
 fingerling (Table  9).   The maximum value was associated with an experi-
 ment  in which  fish were under stress, whereas the minimum value was
 from an experiment in which  the  density  of salmon planted was low and
 their  survival poor.    For Experiment VI modifications to North  Pond in
 the summer of  1973 were to deter predation by species of birds  that feed
 along  the  shore, however, very few birds visited either  the modified
 (North) or unmodified  (South) pond, so that  effectiveness of the pond
modifications  could not be assessed from this study.

                                  790

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Table 9.  Estimated percentage of salmon planted  into North pond
          consumed by birds  (from Blankinship  1973).
 Experiment          Species and Size          Percentage of
   Number               of Salmon              Salmon Consumed
     IB               Coho finger ling                  13~~

    I|                Chinook  fry                      ^

   I||                Coho fingerling                  2

                     (Stickleback,  topsmelt)          (8)

    IVA               Coho f ingerl ing                  2*»-

    IVB               Coho fingerling                  12

     V                Chinook  fry                   no study

    V|                Coho fingerling                trace


 I/  Minimum estimate from  limited hours of observation  near  end of
 "~   experiment.

 2/  Maximum estimate as  fish  were under stress during parts  of  the
     experiment  causing  higher predator success than occurs on
     unstressed  salmon.
                                  797

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     A low predation rate was recorded in experiments with chinook fry.
These experiments (II, V) had low survival thus it was not possible to
know if bird predation was due to unavailability of chinook to birds or
due to differences  in behavior of chinook fry as compared with coho
fingerlings.

OVER-REARING

     There was evidence that we over-reared chinook salmon.  During Ex-
periment  I), 1/2-meter ring nets were fished at night  in North pond to
obtain salmon for study of their food habits.  From April  18 through May
2k, the average catch per haul was from 2.8 to 5.5 salmon  (k-6 hauls per
sampling).  On May  31, 7 hauls only produced a mean catch  of 1.0 salmon.
During Experiment IV, studies on fishing  ability of 2-cubic feet wire
mesh traps with a single funnel entrance  caught 28 salmon  when fished
continuously for 2  days  in early April.   From  17 May until 25 May, the
same traps caught no  salmon during this period  (8 days).

     The  exact  reason for the sudden  loss of salmon  in the ponds during
late May  periods  is not  known but may be  correlated with physical  and
chemical  parameters of the water  in addition physiological changes asso-
ciated with  the migratory habits  of the species  (Reimers26).  Hydrogen
ion concentrations  (pH)  tended  to  fluctuate  between 7  and  8 during the
beginning of  these  experiments, then  rose to stabilize around 9.5  units.
Along with this trend, with  the general  increase  in average daily  temper-
ature and increased sunshine water temperatures approached 19°C  in both
Experiments  II  and  V.  Smolts  recovered  in experiments II  and V were
much  larger  than  the  average  size  reported for smolts  of chinook salmon
 in the Mad River  (70  mm  fk.  len., Taniguchi2?).   Chinook salmon  smolting
may  require  a  different  or more abundant  food  source  than  the ponds were
providing.

DISEASE

      Infectious diseases appeared to  be a minor factor as  a cause  of
mortality.   This  opinion was based on the fact of relatively  high  sur-
vival  of salmon reared  in floating  cages  and exclusion pens during ex-
periments when the  same  stock of  salmon  reared in the ponds showed low
survival.  Richness of  the  diet  produced  in  the ponds for  salmon may  be
a  possible cause  of mortality (nutritional  disease).

 FOOD

      Gammarid amphipods  have been the major food item in the  diet  of  our
 pond-reared salmon (Powers30).   Tube-dwelling amphipods (Corphium) un-
 doubtedly will become a  major component  in  the pond fauna when  some form
 of tidal or flow-through operation occurs.   Although productivity  of  the
 ponds in terms of salmon biomass appears to be declining  (Table 4),  re-
 sults from Experiment VI should be discounted.  Lack of amphipods  during
 Experiment VI  resulted from complete drying of all  areas of the pond
 during a long summer of repair and construction,  the elimination of

                                   792

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holding areas for amphipods, by replacing the wood construction of fish
collecting basin with concrete construction, and finally from a virtual
absence of amphipods  in Humboldt Bay  in September when the ponds were
filled for Experiment VI.  Undoubtedly the successful management of our
wastewater aquaculture system will  require as much understanding of the
biology of the amphipods as that of the salmon.

                            CONCLUSIONS

     Coho fingerling  and chinook fry  can  be  successfully reared  in ad-
vanced secondarily  treated domestic wastewaters mixed with saltwater.
The best results  in our studies were  in mixtures producing 10-15 o/oo
salinity (roughly two parts effluent  to one  part saltwater).

     Experiments with coho fingerling reared in  late-fall to winter
have not carried  these salmon  to complete smolting  of all fish  because
of our need  to  have the ponds  ready for chinook  salmon  experiments,  our
primary  target  species.  Results of these rearing  experiments  suggest
the management  possibility  of  enhancing estuarine  survival through such
an  intermediate period of  brackish-water  rearing.   With completion of  a
recirculating aquarium system  next to our ponds  for fish holding and
marking, we  will  begin to  test this hypothesis by  marking all  coho sal-
mon  reared,  and by  holding  them for release under  the most favorable con-
ditions  possible.

     The single experiment  with coho fingerling  in the  summer  indicated
that  our fish had not grown to sufficiently large  size  prior to planting
to harvest  the  marine fishes which had grown rapidly from eggs and  lar-
vae  introduced  from Humboldt Bay.   Hayes25 noted that  steelhead in  Big
Lagoon were  mainly yearling fish that had moved into the lagoon during
spring months.   Future summer  rearing will use larger fish  (yearling
 rainbow, steelhead, or coastal cutthroat trout).

      Chinook fry have grown rapidly and smolted, although the overall
 survival achieved would not make the system as a feasible alternative
 to existing culture methods.  The  higher growth and survival of ch.nook
 reared in pens and live-cars than  fish reared in the open ponds, indi-
 cate that the full productive capacity of the system has not yet been
 attained.   Recent  laboratory studies on bluegill sunfish have  indicated
 the feasibility of increasing the  ratio of  surface area to water volume
 for development of more fish food  organisms (Pardue^ ).  The effect has
 been noted during  studies of artificial  reefs in marine waters of Hum-
 boldt Bay (DeGeorges32j  prince33)  Lambert^).  Any such underwater
 structures may also  have utility  in  decreasing  interspecific competition
 that we can expect in the ponds (Payne35} Reimers  ).

      The saltwater-wastewater mixture may have some therapeutic effect
 as gross observations of our mortalities  have not  indicated any  ,nfec-
 tioCs disease.  Vibrio can be especially serious  in west-coast  man-
 culture (Fryer20)^

                                    793

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      Reliable  aeratfon  systems  are  essential  in wastewater mariculture
 because  of  the rapidity of  oxygen depletion which  can  occur under  cer-
 tain  environmental  conditions  (heavy  fog  or cloud  cover  in particular).
 Forced-air  aeration appears most favorable, and probably will  improve
 pond  conditions by  stripping ammonia  produced  by salmon metabolism.

                            FUTURE  PLANS

      With recent  passage in California  of initiative  legislation set-
 ting  up  stricter  controls on coastline  development, the area available
 for mariculture could be sharply curtailed, especially as we are lack-
 ing comparative data on economic returns  with  other possible types of
 land  uses.   Future  expansion of mariculture within the confines of the
 oxidation pond,  or  into the remaining portion  of a now abandoned land-
 fill  refuse site  immediately to the west  of the Arcata oxidation pond,
 could take  place  with land-use  conflicts.  A master plan for the oxi-
 dation pond area  has been submitted to  the City of Arcata which in-
 cludes expanded mariculture facilities  (Figure 1), developing  a simi-
 lar plan for the  land-fill  refuse site.

      Wastewater stabilization  lagoons,  fertilized  fish ponds,  and eu-
 trophic  natural waters  all  contain  high numbers of algal cells.  Where
 lagoons  and ponds are discharged into receiving waters, water  quality
 standards on BOD  and particulate matter may not be attainable without
 removal  of  algal  cells.  We are undertaking studies on mussels as a
 possible method of  biological scrubbing to remove algal cells  and
 pathogens.   Successful  bivalve  scrubbing  could reduce  the high cost of
 chlorination required to meet the California State Water Resources Con-
 trol  Board22 standards  for  a receiving  water where shellfish harvest
 for human consumption is likely (median total  coliform concentration
 shall  not exceed  70 per 100 ml, and not more than  10 percent of the
 samples  shall  exceed 230 per 100 ml).   Recently, a standard of 0.2 ppm
 residual chlorine has also  been applied,  thus  increasing the costs of
 treatment by standard physical-chemical means  considerably.

     We  have become  interested  in the possibility that reducing the
 amount of treated wastewaters into  Humboldt Bay might  actually reduce
 the biological productivity of  this estuarine  system.  Estuaries in
 northern California  are  subjected to hugh freshwater inflows during
 the winter  periods  of heavy rainfall.   They are, therefore, ecological-
 ly adjusted  to  intermittent periods of  reduced salinity.  Humboldt Bay
 has an extremely  high flushing  volume (Skeesick^°).  Near-shore water
of the Pacific Ocean are generally  high in nutrients,  especially during
 periods  of  off-shore upwelling  (Allen*?)_  Thus the addition of well-
 treated wastewaters  into the system would have beneficial effects.
 Fortunately, the possibility of reducing a beneficial  effect of a well-
 treated wastewater on a  receiving body  of water has been recognized by
 the California State Water  Quality  Control Board^°).

      In  summary, our future program in wastewater maricultre falls

                                 794

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into four broad categories.  First, we will continue empirical studies
to obtain the maximum production of salmon smolts to allow a feasibility
study of a production system.  Second, we plan to obtain permission for
expansion of mariculture ponds into areas adjacent to our present loca-
tion.  Third, we have formed an  interdisciplinary team of specialists
on the university staff to undertake  investigations within their area
of competence as an effort in applied ecosystem analysis.  Finally, we
will expand our preliminary studies on biological methods for removing
algae and particulate matter in our effluents through polyculture.

                          LITERATURE CITED

1.  Allen, G. H.  1969.  A preliminary bibliography on the utilization
         of sewage in fish culture.  FAO, Fish. Cir. No. 308, 23 pp.

2.  	.  1970.  The constructive use of sewage, with particular
         reference to fish culture.  FAO Tech. Conf. on Marine Pollution
         and its Effects on Living Resources and Fishing.  Rome 9/18
         Dec. 1970.  FIR:  MP/70/R-13.   (_m Marine Pollution and Sea
         Life.  Survey of Problems and Treatments, Fishing News (Books)
         Ltd, Surrey, England, 1973).

3.  Mortimer, C. H. and C. F. Hickling.  195^.  Fertilizers  in fish
         ponds.  London, Her Majesty's Stationery Office, Colonial
         Office, Fish. Publ.:  No. 5,  155 pp.

4.  Law, J. P.  1968.  Agriculture utilization of sewage effluent and
         sludge.  Project Report.  Water Poll. Control Admin., EPA,
         Water Quality Office, Washington, D. C.  89 pp.

5.  Wilson, C. W. and F. E. Beckett, Eds.  1968.  Municipal sewage ef-
         fluent for irrigation.  Ruston, Louisiana, The Louisiana Tech
         Alumni Foundation.  169 pp.

6.  McKinney, R. E., J. N. Dornbush, and J. W. Vennes.  1971.  Waste
         treatment lagoons - state of the art.  EPA, Water Poll. Cont.
         Res. Series, 17090 EHX 07/71,   152 pp.

7.  Ryther, J. H., W. M. Dunstan, K. R. Tenore and J. E. Huguenin.
         1972.  Controlled eutrophication-lncreasing food production
         from the sea by recycling human wastes.  Bio Science, 22(3):
         144-152.

8.  Huggins, T. J. and R. W. Backman.  1969.  Production of channel
         catfish (Ictalurus punctatus) in tertiary treatment ponds.
         U. S. Clearing House, Fed. Sci. Tech. Info. PB Report No.
         190165, 122 PP.

9.  Hal lock, R. J. and C. D. Ziebell.  1970.  Feasibility of a sport
         fishery in tertiary treated wastewater.  Jour. Water Poll.
         Cont. Fed., 42(9): 1656-1665.

                                  795

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10.  Trimberger, J.  1972.  Production of fathead minnows (pimephales
         promelas) in a municipal waste water stabal izat ion system.
         Michigan Dept. Nat. Res., Fish Div., Unpublished.   5 pp.

11.  Anon.  1972.  Fish raised inwastewater lagoons.  The American
         City, 87(6):
12.  Deaner, D. G.  1970.  Public health and water reclamation.  Re-
         print of paper presented to Annual Meeting of the Sacramento-
         San Joaquin, Calif. Water Poll. Control Assn., June, 1970.
         7 PP.

13.          .  1971.  Cal ifornia Water Reclamation Sites 1971.  Calif.
         State Dept. Public Health, Bur. Sanit. Eng., 63 pp.

}k.  Jopling, W.  F., D. G. Deaner, and H. J. Ongerth.  1971 .  Fitness
         needs for wastewater-reclamat ion plants.  Jour. Amer, Water
         Works Assoc., 63(10): 626-629.

15.  DeWitt, J. W.   1969.  The pond,  lagoon, bay estuarine,  and  im-
         poundment culture of anadromous and marine fishes, with empha-
         sis on the  culture of salmon  and trout, along the  Pacific
         coast of the  United States,  U. S.  Depart. Comm., Econ.  Develop,
         Adm., Tech. Assist. Project,  36 pp.

16.  Hansen, R. J.   1967.  A study of  some  physical and chemical en-
         vironmental features of  a large sewage oxidation pond.  MS
         Thesis  in Fisheries, Humboldt State College,   133  pp.

17.  Hazel, C. R.  1963.  Chlorophyll  and  non-astacin  pigment  concen-
         trations and  photosynthetic  oxygen production  in a sewage
         oxidation pond.  MS Thesis  in Fisheries,  Humboldt  State Col-
          lege,  119 pp.

 18.  Allen, G. H. , Guy Conversano, and B.  Colwell.   1972.   A pilot
         fish-pond system for utilization  of sewage effluents,  Hum-
         boldt  Bay,  northern California.   Calif.  State Univ.,  Hum-
         boldt.   HSU-SG-3,  25 pp.

 19.           t and  P.  J.  O'Brien.  1967.   Preliminary  experiments  on
     - th~e~accl i mat izat ion of  juvenile  king  salmon,  Oncorhynchus
          tshawytscha,  to saline  water mixed with  sewage  pond effluent.
         Calif.  Fish and Game, 53(3):  180-184.

 20.   Fryer,  J.   1973.   Need for  pilot demonstration  projects in aqua-
          culture.  Proc.  Assoc.  Sea  Grant  Program Inst.,  Nat.  Conf.,
          Univ.  Delaware,  October, 1973.   In press.

 21.   Leitritz,  E.  1959.  Trout  and  salmon culture -  hatchery methods.
          Calif.  Dept.  Fish  and Game,  Fish. Bull.  No.  107.   169 pp.

                                  796

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22.  California State Water Resources Control Board.  1972.  Water
         quality control plan for ocean waters of California, July 6
         1972.  13 pp.

23.  Allen, G. H.  1973.  Rearing Pacific salmon in saltwater ponds
         fertilized with domestic wastewater July 71-June 1972.  Hum-
         boldt State University Coherent Area Sea Grant Program.
         Data Report, 88 pp.

2k.  Joseph, J.  1958.  Studies of Big Lagoon, Humboldt County, Cali-
         fornia, 1956-58.  MS Thesis  in Fisheries, Humboldt State Col-
         lege,  137 pp.

25.  Hayes, J. M.  I960.  A study of  the salmonids and some other fish-
         es of Big Lagoon, Humboldt County, California.  MS Thesis in
         Fisheries, Humboldt State College,  146 pp.

26.  Reimers, P. E.  1973.  The length of residence of juvenile fall
         chinook salmon in Sixes River, Oregon.  Oreg. Fish Comm.,
         Res. Repts., 4(2): 43 pp.

27.  Blankinship, T. E.  1973.  Impact of birds on salmon populations
         at a rearing pond, Humboldt  County, California.  MS Thesis
         in Wildlife, Humboldt State  University,  83 pp.

28.  Gross, Craig.  1972.  The distribution of acid-soluble Cu, Cr,
         Fe, Na, Ni and Zn in sediments from various environments of
         deposition in Humboldt County, California.  Senior Thesis.
         Department of Geology, Humboldt State University.  23 pp.

29.  Taniguchi, A. K.  1970.  Interval of estuarine residence and out-
         migration of the juvenile chinook salmon in the Mad River,
         California.  MS Thesis in Fisheries, Humboldt State College,
         87 pp.

30.  Powers, J. E.  1973.  The dynamics of a population of Anisogamma-
         rus confervicolus (Amphipoda): a computer simulation"!  MS
         ThesTsin Fisheries, Humboldt State University, 111 pp.

31.  Pardue, G. G.  1973.  Production response of the bluegill sunfish,
         Lepomis macrochirus Rafinesque, to added attachment surface for
         fish-food organisms.  Trans. Amer. Fish. Soc., 102(3):
         622-626.

32.  DeGeorges A.  1972.  Feasibility of artificial reefs in interti-
         dal waters.  MS Thesis in Fisheries, Humboldt State Univer-
         sity.  102 pp.
                                  797

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33.  Prince, E. D.  1972.  The food and behavior of the copper rockfish,
         Sebastes caurinus Richardson, associated with an artificial
         reef  in South Humboldt Bay, California.  MS Thesis in Fisher-
         ies, Calif. State Univ., Humboldt.  102 pp.

3^.  Lambert, R. T.  1973.  Seasons of attachment and settlement on
         various substrates and abundance of colonizing organisms on
         an artificial reef in Humboldt Bay, California.  MS Thesis
         in Fisheries, Humboldt State University.  118 pp.

35.  Payne, T. R.  1972.  Study on the development of the prior resi-
         dence effect in rainbow trout (Salmo gairdneri).  MS Thesis
         in Fisheries, Humboldt State University"!  25 pp.

36.  Skeesick, D. G.  19&3.  A study of some physical-chemical char-
         acteristics of Humboldt Bay.  MS Thesis  in Fisheries, Hum-
         boldt State College.  H*8 pp.

37.  Allen, 6. H.   196*+.  An oceanographlc study  between the points of
         Trinidad Head and the Eel River.  Calif. State Water Quality
         Control  Board,  Publ. No.  25:  125 pp.

38.  California  State Water Resources  Control Board.   1973.  Research
         needs for water  resources control  In California.  Publ. No.
         **8:   *+8 pp.
                                  198

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         AQUACULTURE AS A MEANS TO ACHIEVE EFFLUENT STANDARDS

                               by

                Mark S. Coleman, James P. Henderson,
              H.G. Chichester and R. LeRoy Carpenter*


                          INTRODUCTION

Effluent Standards

      The Federal Water Pollution Control Act Amendments of 1972**
required that all publicly owned treatment works achieve effluent
limitation based on secondary treatment as defined by the Administrator
of the Environmental Protection Agency.  The Act also required that
the State classify all stream segments as either effluent limited or
water quality limited.  Discharges to effluent limited segments must
meet the defined^ secondary treatment requirements whereas discharges
to water quality limited segments must generally meet more stringent
treatment requirements.  Subsequent to publication of the effluent
limitations associated with the definition of secondary treatment,
the Environmental Protection Agency determined2 that waste stabiliza-
tion ponds (lagoons) as presently designed would meet the BOD5
limitation of 30 mg/1, but would not meet the suspended solids limi-
tation of 30 mg/1 or the fecal coliform limit of 200/100 ml without
improved pond design and operation and additional treatment steps
for algae removal and disinfection.  Additionally, specific stream
segments in many states were designated3 as water quality limited
and require that effluent discharges to those streams have BOD5
concentrations of significantly less than 30 mg/1^.  As several
thousand of these inexpensive and simply operated waste treatment
facilities exist, particularly in smaller communities in the less
populated areas of the country, it was highly desirable that a means
be developed whereby these facilities would meet the various upgraded
requirements.

Lagoon Treatment

      Lagoon treatment systems rely on biological systems which de-
grade and stabilize organic compounds in wastewater.  A conventional

*0klahoma State Department of Health, Oklahoma City, Oklahoma

**Public Law 92-500

                                  799

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scheme of this process is demonstrated in Figure 1.  Prior to lagoon
treatment, sewage may receive no. treatment at all, mechanical treat-
ment ranging from primary to a high degree of secondary treatment, or
extensive biological treatment.  Numerous modifications of lagoon
design exist and a wide range are in current use.5,6

      The gross biological steps in this type of common treatment
system are: (1) bacterial (anaerobic and aerobic) decomposition of
organic wastes resulting in component molecules (plant nutrients)
being released into the water; and (2) algal utilization of nutrients
producing rapid growth and large populations.7,8,9,10  The algal
phase of the process constitutes the basic mechanism of separation
of the nutrients (primarily nitrogen, phosphorous, and carbon) from
the water.  Proposed mechanisms (Figure 2) to upgrade effluent quality
from lagoons tend to be physical in scope and include settling, flocu-
lation, and filtration aimed toward the removal of the algal cells.
Removal of the algae results in lower concentrations of suspended
solids and removal of the elemental constituents comprising their
organic mass."

      Proper design and maintenance of such a system should provide
a high degree of waste treatment.  However, these types of treatment
mechanisms have some undesirable attributes which are of concern.
The algal removal processes involve greatly increased construction,
operational, and maintenance costs.  In addition, waste products
are produced requiring further disposal.11,12

      Personnel of the Oklahoma State Department of Health have been
conducting a preliminary research project since 1970 in an attempt
to attack and ameliorate the problems of algal removal and to upgrade
the quality of lagoon effluents.  The basic assumption of the project
is that a biological means of removal exists for the biological
phenomenon of algal production.  Current systems are biological to
the algal removal point.  The physical-chemical treatment phase for
algal removal is replaced in the system undergoing study by employing
a further biological segment based on herbivorus or filter-feeding
fishes.

      This biological removal concept is presented in Figure 3.
Although either an advanced biological treatment system or an ad-
vanced physical-chemical treatment system will produce an effluent
of high quality, a significant point of this alternative is that the
removal of algae through the ecological food chain will produce a
useful product in the form of fish as opposed to a product requiring
further disposal.  Many alternatives for the use of fish are avail-
able ranging from reduction to animal food, to sale of live fish
for bait, restocking or forage.13
                                  200

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' I
                     HAW WAS F»
                                   BACTF RIAL
                                   Ot COMPOSI TION
NUTRIENT
RICH
WATER
               PRIMARY
               PRODUCTION
ALGAE
LADEN
EFFLUENT
                    Figure 1  Schematic  Representation of  a Typical Waste Oxidation Pond

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LAOOON NO. !
                                LAQOON NO. 2
                                                  ALGAE
                                                  LAOEN
ENT






1 1

MECHANICAL
FLOCl/LATION
AND
SEPARATION
                                                      CHEMICAL
                                                      FEED
                                                                           POLISHED
                                                                           EFFLUENT
                                                               • CHLORINATION
        Figure 2  Schmatic  Representation of a Typical
       Physical-Chemical Advanced Waste Treatment System

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i -
•
•
                                                                                              OUTLET STRUCTUK1

                     KAW WA\ I I
                                  BACTERIAL
                                  ULCOMPOSITION
NUTRIF.MT
WAII II
PRIMARY
PHOOUCTION



                                                  ADVANCED
                                                  HU ILOCilCAL
                                                  1 l!( Al MI.NT
                                                F IStt PRODUCTION
, POLISHED
 EFFLUENT
      Figure  3   Schematic Representation of Operation of an Advanced  Biological Treatment System.

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Project History

      As the project began to take form in 1970, several species of
fish were cultured in the laboratory to determine viability.  Thirty
gallon aquaria containing wastewater at various levels of treatment
were utilized in this preliminary effort.  Water was changed on a
weekly basis.  Species cultured successfully included carp, goldfish,
fathead minnow, golden shiner, black bullhead, channel catfish,
mosquitofish, bluegill, green sunfish, largemouth bass and Tilapia
nilotica.

      In the spring of 1971  it was decided to apply a larger scale
test of fish viability in actual oxidation ponds.  Arrangements
were made with the City of Oklahoma City  to use the Quail Creek
Sewage Lagoon system  located northwest of Oklahoma City.  A diagram
of  this system is presented  in Figure 4.  It consisted at that time
of  a two-cell, serially operated system receiving approximately
750,000 gallons/day of raw  domestic sewage.  The facility utilized
the'fiinde  Air-Aqua  treatment process whereby diffused air was  intro-
duced  through  subsurface  air lines.  Fish cages were placed near  the
effluent  end of  the second  cell  and anchored to a cable  stretched
the width  of the pond between two  air  lines.  Approximately 200
individuals of promising  species,  including  channel  catfish, bluegill,
and largemouth bass were  placed  in the cages where  the bench scale
viability  studies were verified.


                    SYSTEM DESIGN AND OPERATION
 Facility Description

       When the facility was originally built, it was planned to
 eventually utilize three separate sets of the two cell Hinde system.
 Sufficient funds existed to construct all needed cells in the initial
 construction.  The plan was to add on first facultative lagoon capa-
 city and add aeration as the collection system expanded.  By early
 1973  about 1 mgd of raw domestic waste was receiving conventional
 aerated treatment in the first two cell set of the three set system.
 For experimental purposes, it was determined to utilize the four non-
 aerated and unused ponds in conjunction with the existing aerated
 system to form a six cell, serially operated system.  (Figure 4)
 Accordingly, in May, 1973, 25,000 two to four inch channel catfish
 were stocked in the third cell and an additional 25,000 into the
 fourth cell.  The fifth and sixth cells received 85 pounds (about
 1500) Rolden shiner adults equally divided between the two cells.  An
 additional stocking of approximately 5 pounds of fathead minnows were
 Introduced into the third cell in July, 1973.  Also in July, 175
 three inch Tilapia nilotica were placed in the third  cell.
                                   204

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Nj
                    Figure 4  Schematic Representation of Quail Creek Lagoon System

-------
      Problems arose in the system when black bullhead, green sunfish
and mosquitofish already present in the aerated cells, contaminated
the remainder of the system.  However, the resulting polycultural
situation lent itself to gleaning data on a wider food web than
originally intended.

Analytical Methods

      Water sampling consisted of removing 2 liters of the raw
waste and effluent of each cell on a weekly basis.  Standard labora-
tory analyses^ were conducted by the Oklahoma State Department of
Health and City of Oklahoma City.  Bacteriological sampling consisted
of removing 100 ml of water from the raw waste and effluent of each
cell on alternate weeks.  Fish population analyses consisted of a
preliminary food habits investigation and biomass estimates.  Estimates
of biomass were made from seine hauls encompassing a  closed known
area of each  cell.  Mean weights were taken  for samples of 100 fish
and applied to estimated numbers.  Food habit data were taken by
excising  stomachs  of various  species  at the  isthmus and duodenal
sphincter.  Identification  of stomach contents was made in the labora-
tory utilizing standard methodology.
                      RESULTS AND DISCUSSION
 Analysis  of Water
       A summary of water analyses are presented in Table 1 and are
 the average of weekly samples from June 6,  19-3 to October 3,  1973.
 The secondary treatment standard for BOD5 of 30 mg/1 was met  in the
 effluent from the second cell and fell significantly throughout the
 remainder of the system.  The remaining secondary standards for
 suspended solids of 30 mg/1 and fecal coliform of 200/100 ml  were
 met in the effluent of the fifth cell.

       With respect to suspended solids, it is important to note that
 from the second cell through the remainder of the system approximately
 half of the suspended solids were non-volatile.  As the newly filled
 lagoons have sustained considerable bank erosion due to wind action,
 it is felt that this, when stabilized, will reduce suspended solids.
 Turbidity data substantiate this contention.  Since the volatile
 portion of the suspended solids is primarily composed of algal cells,
 a primary target of this research, it is anticipated that the volatile
 fraction will decrease more rapidly as the system is optimized.

       The nitrogen forms are of special  interest due to the overall
 removal efficiency by this system.  Ammonia was reduced below  1.0 mg/1
 N  in effluent from the first cell.  It is also significant that of
                                   206

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      Table 1  Mean Values of Weekly  Analyses of Water  Samples from the Quail Creek Lagoon  System.


                        Samples were Collected June 6, 1973 through October 3, 1973.
            Parameters
Raw
o
NJ
Biochemical Oxygen Demand
(5 day) 184
Suspended Solids
Volatile Suspended Solids
Total Nitrogen (as N)
Nitrite Nitrogen (as N)
Nitrate Nitrogen (as N)
Ammonia Nitrogen (as N)
Organic Nitrogen (as N)
Total Phosphorous
(asP)
197
131
18.94
0.07
0.20
12.67
6.10
9.01
Fecal Col. form/1 00 ml 3.05x106
Turbidity
pH (standard units)
55
7.3
47
79
54
10.50
4.54
1.00
0.91
4.05
9.87
10880
15
7.8
24
71
45
7.04
0.96
2.31
0.40
3.37
7.97
1380
23
8.2
17
52
34
6.65
0.86
1.79
0.31
2.69
5.80
322
25
8.6
14
54
27
3.97
0.34
0.79
0.28
2.56
3.66
15
42
8.9
9
26
13
3.13
0.34
0.31
0.10
2.28
3.01
15
17
8.4
6
12
6
2.74
0.16
0.29
0.12
2.17
2.11
20
9
8.3
                                        'Data reported in tng/1 except where noted.

-------
total nitrogen remaining  in the system, the majority from the third
through sixth cell was organic nitrogen.  This organic fraction was
likely present in algal cells.  Thus with further refinement of the
system, this should ultimately be reduced to even lower levels.

      Phosphorus showed good reduction throughout the system to a
low of 2.39 mg/1 in the final effluent.  There were doubtless a
variety of factors involved in this reduction, including removal of
algal cells.  It is likely that at least a part of this removal was
due to the formation of a calcium-magnesium-phosphate sludge under
the conditions of the high pH values in the third through sixth cell.

      The overview of these data indicate a high grade effluent.
These preliminary efforts are not seen as maximum efficiency, however
further research efforts  should attain greater removals.

Food Habits

      The preliminary data on food habits allowed general classifica-
tion of the fishes according to position in the food web.  A dia-
gramatic representation is presented in Figure 5.

      Tilapia nilotica, fathead minnows and golden shiners were
found to consume phytoplankton directly and microcrustacea and insect
larvae to a lesser extent.  Channel catfish  were found to utilize
microcrustacea and insect larvae as their principal foods.

      This analysis was intended to provide preliminary information
regarding the food niche  of each species.  Future studies should
substantiate these preliminary conclusions on food habits.

Biomass

      Since no supplemental feeding of fishes occured during the
experiment, it follows that fish biomass must have been elaborated
from organic constituents present in the system.  It is then rea-
sonable to assume that all biomass gained during the observation
period represents entrapment of elemental constituents that would
possibly have been discharged to receiving waters if the fish had
not been present.  Thus,  the increase in fish biomass was viewed
with special interest.

      Tilapia nilotica biomass increased from the initial 4 pounds,
consisting of 175 individuals (averaging three inches stocked in
July 1973) to 163 pounds  in October 1973.  The harvest consisted of
2339 fish with some individuals attaining lengths of ten inches.  It
was felt that their planktivorous food habits, the presence of abun-
dant algae, and rapid reproduction rate were the major reasons for
this increase.  The total biomass of Tilapia was not determined due
to procedural difficulties coupled with a sudden temperature drop

                                  205

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                                                   NUTRIENT INPUT.
Nj
                 BIOLOGICAL

                 DECOMPOSITION
                                                                  SLACK BULLHEAD

                                                                CHANNEL CATFISH

                                                                GREEN SUNFISH
                        BACTERIA
CHEMICAL
DECOMPOSITION
                        Figure 5  General  Schematic Food Web of Organisms  Inhabitins
                             Cell Three  in the Quail Creek Lagoon System,  1973.

-------
which resulted in a large mortality.  The biomass reported was deter-
mined from Tilapia removed.  It is likely that a significantly greater
amount of Tilapia were produced and not recovered.

      The biomass of channel catfish increased from the initial 600
pounds to an estimated 4400 pounds.  However, the channel catfish
were observed to gain the majority of this biomass in a six week
period from late May 1973 to mid July 1973.  Slow growth followed
this initial rapid gain.  Reasons for this phenomenon are unclear
but limited information suggests stunting due to limited food supply.
After the virtual stoppage of growth, the system was not operating
at maximum efficiency for either water quality improvement or pro-
duction of fish biomass.

      From the initial stocking of 85 pounds of golden shiner minnows,
only an estimated 535 pounds were recovered probably due to bullhead
catfish predation.  It should be noted that only the production of
golden shiners in cells five and six was estimated.  However, this
species was found in moderate numbers in other cells.

      Further studies in this area should be oriented toward and
include detailed niche identification and utilization to maximize
production and resultant upgraded effluent quality by varied species
at varied stocking rates.
                         COST  EVALUATION
      The  replacement  of  the  physical-chemical  technology with a
biological system producing a marketable  product  can help defer the
cost  of  treating  sewage.  Using  this  concept, cost-benefit analyses
were  a part of  the overall  project.   Cost figures for  the project
are presented in  Table 2.   The cost of  additional operation and
equipment  (line 2)  can reasonably  be  expected to  decrease after
initial  purchases of durable  equipment  are made.   Total  estimated
cost  is  15C/1000  gallons  of domestic  sewage.

      It is appropriate to  note  that  this comparatively  low figure
is for an  effluent of  high  quality, surpassing  secondary standards
usually  attainable only through  mechanical advanced waste treatment
technology. Costs of  40c to  45C/1000 gallons have been  reported in
mechanical separation.6  If no monitary recovery  at all  were made
from  the fish in  tae system,  this  would remain  an excellent effluent
at a  comparatively low price.

      The  calculations in the lower body  of the table  project possible
cost  recovery based on local  market value.  However, the production
estimates  are based both  on our  data  and  literature sources which
suggest  the average yield in  fertilized systems.15

                                   210

-------
                             TABLE 2
              ESTIMATES OF PROJECTED COSTS & INCOME
          FROM AN ADVANCED BIOLOGICAL TREATMENT SYSTEM
Costs

  180 X 106 gallons/six months X  .08C/1000  gallons     =   14,400.00

  Additional operational & equipment  costs  @  rate
                               of $20,000.00/year      =   10,000.00

  Fish   50,000 channels at 4c apiece                 =    2,000.00
         50 gallons minnows at $15.00/gallon           =      750.00
         250 Tilapia at $1.00 each                     =      250.00
           $27,400.7180 X 10& = 15c/l,000 gallons        $ 27,400.00


Income

  50,000 channel catfish at  .250  each                 =   12,500.00

  * 1,200 gallons minnows at $15.00/gallon             =   18,000.00

  * 18,000 pounds of Tilapia & bullheads at 6C/pound  =    1,000.00
           31.500./180 X 106 = 17
-------
      Using these figures, a return of 2.3e/1000 gallons is possible.
However, even if these figures are high, any sale will be of benefit
to the community involved.

      As the project proceeds, data will be obtained to more accurately
determine the benefits accured from such a biological system operating
toward maximum efficiency.  The next phase of the project will be to
repeat the experiment with no fish present throughout the system.
                                  272

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                           REFERENCES
 1.  Anon.   Water Programs, Secondary Treatment Information.
     U.S.E.P.A.  Federal Register.  38^22298-22299,  August   1973.

22.  Cahill, H.P. Jr.  Waste Stabilization Ponds.  (Memorandum de-
     fining secondary treatment standards) U.S. Environmental
     Protection  Agency.  September 1973.

 3.  Anon.   Fiscal Year-74 Water Pollution Control  Program for
     State of Oklahoma.  Oklahoma Department Pollution  Control.
     (mimeo), 1974, 103p.

 4.  Anon.   Permit Management Plan for Planning Basin  One  - State
     of Oklahoma.  Oklahoma Department Pollution Control,  (mimeo),
     1974.

 5.  Anon.   Bacterial Reduction, Phosphate and Nitrogen Variations
     and Algae Prevalence  in Oklahoma Waste Oxidation  Ponds.
     Oklahoma State Department of Health.   (No date).

 6.  Anon.   Upgrading Lagoons. U.S. Environmental Protection Agency,
     Technical Transaction Series,  pp 43, 1973.

 7.  VanHeuvelen, W. and J.H. Svore.  Sewage Treatment by  the Lagoon
     Method.  Transactions Fourth Annual  Conference Sanitary Engineer-
     ing.  Bull.#30:5-8, 1954.

 8.  Hopkins, G.J. and J.K. Neel.  Sewage Lagoons in the Midwest.
     Missouri Department of Health, Education and Welfare,  pp 23,
     1956.

 9.  French, D.E. Sewage Lagoons in the Midwest. Water and Sewage
     Works,  pp  537-540, December 1955.

10.  Kincannon,  D.F.  The  Role of Oxidation Ponds in Waste Treatment.
     Journal Missouri Water and Sewage Conference 1966.  pp 6-11.

11.  Parker, D.S., J.B. Tyler and T.J. Dosh.  Algae Removal.  Wastes
     and Wastewater Engineering, pp 26-29, January  1973.

12.  Kothandaraman V. and  R.L. Evans.  Removal of Algae from Waste
     Stabilization Pond Effluents—State  of the Art.  Illinois Water
     Survey.  Cir. 108:9,  1972.
                                  213

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13.  Trimberger, J. Production of Fathead Minnows (Pimepheles
     promelas) in a Municipal Waste Water Stabilization System.
     Michigan Department National Research,  pp 4, 1972.

14.  American Public Health Association.  Standard Methods for the
     Analysis of Water and Wastewater, 13 Edition.  A.P.H.A.  New York.
     pp 873, 1971.

15.  Altman, W.R. and W.H. Irwin.  Minnow Farming in the Southwest.
     Bulletin  Oklahoma Department of Wildlife Conservation,   pp 35,
     (No date) .
                                  274

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              PART it   AGRICULTURE
                SESSION CHAIRMAN

                RICHARD E. THOMAS
             RESEARCH SOIL SCIENTIST
ROBERTS. KERR ENVIRONMENTAL RESEARCH LABORATORY
      U.S.  ENVIRONMENTAL PROTECTION AGENCY
                 ADA,  OKLAHOMA

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    AN EXPERIMENT IN THE EUTROPHICATION OF TERRESTRIAL ECOSYSTEMS
            WITH SEWAGE: EVIDENCE OF NITRIFICATION IN A
                      LATE SUCCESSIONAL FOREST

                                 by
                           W            "jV            *fc
             G. M. Woodwell , J. Ballard , J. Clinton ,
                     M. Small** and E. V. Pecan*

                            INTRODUCTION

Present practice in treatment of sewage is to release partially
treated effluents into water bodies and to rely on natural pro-
cesses for completion of the treatment.  This system of disposal is
reaching clear limits as water supplies are exhausted and inland
and coastal waters become increasingly polluted.  One alternative to
this "tertiary" treatment is to use natural or lightly managed
terrestrial and aquatic ecosystems singly or in combination as
living filters to remove nutrients and release potable water to
either ground or surface water channels.  The system has the poten-
tial for enabling reuse of both water and nutrients.

A series of experiments has been designed at Brookhaven National
Laboratory to explore the potential of terrestrial and aquatic
systems for recovery of water and nutrients in effluents from primary
and secondary sewage treatment plants.  Aquatic systems are a pond,
a freshwater marsh, a pond-marsh complex and two  Phalaris arundinacea
meadows .  The terrestrial research has been designed around the
field-to-forest sere of central Long Island1"3.   The questions are
superficially simple:  what  is the potential for  each of the major
communities of the sere for absorption of the solids and nutrient
elements in sewage and  for the release of "clean" water?  How can
terrestrial and aquatic systems be manipulated  to offer the greatest
degree of treatment with  the  least commitment of  fossil fuel energy
and the least management?
   *Biology  Department,  Brookhaven National Laboratory,  Upton, New
 York  11973.
   **Department  of Applied  Sciences, Brookhaven National  Laboratory,
 Upton, New York 11973.
  ***Research carried  out at Brookhaven National Laboratory under
 the auspices of the U.  S.  Atomic Energy Commission with  partial
 financial  support from  the Town of Brookhaven, Long Island, New
 York.

                                   275

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                       THE BROOKHAVEN PROGRAM

DESIGN

The design of the experiments was based on the assumption that the
hydrologic cycle for Long Island can be evaluated continuously if
precipitation and solar radiation are known^.  Porous cup lysimeters
were used to sample the quality of the percolate that moves below
the rooting zone into the ground water; these data will ultimately
be used to appraise the water and nutrient budgets of the various
plant communities.  We are reporting here on the design of the
terrestrial segment of the experiment and the early results, some of
which have been reported elsewhere^.

The experiments were established in a section of the Brookhaven
National Laboratory site called the Biology Farm (Fig. 1).  The
terrain is flat throughout the area.  Soils are thin, well-to-
excessively drained, coarse textured, and podzolic.  They have been
derived from Wisconsin glacial outwash.  Depth to the water table
ranges from 5-8 meters.  The ground water flows southeastward at a
rate of about 13.4 cm/dayl.

Terrestrial plots included an agricultural field, and three commun-
ities that together span the field-to-forest sere of central Long
Island.  The agricultural field was planted in the spring of 1973
with timothy (Phleum pratense).  The sere was represented by old
field, pine forest, and oak-pine forest.  The old field community
was abandoned after harrowing in the fall of 1972.  Most of the
field had lain fallow for several years previously.  The pine forest
was a naturally seeded stand of pitch pine (Pinus rigida) that was
about 25 years old in 1972.  The late successional forest was
representative of oak-pine (Quercus alba. (}. coccinea, Jp. rigida)
stands on Long Island^"?.

Each of these plant communities was divided into three plots, a
control and two experimental.  The agricultural control plot was
fertilized with 1680 kg/ha of commercial 5-10-10 fertilizer.  No
other control plot received any treatment.  The irrigated plots
received either primary or secondary sewage at the rate of about
5 cm/week supplementing the normal rainfall.

COMPOSITION OF SEWAGE

The sewage was a mixture of cesspool pumpings (scavenger wastes)
obtained from the Town of Brookhaven and sewage from Brookhaven
National Laboratory.  Two blends were used;  one to approximate
the effluent from a primary treatment plant;  the second, from a
secondary treatment plant.  A comparison of these blends with other
sewage appears in Tables 1 and 2.


                                  276

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Table 1.  Comparison of Brookhaven National Laboratory Primary Sewage Blend with Sewage from
          Primary Treatment Plants in Various Locations.

Reference
MBAS
BOD
COD
S. Solids
D. Solids
Total Solids
NH3-N
NO.-N
NOo-N
ci-
Fe
Mn
P04
S04
K"*"
Na+
Ca2+
Mg2+
Cu
Zn
BNL
"Primary"

0.368
372
813
506
210
716
10.91
0
0.516
38.1
1.97
0.08
4.23
14.08
8.23
33.9
16.2
4.13
0.47
1.33
Muskegan
Mich.
(8)






17.21

0.89
92.0
0.99
0.15
3.04
22
9.63
65.6
85.05

0.93
1.18
Lancaster
Calif.
(9)

45
392
90
553
643
27
0
0.1







32
3


Allentown
Pa.
(10)

84

88
482
570
13.5
0.09
0.60
92










Port Jeff. Holbrook
N.Y. N.Y. x
(11) (11)
1.0
73 180

54 75
256 482
310 557
11.50
0.32
0

1.0
0.04

60




0 0.15
2.4
Selden
Domestic N.Y.
(12) (13)

130 240

120
560
680 228
15














-------
Table 2.  Comparison of Brookhaven National Laboratory Secondary Sewage Blend with Sewage from
          Secondary Treatment Plants in Various Locations.
BNL Muskegan Average
"Secondary" Mich, municipal
Reference
MBAS
BOD
COD
S. Solids
D. Solids
Total Solids
NH3-N
N02-N
NOo-N
ci-
Fe
Mn
po4
soj

Na+
Ca2+
Mg2*
Cu
Zn

0.512
131
280
168
172
340
5.29
0
0.931
33.9
1.08
0.03
1.93
10.59
5.27
28.46
12.35
3.37
0.24
0.70
(8)






27.4

0.12
122
0.73
0.04
3.32
29.6
12
80
129.6

0.08
0.11
(14)
6
25


730

15.5
0.3
3.5
130


8.3
33.3
15
135
60
25


Phoenix
Ariz.
(15)

15
45



24

0.2
213


13
35.7
8
200
82
36


Bay Park
N.Y.
(16)
0.77

69.9
5-20

384
34
0.045
0.06
101
0.22
0.03
6.2
165.3


11.2

0.02

Penn State
Pa.
(17)
0.54





12.8

5.1
4.44

0.10


13.8
52.8
32.0
16.3


Allentown
Pa.
(10)

35

54
489
543
1.23
0.47
5.12
93










Hoi brook
N.Y.
(11)

60

30
490
520
25.0
0.06
0









0.15

Hauppauge
N.Y.
(11)

20

1
399
400
3.0
0.3
0.5












-------
The two blends differed appreciably from one another and from other
sewage.  Concentrations of phosphorus, sodium, potassium, calcium,
magnesium, chlorine, sulfate, and inorganic nitrogen (NH3, N02, N03)
were slightly lower than in other sewage.  BOD and COD loadings were
high in both blends used in the Brookhaven work.  Sediment content
was also high.  The ratio of dissolved to suspended solids was very
much lower than in municipal effluents.  Suspended sediments con-
tained approximately 6% nitrogen and  1% phosphorus.  Of the dissolved
and suspended sediments, 40 and 807= were combustible and presumably
organic.

OPERATION

Two types of spray irrigation equipment were used.  In the Primary
Agricultural Field sewage was applied by a rotating Aquatower con-
structed by MacDowell Corporation, DuBois, Pennsylvania.  The tower
was designed to spray sewage under low pressure  close to the surface
of the ground.  The area covered was  4087 m^.  This equipment has
the advantages that the land is not cluttered with piping and is
easily accessible to farm machinery,  and the low pressure application
reduces aerosols.  The tower has the  disadvantage that it is complex
and easily disabled.  Conventional aluminum  irrigation pipe and
fittings were used in all other sprayed plots.   Sprinklers were
standard Rainbird equipment.

The application rate was about 1 cm  (40.4 m3/ha  or 10,691 gal/acre)
per day; plots were sprayed Monday through Friday to apply about
5 cm (2 in.) per week.  These operations required no more than
75 min/plot/day.  Spray operations were conducted throughout the
year.  In the first winter of operation difficulties encountered
during freezing weather prevented operation  for  several weeks, but
improved design will probably allow operation  at temperatures well
below  freezing.

The annual application  rates  for the  chemical  and sedimentary
constituents of sewage  can be approximated  from  the  following
relationship:
                      R = K  S C
where  R = the application rate  in kg/ha/yr  or  Ib/acre/yr; K  =  a
constant  (5.2 for kg/ha/yr and 4.6  for Ib/acre/yr);  S =  spray  rate
in cm/week; C = concentration  of the  substance in question  in
ing/liter.

Applications rates  in the Brookhaven  study  for N, P  and  K were  2100,
240, and 215 kg/ha/yr  for primary plots and  425,  95,  and  140  kg/ha/yr
for*secondary plots.  The total  sediment  deposit was approximately
3700 and  1750 kg/ha/yr  for primary  and secondary plots  for  the
spraying  regime in use  through  1973-74.   Spraying was  begun in the
pine forest and oak-pine  forest  on  July 16,  1973 and in the  old
field  and timothy  plots on October  2, 1973.

                                  279

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SAMPLING

Sewage was sampled for chemical composition from the irrigation pipes
at the time of application.  Amounts applied were measured with
buckets distributed in the treated plots1.  Ground water was sampled
for data reported here using 16 wells that tap the upper segments of
the ground water1.  Well pipes were plastic and slotted.  A dye
study showed that the water moves through the wells with the flow of
the ground water.

Percolate was  sampled with porous cup lysimeters placed in each plot.
A  central vacuum system maintained about  1/10 atmosphere tension on
each  lysimeter continuously.  Difficulties with suction equipment
caused occasional increases  in  tension  to as much as 6/10 atmosphere.

                        RESULTS  AND DISCUSSION

GROUND WATER

The quality of the  ground water varied  greatly  among the  series  of
ecosystems examined before spray irrigation was started.  The  most
 important variation was in nitrate-nitrogen and calcium contents
 (Fig  2).  Although there was appreciable change  in the  nutrient
 concentrations in the ground water through the  year of sampling
before irrigation,  the greatest differences were  between the differ-
 ent communities.   The mean concentration of nitrate-nitrogen under
 the forest was about 1/1000 that under the agricultural community;
 the concentrations  of nitrate-nitrogen under the intermediate
 successional stands were intermediate.   The data for calcium followed
 a similar pattern (Fig. 2).  The forest was clearly less of a source
 of both calcium and nitrogen than the less mature communities.
 Similar relationships were shown by other major nutrient elements,
 although in lesser degree.

 PERCOLATE

 The data on percolation through the first six months of the experi-
 ment corroborated the data from the wells.  The forest appeared less
 leaky for most elements than the successional communities.  The
 exceptionwu  total nitrogen (Fig. 3), whose concentration in  the
 percolate  of  the primary  plots after five months rose abruptly to
 ?he  concentration  in  the  spray.  A  similar  Pattern was shown  in the
 plots treated with secondary effluent  (Fig. 4), but the concen-
 trations  reached a maximum  after three months  and did not match
 tJe  concentration  in  the  spray.  Examination of the relationship
 between  the three  forms  of  nitrogen (Fig.  5) shows that nitrogen
 in^he percolate is  in the  nitrate  form  and that the  total  removed
 in percolation is  approximately the  total  nitrogen applied.
 Apparently there  is  nitrification within the more  mature  forest
 during  the fall  and winter.  Whether the nitrification was  triggered
                                    220

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by the treatment remains to be seen.  It did not occur in the pine
forest (Figs. 3,4) in either the primary or secondary treated plots.
This relationship simply emphasizes the complexity of the nitrogen
cycle and the need for greater emphasis on this type of study.

                             CONCLUSIONS

1.  The quality of ground water on Long Island is affected profoundly
by the vegetation.  The more mature, late successional forest pro-
duces percolation into the ground water that is normally low in the
major nutrients, especially nitrate-nitrogen.  Agricultural commun-
ities and plant communities of early succession are leaky by
comparison.

2.  The conversion of ammonium-ion nitrogen to nitrate appears
to occur in the irrigated stands of oak-pine forest during winter
but not in the pine stands.  The question of whether irrigation has
triggered nitrification is unresolved.

3.  There is little question that natural and agricultural commun-
ities can be used to treat sewage.  There is need, however, to
examine how harvest of nutrients is to be accomplished most effect-
ively to avoid the accumulation of salts to the point where losses
equal inputs.  The assumption seems justified that any treatment
system will require more than one plant community but there is little
basis at present for speculation as to the combinations that will
prove most useful.
                                  227

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                             REFERENCES
1.  Woodwell, G. M., J. Ballard, M. Small, E. V. Pecan, J. Clinton,
    R. Wetzler, F.  German and J. Hennessy.  Experimental Eutrophica-
    tion  of Terrestrial and Aquatic Ecosystems.  Technical Report,
    Brookhaven National Laboratory, Upton, N. Y., in press.
2.  Woodwell, G. M., B. R. Holt and E. Flaccus.  Secondary succession:
    structure and production in the field-to-forest sere at Brook-
    haven, Long Island, N. Y.  Ecology,  submitted.
3.  Ballard, J. and G. M. Woodwell.  The flux of water and nutrients
    to the ground water under a field-to-forest sere.  In Proc.
    IBP Grassland Biome   Symp., Fort Collins, Colo., in press.
4.  Woodwell, G. M. and A. L. Rebuck.  Effects of chronic gamma
    radiation on the structure and  diversity  of an oak-pine forest.
    Ecol. Monogr. _37:  53-69, 1967.
5.  Whittaker,  R. H. and  G. M. Woodwell. Structure, production,  and
    diversity of an oak-pine  forest at Brookhaven, New York.  J.
    Ecol. 17: 157-174,  1969.
6.  Harshberger, J. W.   The Vegetation of the New Jersey Pine Barrens
    An Ecologic Investigation.   Christopher  Sower Co., Philadelphia,
     1916. (1970 Edition by Dover Poublications, New York).
7.  Conard,  H.  S.   The plant associations of central Long Island.
    Amer. Midi.  Nat. 16:  433-516,  1935.
8.  Engineering Feasibility Demonstration Study  for Muskegan  County,
    Michigan Wastewater Treatment-Irrigation System.  Muskegan
     County  Board and Department  of Public Works.  Water  Pollution
     Control  Research Series  11010FMY  10/70,  1970.
9.   Dryden,  F.  D.  and G.  Stern.   Renovated  wastewater  creates
     recreational lake.  Environ.  Sci.  Technol. JZ:  268-278,  1968.
10.   Steel,  E. W.   Water Supply and Sewerage.  New York,  McGraw-Hill
     Book Co.,  Inc., 1960.
11.   Matzner, B.  Chief, Operations Division, Suffolk  County Depart-
     ment of Environmental Control, personal communication,  1973.
12.   Clark,  J.  W.,  W. Viessman and M.  J.  Hammer.   Water Supply and
     Pollution Control, 2nd ed.  Scranton, International  Textbook
     Co., 1971.
13.   Haack,  C.   Levitt Residential Communities,  Lake Success,  New
     York, personal communication, 1974.
14.   Weinberger, L. W., D. G.  Stephan and F. M.  Middleton.
     Ann. N. Y.  Acad. Sci. 136; 131-154, 1966.
15.   Bouwer, H., et al.  Renovating Secondary Sewage by Groundwater
     Recharge with  Infiltration Basins.  EPA  Water Pollution Control
     Research Series, 16060DRV 3/72, 1972.
16.   Environmental  Protection Agency.  Environmental Impact State-
     ment on Wastewater Treatment Facilities  Construction Grants  for
     Nassau and Suffolk Counties, New York, 1972.
17.   Kardos, L. T.  Unpublished results.
                                    222

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                           FIGURE LEGENDS

Fig. 1.   The Brookhaven National Laboratory site.  The experiment
is at the Biology Farm in the southeastern corner of the site.
Sewage is prepared from scavenger wastes and untreated Laboratory
sewage and pumped to holding ponds at the experiment.

Fig. 2.   Nitrate--N and Ca content of ground water under major plant
communities of the field-to-forest sere, Brookhaven, New York.
The ground water flows laterally at about 13 cm/day.  Each curve
represents a single well.

Fig. 3.   Total nitrogen concentrations  in primary sewage and  in
soil percolate under the plant communities of the sere.

Fig. 4.   Total nitrogen concentrations  in secondary  sewage and in
soil percolate under the plant communities of the sere.

Fig.  5.   Concentrations  of  ammonium-nitrogen,  nitrate-nitrogen and
total nitrogen  in  primary  sewage  applied  to terrestrial  communities
and in percolate  leaving the  rooting zone.   Key:  S  = sewage, P  =
pine  forest  percolate, F =  oak-pine  forest  percolate,  A = agri-
cultural  field  percolate, 0 = old  field percolate.
                                   223

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;,
                                                                                                                           BROOKHAVEN  NATIONAL LABORATORY



                                                                                                                                          PHYSICAL FEATURES
                                                                               FIGURE 1

-------
                       NITRRTE - N
                                                                            CRLC1UM
  .
  :

 -
        .056
                                                       3.22
                                                       2.T6
                                                       2.30



                                                        .

                                                       0.00
                                                                           BU  MM   1C
«
z
a
                                                                  ja ' *c ' *r '  a ' ** ' oil
Q
O
   2.60
   2.23
   1.86
K
u  1.49
5  ••"
8  ...3

   0.00
                    1972
                                            1973
i.n
5.96


2.39
 .
                                                                        «r ' I, '
                                                                        1972
                                                                                          1973
                                                  WELLS   CONCENTRRTIONS
                                                        FIGURE  2
                                                          225

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o
LU
LJ

O
LJ
        INPUT  flND  PERCOLflTE  CONCENTRflTIONS
         PRIMRRY  PLOTS  VEGETRTED
               TOTflL N (SUM)
RUG
                  SEP
NOV
                      FIGURE 3

-------
          INPUT  nND PERCOLRTE  CONCENTRATIONS
            SECONDflRY PLOTS VEGETflTED
                 TOTflL N  (SUM)
  £
Kj
Nj O
CJ

O
LJ
              HUG
                  SEP    OCT

                    1973
                                      Other
                                     •'' plots
NOV
                        FIGURE 4

-------
              RMMQNIUM - N

            rF|FFPPfPB—B B| B B
_-

:
•
.
h
Z
—
0
-

7.60-
6.51-
5.43
4.34
3.26-
2.17
1.09
o.oo-
           RUG
SEP
              NITRRTE - N
OCT
NOV
      SeWfC
           RUG
SEP
              TOTRL N (SUM)
                              •
           BUG
SEP
OCT
                   1973
INPUT RND  PERCOlflTE CONCENTRflTIONS
          PRIMflRY PLOTS  VEGETRTED
                 FIGURE 5
                   228

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  IRRIGATION WITH WASTEWATER AT BAKERSFIELD, CALIFORNIA

                            by
                     Ronald W. Crites
INTRODUCTION

Wastewater has been used to irrigate cropland at Bakers-
field, which is located at the southern end of the San
Joaquin Valley in the central part of California, for more
than 60 years.  The purpose of this paper is to document
the principal features of this successful operation by
tracing its historical development, current operation, and
environmental effects.  Crop yields are related to the
characteristics of the effluent applied and compared to
typical yields in Kern County.  Future plans are discussed
and the applicability of the data and experience gained
from this long-established operation to other similar
operations is delineated.

Normal annual precipitation in the Bakersfield area is
about 6 inches.  Because it occurs mostly from December to
February, irrigation is necessary for summer crop produc-
tion.  Since irrigation began in the area, agriculture has
been a major industry.

HISTORICAL DEVELOPMENT

Although the city has three wastewater treatment plants,
most of the domestic wastewater from the city is treated
at Plants No. 1 and No. 2, located southeast of the city.
The farmland adjacent to these two plants has received
wastewater since 1912, when the first portion of the
city's sewerage system was constructed.1  The outfall
sewers ended in an open ditch from which untreated waste-
water was taken for irrigation.

In 1933, the California Department of Health conducted a
state-wide survey of wastewater irrigation systems.2  The
report for Bakersfield was that 58 acres of cotton, 480
acres of wild grass, and an unspecified acreage of pasture
were being irrigated with untreated wastewater.
*Project Engineer, Metcalf & Eddy, Inc., Palo Alto,
 California
                             223

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In 1939, a 9.0 mgd primary treatment plant (Plant No. 1)
was constructed.  In 1952 the plant was severely damaged
by an earthquake and the effective capacity was reduced
to 4 mgd.$  In the same year, a 16 mgd primary plant
(Plant No. 2) was constructed approximately 2 miles to the
south.  The city continued to dispose of the effluent from
both plants on adjacent farmland, now owned by the city
and leased to a grower.  In 1948, a secondary treatment
plant was built adjacent to city Plant No. 1 by the Mount
Vernon Sanitation District,  Although the effluent is
also used nearby to irrigate crops  (pasture, alfalfa, and
barley), the Mount Vernon operation will not be discussed
further.

In 1956, Merz4 made a field visit and reported corn,
cotton, alfalfa, sorghum and grass being grown on  2,000
acres.   In 1959, ScottS reported cotton, field corn,
milo maize,  sugar beets, barley, and permanent pasture as
crops on the  2,500 acre farm.  Finally in 1973, the crops
grown were cotton, corn, barley, alfalfa, and pasture
grass.6  Although the  total  farm acreage is 2,500  acres,
only about 2,400 acres  can be  irrigated for crop production,

CURRENT OPERATION

Presently the city of  Bakersfield  leases the farmland to
Mr. Joe Garone  for $40,000.00  annually or $16.00 per acre.
Mr. Garone takes all the 13  mgd of  effluent from the two
primary plants  throughout  the  year.  He rotates his  crops,
maintains ditches, and  controls the  tailwater  from
irrigation.   The major  summer  crops  are field  corn and
cotton, with  only 80 acres being planted to alfalfa.  Mr.
Garone  is in  his fourth year as leasee; consequently, he
is still releveling certain  areas  and  leaving  some plots
fallow  to reduce weed  problems that  he inherited.

Wastewater Characteristics

The wastewater  is primarily  domestic  in nature, with only
a  few poultry-processing plants discharging high-BOD
wastes  to Plant No.  1.  The  characteristics of  effluents
from  Plant No.  1  (3.5  mgd) and Plant No.  2  (9.5 mgd) have
been  combined and a  typical  blend  of constituents  that
would be  found  in the  irrigation water is given  in Table  1.

The domestic  water  supply  for  Bakersfield is obtained  from
deep  groundwater wells.  Nitrate concentrations up to  18
and  19  mg/1  as  nitrate  are found  in some of  these  wells.
                             230

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      Table 1.  Characteristics of Treated Effluent
                       at Bakersfield
                                   Values in mg/1
                Characteristic      except as noted

            Flow, mgd                    13.0
            BOD                          150
            Suspended solids              48
            Total nitrogen as N           28
            Ammonia nitrogen as N         25
            Organic nitrogen as N          3
            Nitrate nitrogen as N          0
            Phosphorus as P                6.2
            pH, units                     7.2
            Total dissolved solids         380
            Chloride                     60
            Sulfate                      90
            Bicarbonate                  220
            Calcium                      15
            Magnesium                    18
            Sodium                       112
            Potassium                    12
            Percent sodium                65
            Sodium adsorption ratio         4.4
            Boron                         0.5
The mineral  quality of the combined primary effluent  is
quite suitable  for irrigation.   The total nitrogen  level
of 28 mg/1 causes some problems  in the growing of cotton,
as will be discussed later.   The.percent of sodium  is
relatively high at 65 percent; however, it is not critical
The total dissolved solids  (TDS)  concentration is not  a
problem for  any of the crops  grown.
Site Characteristics
The topography  is very flat,  with the fields graded for
surface irrigation.  General  drainage is from north to
south with sump pumps along the  southern end to return
tailwater to storage ponds  (See  Figure 1).   Soils range
from fine sandy loams to clay loam.  The soils are
                              237

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              WELL  NO.2
                         PLANT NO. 1
                           •RESERVOIR
• CLL
Nt.5
fILL
M.4-
                             BARN
             PLANT NO.2
m
                        \
                             WELL
                             NO. 3
           -
RESERVOIR
                       [STORAGE
                        POND
                            PASTURE
               SOUPS
                                       SUMP-
 FIGURE  1   SOUTHEAST  BAKERSFIELD  CITY  FARM
                      232

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generally alkaline and poorly drained  with  dense  clay
lenses at depths ranging from 10  to  15 feet below the
surface.  This clay barrier produces perched water in
areas where it is continuous and  reduced  percolation in
areas where it is not.

Permanent groundwater aquifers exist at approximate depths
of 100 to 200 feet and at  300 feet.  The  two are  separated
by a clay barrier, and the confined  lower aquifer is used
for water supply.  The deep wells on the  farm,  as shown  in
Figure 1, produce water for supplemental  irrigation water.
The quality, however, is inadequate  for potable uses as  a
result of high TDS and nitrate content.

Crop Irrigation

All crops are irrigated by surface methods.  Corn and
cotton are irrigated using the ridge and  furrow method;
and pasture, barley, and alfalfa  using the  border strip
method.

The nitrogen needs of the  crops,  the usual  amounts added
by commercial fertilizer in Kern  County,  and the  actual
nitrogen loading rates are given  in  Table 2. -As  can be
seen, the nitrogen applied more than meets  the  nitrogen
uptake of all crops.  For  both cotton  and alfalfa,
nitrogen loading is more than
utilized.
fertilized
fertilizer
expense of
descreased.

  Table 2.
                                         the
                   twice  that which  can  be
Because alfalfa is a legume, it  is not usually
with nitrogen.  For cotton,  excess nitrogen
results in excess vegetative growth  at the
fruitive growth, and subsequent  yields are
 Nitrogen Loadings at Bakersfield Compared  to
 Typical Fertilizer and Uptake Rates
Crop
Alfalfa
Barley
Corn
Cotton
Pasture grass
Actual
Nitrogen
loading
rate from
effluent,
Ib/acre/yr
371
139
252
277
371
Typical
commercial
fertilizer
rate,
Ib/aere/yr
_.b
80-90
200-250
100
100+
Nitrogen uptake,
Ib/acre/yr
100-150
75
150
100
150-250
            a.  Average for Kern County.

            b.  50 Ib/acre/yr of P70r added by some growers.
                             233

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Liquid loading rates and loadings of BOD, potassium, and
phosphorus are listed in Table 3.  The irrigation and
agricultural practices will be discussed individually for
each crop.

           Table 3.  Loading Rates for Primary
                 Effluent at Bakersfield
Crop
Alfalfa
Barley
Corn
Cotton
Pasture grass
Liquid
loading
rate,
ft/yr
4.9
1.8
3.3
3.7
4.9
BOD
loading
rate ,
Ib/acre/yr
1,985
730
1,500
1,500
1.98S
Potassium
loading
rate,
Ib/acre/yr
159
58
107
120
1S9
Phosphorus
loading
rate,
Ib/acre/yr
82
30
55
62
82
Alfalfa  -  Alfalfa  is  a  perennial  crop  that  is  irrigated
from March to  October at  about  2-week  intervals.  Cuttings
are made approximately  monthly  during  this  period.  As
noted  in Table 2,  some  growers  in Kern County  fertilize
with 50  pounds of  P20s  per  acre,  which is equivalent to
about  11 pounds  of elemental  phosphorus.

Alfalfa  will take  up  to 21  pounds annually  of  phosphorus
and 110  pounds of  potassium.7   Both  uptakes are more than
met by the application  of effluent.

Barley -  Planted in mid-December,  barley can be harvested
for grain in mid-May  or early June,  or it can  be  grazed.
Mr. Garone allows  his cattle  to graze  about 160 acres of
barley in rotation with other pastureland and  the feed  lot
Barley is pre-irrigated in  November, and then  irrigated
once every 2 weeks from mid-March to harvesting.  Barley
that is  grazed is  generally disced under by June  1  to
allow  the field to be prepared  for planting of corn.

Corn - Planted in  early June  or late May, corn requires a
great  deal of  water during  its  90 to 100 days  of  growth.
Corn is  generally  irrigated at  a rate  of nearly 3 in./wk.
Mr. Garone harvests his field corn in  September for
ensilage and hauls it to his  feed lot.

Cotton - Cotton is an annual  crop, planted  in  mid-March
and harvested  in mid-October.   Land  to be planted is
pre-irrigated  with about 15 inches of  effluent in
February.  Irrigation of the  cotton  begins  in  June  and  is

                              23ft

-------
 ceased by September 1.  Mr. Garone has problems growing
 cotton because irrigation with 3.7 feet of effluent adds
 nearly 3 times as much nitrogen as needed.  This ?esultl
 in excessive plant growth and reduced yields   To com

 ?oenme(hinr?hUl mana*ement is ^quired, including steps
 to (I)  thin the cotton more than usual, (2) stress the
 plants  between irrigation periods to stimulate fruitive
 growth  and (3) blend effluent with well wa?e? to deduce
 the  total nitrogen concentration.  Despite the use of

 less%h™ ?iqUCSf ^°tt0!? ylelds are g^erally 20 -percent
 less  than the  county-wide average.

 Pasture  - Mr-  Garone raises pasture grass  on the remaining
 portions of the farm.   These 750  to loo ac?es represent  g
 the poorest soils and  also receive the tailwater from
 other  areas.   Pumps and small  sumps exist  at the south
 51 5 ° ^   Pastureland to return tailwater to a storage
 pond.  The  pastureland supports between 1.5 and 2 animal
 units per acre.   The land is irrigated every 10 to 14  days
 throughout  the  year   In the past, excessive irrigation has
 led to ponding  in the  southern portion of  the pastureland,
 with problems  from  odors and mosquito  propagation resulting

 Crop Yields

 Yields resulting  from  irrigation  with  primary effluent
 typical  prices  commanded by  each  crop  in 1973   and the'
 economic  return per  acre are presented  in  Table  4   The
 double-cropping practice of  barley followed by  field corn
 will yield the highest  gross economic  return  but  will also
 require  the most  field  preparation, cultivation   and
management.

        Table 4.  Crop  Yields  and  Economic  Return
Crop
Alfalfa
Barley
Corn
Cotton
Yield,
Ib/acre
16,000
3,000-5,000
36,000-60,000
600-800
Typical
price ,
$ per pound3
0.025
0.045
0.0075
0.35
Economic return,
$ per acre
400
135-225
270-450
210-280
    a.  Based on 1973 prices.
                             235

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Alfalfa yield is equal to, if not slightly higher than, the
county-wide average of 6 to 8 tons per acre.  Barley yield
is also approximately the same as the county-wide average.
Corn yields on a county-wide basis are 20 tons per acre.
Mr. GaroneS finds that up to 30 tons per acre are possible
on the heavier loam soils, while 18 tons per acre is a
typical yield on the sandy alkali loam soil.

Yields for cotton are typically 20 percent less than
county-wide averages as a result of the excess nitrogen
and the compensatory measures required, and because of
the relatively poor quality alkali soil.  To increase
yields, Mr. Garone is applying gypsum to areas that are
strongly alkaline.  When Merz visited in 1956, he reported
that gypsum was applied at 4 tons per acre.4  He also
reported a cotton yield of 1.82 bales per acre for 1955
compared to the current yield of approximately 1.5 bales
per acre.

E c o n om i c C o n s i d e r a t i o n s

Although the original cost of the land was probably less
than $100.00 per acre because of the poor quality of the
soil, it is now worth close to $1,000.00 per acre. Mr.
Garone?s lease from the city amounts to about 20 percent
of the operation and maintenance costs for the two treat-
ment plants, which are approximately 5 cents per thousand
gallons.7  Thus the city's property has appreciated in
value, while 20 percent of their annual budget for two of
their plants is repayed from the farming operation.

The cost of irrigation water from the local canal is
about $5.00 per acre-foot at the canal side, and the
cost of pumping groundwater is typically $15.00 to $20.00
per acre-foot.  If the $40,000 lease applied only to the
14,500 acre-feet of effluent supplied, the cost of
effluent would be $2.76 per acre-foot.  This economical
price must be balanced against the requirement of managing
excess flows in the winter.

HEALTH EFFECTS

Merz4 reported that no diseases have been traced to
effluent use, although there have been problems with flies
and mosquitoes.  The current Superintendent of the city's
treatment plants, Mr. James Groves,9 reiterated that no
diseases have been associated with the operation.
Mosquitoes are a problem wherever water is allowed to pond
and stagnate, so that keeping the excess water moving is  a
major management problem.  The two equalizing reservoirs


                             236

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(5 and 10 acres in size) and the storage pond for tail-
water are periodically sprayed to control mosquito
propagation.

Current public health regulations for irrigation of fodder,
fiber, and seed crops are that the quality of reclaimed
water shall be equivalent to primary effluent.10  No
disinfection of the effluent is required, and none is
provided at the two plants.  Dairy cows are not allowed
to graze pastures irrigated with nondisinfected effluent;
however, beef cattle are not restricted.

ENVIRONMENTAL EFFECTS

A complaint by the Regional Water Quality Control Board
that the irrigation operation was contaminating the
groundwater with nitrates led to an  investigation of the
groundwater quality by Metcalf $ Eddy.°  Six  shallow wells
were drilled and sampled in late 1972.  Perched water was
found at the 11- to 12-foot depth in 3 wells  and not at
all in the other 3 wells (drilled 30 feet deep) .  Nitrate
concentrations ranged from 4 mg/1 as nitrate  in the center
of the farm to more than 600 mg/1 nitrate directly beneath
the old site of the sludge drying beds  at the Mount Vernon
plant.

The Regional Board conducted a sampling program in
September  1971 and found nitrate concentrations ranging
from 0.4 mg/1 to 68 mg/1 as nitrate. These wells were
along the  western edge  of the farm,  which is  in the
direction  of flow of the unconfined  aquifer.  These wells
were sampled at depths  of 80 to  170  feet.

No firm conclusions relating effluent irrigation  to
groundwater nitrate levels could be  drawn from  either
investigation.  The confined aquifer below  the  farm
 (300 feet  deep) has nitrate  concentrations  of 50  to
60 mg/1 as  nitrate.6  However, many  wells,  both deep and
shallow throughout the  Balcersfield  area have  high nitrate
and TDS concentrations.  The graundwater hydrology of  the
area  is complex and groundwater  quality is  influenced  not
only by irrigation practices, but  also  by oilfield well
injections, natural occurrences  of  salt, and  cattle  feed-
lots.  A more  detailed  investigation of groundwater
quality is in  the planning  stage  for 1975.

FUTURE PLANS

Because of the problems with mosquitoes,  the  ponding of
excess  irrigation water in  the winter,  and  the  poor

                             237

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quality  of  effluent  from  Plant No.  1,  the  state has
required that  the city  investigate  alternative treatment
and  reuse systems.   Metcalf  § Eddy  conducted  this  investi-
gation and  prepared  a project report.3  Treatment  and reuse
alternatives included  (1)  overland  flow  treatment  and dis-
infection with effluent discharge to  an  irrigation canal,
(2)  treatment  and continued  irrigation using  more  land,
(3)  treatment  and reuse in recreational  lakes, (4) treat-
ment and groundwater recharge followed by  withdrawal for
discharge to irrigation canals, and  (5)  advanced waste-
water treatment with discharge to the  irrigation canal.
It should be noted that for  Alternative  2  the Regional
Water Quality  Control Board  proposed  a requirement of
secondary treatment  prior  to irrigation, which is  more
restrictive than the State Department  of Health regulations
require.  Although all alternatives would  involve  benefi-
cial reuse, Alternative 1  was the most cost-effective.

Current  plans  (provided the  regulatory agencies approve
the  concept) are to  consolidate Plant  No.  1 and No. 2 and
construct a pilot overland flow (spray-runoff) system.
The  design  of  this first  stage could  begin as early as
July 1974.

SUMMARY

Much can be gained by studying the  successful operation of
wastewater  irrigation at Bakersfield.  Storage of  effluent,
control  of  tailwater, and  circulation  of water to  avoid
shallow  ponding are  successful management  techniques that
have been developed.  Control of the  farming operation by
an experienced grower who  applies proper amounts of water
for  crop needs, adds soil  amendments  as needed, and com-
pensates for excess  nitrogen (in the  case  of cotton) is
another  lesson.

Crop yields vary from slightly below  to well above the
county-wide average.  These  yields are greatly affected
by the soil characteristics  as well as the effluent
characteristics.   The alkaline soil is still being
reclaimed by additions of  gypsum in certain areas.

ACKNOWLEDGEMENTS

The  cooperation of Mr. Joe Garone and  Plant Superintendent
Mr.  James Groves is  gratefully acknowledged.   The  typical
county-wide yields and prices were provided by Mr. Dave
West of  the Agricultural Extension Service in Bakersfield.
                             238

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REFERENCES

1.  Stevens, R.M.  Green Land--Clean  Streams:  The
    Beneficial Use of Waste Water  through Land Treatment.
    Center  for the Study of Federalism, Temple University.
    Philadelphia, Pennsylvania.  1972.

2.  Files of the  Bureau of Sanitary Engineering, California
    Department of Health.  Berkeley,  California.

3.  Metcalf § Eddy,  Inc.  Project  Report for  the Bakers-
    field Sub-Regional Wastewater  Management  Plan.  Pre-
    pared for the City of Bakersfield and Mount Vernon
    County  Sanitation District.  February 1974.

4.  Merz, R.C.  Continued Study  of Waste Water Reclamation
    and Utilization.  State Water  Pollution Control Board
    Publication No.  15.  Sacramento,  California.   1956.

5.  Scott,  T. M.  Effluent Grows Crops on "Sewer Farm".
    Wastes  Engineering, 3£:486-489,  1959.

6.  Metcalf § Eddy,  Inc.  Preliminary Investigation of
    Wastewater Treatment and  Disposal for Southeast
    Bakersfield.  Prepared for the City of Bakersfield
    and Mount Vernon County  Sanitation District.
    January 1973.

7.  Pound,  C.E. and  R.W. Crites.  Wastewater  Treatment  and
    Reuse by Land Application, Volumes I  and  II.   Office
    of Research and  Development, Environmental Protection
    Agency. August  1973.

8.  Garone, J.  Personal Communication.  February  1974.

9.  Groves, J.  Personal Communication.  February  1974.

10.  California  State Department of Health, Water  Sanitation
    Section.   Statewide Reclamation  Criteria  for Use  of
    Reclaimed water  for  Irrigation and Recreational
     Impoundments.  February  1974.
                              239

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              NUTRITIVE VALUE OF AEROBICALLY TREATED

                  LIVESTOCK AND MUNICIPAL WASTES *

                                 by

                    D. L. Day and B. G. Harmon **

Numerous methods of utilizing livestock wastes (manure) have been
studied, including spreading it on the land as a fertilizer; using it
to produce fuels, make building materials; composting it for a soil
annnendment; and various methods of processing for refeeding.  Using
livestock wastes to produce feedstuffs offers two obvious advantages:
minimizing environmental pollutants and realizing a new source of
nutrients.

The acceptance of procedures that utilize treated wastes as nutrients
requires some explanation and education.  However, refeeding animal
excrement, either inadvertently or intentionally, has been practiced
to some extent since  the domestication of animals.

Before vitamins and amino acids were understood, livestock producers
knew that the fastest-growing and most-efficient hogs were those that
"followed" the cattle.  The hogs gleaned the cattle manure for un-
digested grain, but they also utilized the vitamins and monocellular
protein.  Also, young pigs needed more iron than they received from
sow's milk.  Access to the sow's feces containing high levels of
biologically available iron prevented anemia in the pigs.  Modern
livestock rations are nutritionally balanced to avoid such problems;
however, processed wastes can supply some of the nutrients and thus
help resolve the waste-management problem.

This paper deals with the aerobic treatment of metabolic wastes for
utilization in diets  of livestock as a source of various nutrients but
especially protein as amino acids.  Aerobic treatment of sewage is a.
long-known process by which microbial activity is intensified (Arden
and Lockett, 1914).   By its nature, this is a low-odor process; but it
requires external power for the aeration.  In recent years, the aerobic
   *Paper  prepared  for  presentation  at  the  conference on Use of Waste-
   water  in  the  Production  of  Food  and Fiber, March 6-8,  1974 Oklahoma
   City,  Okla.
  **Departments of  Agricultural Engineering and  Animal Science,
   University of Illinois at Urbana-Champaign.
 ***This project  is presently supported in  large part by the Illinois
   Agricultural  Experiment  Station.
                                   240

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treatment of livestock wastes has gained popularity mainly because of
its low-odor characteristic  (Jones et^ al., 1971).  The power require-
ment for continuous aeration is the main disadvantage.

              NUTRIENTS IN MUNICIPAL ACTIVATED SLUDGE

Hurwitz (1957) reported the  amino-acid, vitamin, and mineral content of
dried activated sludge from  the Metropolitan Sanitary District of
Greater Chicago, Table 1.  This product was successfully used as a feed
additive for swine, poultry, and sheep and beef  cattle in experiments
at the University of Illinois in the 1950's (Schendel and Johnson,
1954; Firth and Johnson, 1955; and Hackler et_ al., 1957).  These studies
established the value of vitamin B-_, nitrogen,  and unidentified growth
factors in the activated sludge.  There were, however, some severe
diarrhea problems with steers and lambs (Hackler, 1958).

Work conducted at Bangalore, India in the 1960's showed that half of
the vegetable and animal protein supplements in  the diet for chicks can
be supplied by activated sludge (Pillai et^ al.,  1967).  Table 2 shows
an analysis of the sludge.   The experiment was carried through the egg-
laying period, and significantly more eggs were  laid by the birds on
the sludge-supplemented diet than by those being fed a typical diet.
Pillai et al. (1952) also reported that the purification processes
inherent in the aerobically  treated activated  sludge eliminated patho-
genic bacteria causing typhoid, cholera, and dysentery.

A method of producing organic molasses by the hydrolysis of activated
sewage sludge is reported by Bouthilet and Dean  (1970).  The molasses
had an amino-acid content favorable for animal feeds, Table 3; and it
was successfully fed to weanling rats at the 5-  and 10-percent level;
feeding at the 25-percent level produced detrimental results.

An analysis of grab samples  of activated sludge  from the Urbana,
Illinois Municipal Waste-Treatment Plant is given in Table A.

          NUTRIENTS IN AEROBICALLY TREATED LIVESTOCK WASTES,
                   UNIVERSITY OF ILLINOIS PROJECTS

Since 1963, a research project has been underway at the University of
Illinois at Urbana-Champaign on managing livestock wastes by means of
aerobic treatment in oxidation ditches beneath slotted floors in live-
stock buildings, a modified  form of the Pasveer  oxidation ditch  (see
Figure 1).  The original emphasis was on odor  control  and low-labor
waste management; but in recent years, the microbially enhanced
oxidation ditch mixed liquor (ODML) has been studied as a source of
nutrients for livestock.

An analysis of a sample  of swine ODML in 1967  showed  a high potential
for a protein supplement, Table 5.  The refeeding implications are
obvious.  Methods of isolating and concentrating the  amino acids were

                                   247

-------
       Table  1.   Amino  Acid and  Vitamin  Content
                    of Activated  Sludge  (Hurwitz,  1957)
 Amino Acids, per cent (dry basis)
Total Protein
  (N x 6.25)...
30-35 per cent
Arginine	 1.04-1.26
Cystine  			 0.18
Histidine  	0.41-0.50
Jsoleusine  	 0.91-2.20
Ltusine  	 1.58-2.03
Lysine  	-	 0.92-1.33
Methionine  	 0.45-0.65
Phenylanine  	 1.20-2.00
Threonine ._	  1.15-2.20
Tryptophane 	0.22-0.34
Valine 	 1.1&-2.77
Tyrosine		0.70
Glyciue 	_	 1.55-1.71
Gluta»ic acid ....„	_	 2.89
  Vitamins M gm/gm (dry basis)

                          pgm/gm
 Riboflavin  (Bs)  ..„	  12.7-29.0
Cobalamine (Bu)  	  2.4- 4.0
 Pantothenic acid  	  4.0-
 Niacine 	_	  76.4
 Coline 		212.
•Pyridorine 		  1.2
*Biotin 			  C.7
*Inositol 			6401
•Kibi.  Vitamin 10,433
                         Ions  and Metals
           Common ions
                           Trace Metals
                     (Spectrometric Analysis)
 Aluminum  	 3
 Calcium  	 2  -3
 Iron 	 5  -6
 Magnesium 	 0.5-1
 Silicon  	 5  -6
 Sulfate (SO,)  	 1
 Phosphate  (F.-Oj)  	 5  -6
      per cent (dry basis)
    -4percent  Boron 	 0.002 per cent
                Chromium 	 0.1
                Copper 	 0.1
                Lead	 0.1
                Manganese  	 0.1
                Xickel 	 0.1
                Potassium 	 0.1
                Silver  	 0.1
                Tin  	 0.1
                Titanium  	 0.1
                Zinc  	 0.1
                Cobalt	 8-1 n
                                                              ppm
                                   24(2

-------
Table  2.   Analysis of  Municipal  Activated Sludge
                  (Pillai  et  al., 1967)
      (The results, except vitamin Bt2, are expressed as parcenir-oe on
                           ovea dry basis}.

      Organic matter (loss en ignition)                       $7.5
      Nitrogen (M)                                         6 C
      Crude protein                                      37.5
      Fatty matter (Petroleum ether extractable)                  C.O
      Crude fibre                                         g S
      Minsral matter (residue on ignition)                     32.3
      Silica (S:O.)                                         g'g
      Calcium (C=)                                        1.54
      Phospliorus (P)                                      :^9
      Vitamin B|2:
                  Total activity ;tg,'1CO g                    75.8
                  Truo activity (alkali labils) /ig/100 g          73.5
     Table 3.   Amino  Acid Analysis  of Organic
         Molasses  (Bouthilet  and  Dean, 1970)
  Amino Acid                                Percent dry basis
  Arginine                                        0.79
  Cystine                                         0.17
  Clycine                                         l.gg
  Histidine                                       0.37
  Isoleucine                                      1, gg
  Leucine                                         1.01
  Lysine                                          0.87
  Methionlne                                      0.36
  Phenylalanine                                   0174
  Threonlne                                       1.13
  Valine                                          !.37
  Crude protein                                   20
     (Nx6.25)
                                243

-------
             Table 4.  Amino Acid and Mineral Content of
         Activated Sludge From Urbana Waste Treatment Plant

Amino Acids *
Arginlne
Hlstldlo*
iBoleukimt
LMuslae
lymia*
MBthioaiM
Threo&lxM
Percent
dry matter
1.20
0.56
0.93
1.89
1.37
0.48
1.35

Minerals b
Art
Calcium
Iron
K*gn*«iuB
Phoiphoru*


Percent
dry matter
32.12
3.36
0.80
1.32
3.24


      •.  Average of two grab •••pin. 1971.
      b.  Grab MBpl«, 1974.
reported by Holmes e_t^ al_.,  (1971).  These  studies verified that  the
major portion of protein was  in  the very small-sized particles and
could be passes through a 200-mesh sieve (74 micron  openings),
supporting the theory that  it  is monocellular  protein produced by
microbial enhancement, Table  5.  Screening was not a practical method
of concentrating the high proteinaceous fraction of ODML, but the size-
analysis studies were of interest because  they showed a definite
relation between particle size and protein content-the smaller the size,
the higher the protein.

Other methods of concentrating the proteinaceous fraction were also
tested, including settling  and centrifugation.  Using a centrifuge was
the most effective method.  An amino-acid  analysis of swine ODML
centrifuge cake is given in Table 6.  The  protein concentration  varied
with the depth into the centrifuge cake, the farther into the cake,
the more the G force, and the less the amino-acid content.  Clarified
ODML centrate was also analyzed  for its amino-acid content, but  none
was detected.

Most of the nutrition attention  has been given to amino-acid content,
partly because amino acids  are the most obvious constituent of the
single cells and partly because  they are the costliest of the nutrients
needed to supplement corn.  Lysine is one  of the most used indicators
of amino acid quality because of the low lysine in corn.  ODML also
contains vitamins and minerals,  but these have less economic value
than the amino acids at the present time.

The nutritional value of swine ODML was first  tested by including a
dried product in the diets  of growing rats to  provide the protein of up
to half the soybean meal.   Satisfactory gains  and efficiency values

                                   244

-------
                                      ROTORS




/
/
J
•N
w — - 1-' >-
p—
                 PLAN VIEW  OF AN OXIDATION  DITCH

                         ELEVATION   VIEW

        Figure 1   Totally Slotted Swine-Confinement Building With
        an Oxidation Ditch Beneath the Self-Cleaning Slotted Floors
resulted (Harmon e^t _al. , 1972).  Next, finishing swine were used in
trials where several ODML refeeding schemes were tested (Day and
Harmon, 1972).  Two of the simpler schemes are depicted in Figures 2
and 3.

As shown in Figure 2, ODML was pumped from the oxidation ditch into a
holding tank where it was further aerated between feedings in order to
avoid the possibility of refeeding any unprocessed wastes.  To verify
the nutritional value, ODML from the holding tank or water was mixed
with a 12-percent protein diet in a ratio of two parts liquid to one
part feed (approximately the normal ratio of water to feed that finish-
ing swine consume).  Regular waterers were provided in all pens.
Nutrient composition of the ODML for one experiment is given in Table
7.  Corresponding oxidation ditch operating parameters are given in
Table 8.  In five replications, a total of 76  finishing hogs were fed
twice daily in open troughs.   The gain and efficiency values were
significantly greater  for hogs receiving the ODML, Table 9.

                                   245

-------
Table 5.  Amino  Acid Contents of Aerobically Treated
  Livestock Wastes Compared to Corn,  Soybean Meal,
          Swine  Feces, and Pig Requirements
(Percent dry matter)

Arglnlne
Cystioe
Clycioe
Ri at Id In*
Isoleuslne
Leusine
Lysine
Methlonlne
Phenylalaalae
Threonine
Trypotophao
Tyrodne
Vallne
Aapartlc acid
Clutanic acid
Crude protein
(N«6.25)
a,b Crab i ample
c Avg. of two
d Crab sample
e Fresh swine
Beef
ODML*
1.85
0.56
1.49
1.03
1.19
2.28
1.68
0.62
1.34
1.35
	
0.96
1.72
2.75
3.79
Poultry
ODML t>
1.43
0.43
1.69
0.80
1.40
2.48
1.76
0.69
1.34
1.54
—_
1.15
2.11
3.15
3.61
Swine
ODMLC
3.49
0.51
3.70
1.39
2.96
4.53
3.46
1.38
3.58
3.13
	
1.96
3.30
5.35
9.56
Swine.
OWL d
1
1
2
0
1
2
1
1
1
1

1
2
3
5
.73
.30
.15
.45
.66
.91
.64
.41
.62
.86

.36
.26
.82
.37
Swine
Feces6
0.44
	
—
0.14
0.52
0.92
0.60
___
O.S1
0.53
	
—
0.58
	
—
	 	 	 45.6 	

t passed
grab sea
. 1967.

through a 200

•ash sei
pies passed through a



•een,
200


1971.
mesh act



•en, 1971.

Pig f
Req't.
0.28


0.24
0.27
0.74
0.79
0.53
0.58
0.49
0.13
	
0.50
	
—
16






Corn*
0.
0.

0.
0.
1.
0.
0.
0.
0.
0.
0.
0.
0.
1.
a.




46
12

22
31
04
26
18
42
34
058
36
45
61
61
69






Soybean
Mealh
3
0

1
2
3
2
0
2
1
0
1
2
5
7
45




.09
.42

.00
.21
.69
.69
.63
.39
.93
.69
.73
.36
.82
.74
.7




faces (Covens, 1966).
f Weenling pig (Becker
et at., 1966)









g.h (HarmoD et al., 1969).
      Table 6.  Amino Acid Analysis of Swine ODML

       Screened and Centrifuged    (Holmes, 1971)
                   (Percent dry Batter)
Centrifuge Cake
(Composite)
Arglnlne
Cyatine
Clycine
Hifttldine
Isoleuslne
Leusine
Lyslne
Hethionine
Phenylal anise
Tbreonine
Tyro sine
Valine
Aspartic Acid
Glutamic Acid
1.15
0.41
2.07
1.00
1.24
2.16
1.78
0.54
1.26
1.71
1.15
2.14
3.17
2.90
Varying Radial '
Distance Into Cake
2.52 	 1.15

1.70 	 0.77


2.85 	 1.70







Contained on
20 Mesh Screen
0.566

0.177


0.432







   a.  Sharpes Mark III Centrifuge

-------
         ODML  HOLDING TANK
            ( MIXED  AND  AERATED)
                         FEEDER
                                     WflTERER
                   OXIDATION   DITCH  ~
                                                   -PUMP
Figure 2  ODML  is pumped  from the oxidation ditch into a holding
tank where it is kept mixed and aerated between feedings to pre-
vent the possibility of  feeding any unprocessed wastes.  The ODML
is fed by adding it to a  regular ration in the ratio of 2 parts
ODML to 1 part  dry diet.   Regular water is provided. (Day and
Harmon, 1972).
                             FEEDER
                                       VWTERER
                                                    PUW
                    OXIDATION  DITCH  -^ „-
 Figure 3  ODML is pumped from  the  oxidation ditch directly into
 a watering trough.  No other water is  provided.   (Day and Harmon
 1972).
                                247

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Table 7.  Nutrient Content of Swine ODML April-August, 1971
        [The oxidation ditch had been in operation
           since spring, 1969, without emptying.]


Phenylalanine
Lysine
Histidine
Arginine
Threonine
Valine
Nethionine
Isoleucine
Leucine
Aspartic
Serine
Glut ami c
Pro line
Clycine
Alanine
Tyros ing
Tryptophan
g Means of 13 weekly
Men of 6 weekly
Percent .
dry matter-'
1.48
1.42
.47
1.28
1.96
2.06
.77
1.49
2.79
J.73
2.5S
S.06
1.29
2.29
2.83
1.17
0.28
analyses of ami no acids
analyses of minerals.


Calcium
Phosphorus
Magnesium
Sodium
Potassium
Iron
Copper
Zinc









except for tryptophan
Percent b .
dry matter—
3.33
3.83
1.49
2.75
4.14
.5507
.0071
.1148









(1 analysis).
   Table 8.  Summary of Weekly  Samples, April-August, 1971
         [The oxidation ditch had  been  in  operation
             since  spring,  1969, without emptying.]
Parameter
pH
Dissolved oxygen, Mg./l.
Temperature. *C
Cheaical oxygen coma-nd, mg./l.
Dry matter, pet.
Nitrcgen, pet. dry matter
Ash, pet. dry matter
Mean
7.7
1.3
27
29,423
3.4
7.9
41.7
Range
6.0 -
0.3 -
17.5 -
18,425 -
2.1 -
S.I -
36.1 -
8.0
4.3
37.0
55,300
4.0
10.0
48.7
                                248

-------
      Table  9.  Performance  of 76 Finishing  Swine  Fed
   ODML or Water Mixed  with  Feed  (Harmon et  al.,  1973A)
                                         Wateri/
        Average daily gain
          Replication
              1	1.08
              2	1.01
              3	1.08
              4	  1,41
              5	1.10
                   Average	1.14

        Gain p.?r 1,CC./ pf>;,/.i: „•_" feed
          P.epl ication
                                                pounds
         Source:  Harmon et al., 1973.
         £/  Initial weight, approximately 110 pounds.
         t/  Liquid added  to feed, 2:1 ratio.
1
2 	
3 .
4 	
5 . . . . .
Average 	

.... 218
	 213
	 275
	 283
	 256
	 249

232
249
276
302
270
266

Table 10.   Performance  of 120 Finishing Swine Receiving
             ODML or  Water (Harmon  et_ al. , 1973)
Dallv aain

Tap H?0

Replication
1 1.34
2
3
4
5
6
Av
1.45
1.69
1-32
1.28
- . 1-56
;rage 1.45
Source: Harmon et al., 1973.
ODMl

1.39
1.54
1.98
1.58
1.45
1.74
1.61
Each
Gain oer 1.000
Tap H20
pounds
303
296
244
230
260
309
-Z7JT
value represents 10 pigs
pounds of feed
ODML

308
297
343
278
263
338
~~30~4~

                                    249

-------
Hogs slaughtered from each replication  showed no evidence of liver or
lymphatic  alteration due to feeding  the single-cell protein source
(Harmon je£ al. ,  1973A) .

As shown in Figure 3, ODML was pumped directly from the oxidation ditch
into troughs for 20 seconds out of every 10 minutes.  The initial flow
flushed out the  remaining liquid  in  the trough, while the flow as the
pump stopped remained in the trough.  This provided fresh ODML at all
times.  No other waterers were in the pens.  Control swine had access
to regular water at all times.  A corn-soybean meal diet (12-percent
protein) was available in self-feeders  in each pen.  For three experi-
ments  (two replications per experiment) with 120 finishing hogs, the
weight-gain and  feed-efficiency values  were greater for the hogs re-
ceiving the ODML, Table 10. (Harmon  et^ al., 1973).

The  scheme of Figure 3 uses the processed waste in situ to furnish
water  requirements as well as other  nutrients.  With this scheme, there
is little  if any surplus material for  disposal.  The levels of non-
biodegradable materials will build up,  and eventually have to be re-
moved .

A brief  energetic and economic analysis of the operation depicted in
Figure 3  is given in Table 11.  The  energetics show a slight loss and
the  economics show a considerable gain based on the current price of
soybean meal.  This analysis does not  include expenses of initial
equipment  and maintenance nor does it  include savings from other
nutrients  and reduced disposal.
         Table 11.  Energetic  and  Economic Analysis of Feeding
                            ODML  to Swine3
b
Aeration Expenses
Dffi c K Cal $ d
19.8 17,050 0.40
Otter expenses:
Initial equipment
Maintenance and repair
Protein Saving*
K Cal f $ *
16,920 0.80
Other saving*:
minerals
Vitamins
Water
Reduced disposal
Odor control
Vet
K Cal $
- 130 +0.40


            Finishing phase of production, 100 to 200 Ib. in 60 days.
         b  Operating expenses only.
         c  Electricity requirement, 0.33 WH/Day  (p.38 Jones at al., 1971).
         d  Electricity at $0.02/WH.
         e  Baaed on the OWL supplying 1/5 of the 40 Ib. of soybean seal.
         f  Gross energy of soybean Meal, 4.66 K Cal/Cot (Diggs et al., 1965).
         I  Soybean Mai at $200.00/Ton
                                     250

-------
Studies comparing samples of pork chops and roasts from hogs fed ODML
and from control hogs showed that the taste and odor were not influenced
by feeding the aerobically sustained product  (Wax et^ al., 1972).  Rep-
resentative samples of all tests have passed  state meat inspection in
Illinois.

There have, however, been some health problems and some unexpected pig
deaths.  High nitrate levels in the ODML were suspected of causing
deaths in one experiment in which the mixed liquor had been aerated for
a considerable time without any hogs in the building.  High nitrate
levels would be expected under this condition (no new feed for the
microorganisms).  Another problem has been the survival of intestinal
worm eggs that are excreted into the oxidation ditch and are refed in
the ODML.  No simple method of killing the worm eggs in the ODML has
been found.  The problems of killing the parasites in biologically
treated sewage was discussed by Liebmann (1964).  Heating to a temper-
ature of 144F. for a short time was the most  successful method of kill-
ing worm eggs.

Other studies are underway at the University  of Illinois to test the
nutrition of various forms of ODML for feeding to poultry and cattle,
as well as to swine.  A new building for 200  beef cattle with an oxi-
dation ditch beneath slotted floors is now available (Bauling e^ a^.,
1973).

             SOME OTHER PROJECTS UTILIZING AEROBICALLY
                      TREATED LIVESTOCK WASTES

Vetter et^ al. (1972) reported the use of beef cattle ODML in situ in
the diets for beef cattle.  The results of that study suggest that
aerobically processed livestock wastes have an acceptable nutritional
value and can be effectively used as a partial protein and mineral
supplement.  No animal health problems associated with feeding the
processed wastes or with meat quality were reported.

Studies at Purdue University have shown that  aerobic micro-organisms
can convert the soluble organic matter in dairy cattle wastes into a
biomass containing 30 percent crude protein,  Nye et al. (1972).  This
biomass product was harvested and fed to laboratory rats for up to 20
percent of their diet with no dilatory effect.  However, the rats could
not use the product as their only protein supplement.

Orr et al. (1972) reported amino-acid contents of swine ODML similar to
those in the University of Illinois studies.  They also reported of
various schemes for refeeding the ODML from oxidation ditches that had
been in continuous operation for two years.

Diesch et al. (1971) reported a study at the  University of Minnesota
for detecting and measuring the survival time of leptospires in aerated
beef cattle manure, using a model oxidation ditch.  A maximum survival
time of 18 days was measured at a pH of 6.9.

                                   257

-------
                                 SUMMARY

The aerobic process of treating organic wastes produces a biomass with
a monocellular-type protein high in amino acids.  This makes it of in-
terest as a protein supplement for animal diets that have a major pro-
portion of corn or other grain which is deficient in lysine and other
essential amino acids.  The biomass also contains other nutrients, in-
cluding vitamins, minerals, and unidentified growth factors.  The
health-related aspects of utilizing aerobically processed wastes are of
concern, but the aerobic method inherently retards pathogenic organisms.
The aerobic process is also a low-odor method of waste management that
is of interest to livestock producers.   However, the amount of energy
required for aeration may be discouraging.

This paper reviews some of the major projects of analyzing the amino-
acid content of aerobically treated sewage and  livestock wastes and of
evaluating the product as a protein supplement  in the diets of live-
stock.  Although the  amino-acid content is similar for aerobically
treated municipal wastes and livestock wastes,  extraneous materials can
be more closely controlled  in  livestock wastes  than in municipal sewage.

A method developed at the University  of Illinois in recent years
utilizes oxidation-ditch mixed  liquor (ODML)  in situ, supplying drink-
ing water as well as  protein and  other nutrients.  Crude protein in the
ODML varies from 30 to 46 percent,  the latter value is as high as in
soybean meal.  Also,  lysine and other amino  acids essential to growth
can be as high in concentrated  ODML as in  soybean meal.  This method
avoids the ordinary expenses generally associated with recycling.  It
also offers two obvious advantages:   minimizing pollution and realizing
a new source of nutrients.  The present costs of soybean meal make the
method economically feasible and  energetically  attractive.  However, a
more efficient method of oxidation  is needed.   Even so,  the  aerobic
process offers possibilities for  a  least-cost method  of waste manage-
ment that has  several advantages  over alternate methods.  Obviously,
the acceptance of  the use  of this monocellular  protein product  in the
diets of  livestock will  require some  explanation and  education.
                                    252

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                              REFERENCES

 1.  Arden, E.  and W.  T.  Lockett.  1914.  Experiments on the  oxidation of
    sewage without  the aid of filters.  J.  Soc.  Chem.  Ind.  (London)
    33:523-539.

 2.  Bauling,  D.  B., W.  D.  Boston, and D.  L.  Day.  1973.  A beef  confine-
    ment  building with an  oxidation ditch.  Amer.  Soc. of Agri. Engin.
    Paper 73-4544.

 3.  Becker, D. E.,  A.  H. Jensen,  and B. G.  Harmon. 1966. Balancing
    swine rations.  Circular 866,  Cooperative Extension Service, Uni-
    versity of Illinois at Urbana-Champaign.

 4.  Bouthilet, R. J.  and R. B. Dean, 1970.   Hydrolysis of  activated
    sludge. Proc.,  Fifth International Water Pollution Research Con-
    ference.  July-Aug.

 5.  Day,  D. L. and  B.  G. Harmon.  1972.  A recycled feed source  from
    aerobically  processed  swine wastes. Amer. Soc. of Agri.  Engi.
    Paper 72-954.

 6.  Diesch, S. L.,  B.  S. Pomeroy  and E. R.  Allred. 1971.  Survival and
    detection of leptospires in aerated beef cattle manure.  Proc.,
    International Symposium on Livestock Wastes.  Amer. Soc.  of Agri.
    Engin. Publication PROC 271:263-266.

 7.  Diggs, B.  G. , D.  E. Becker, A. H. Jensen and W. H. Norton. 1965.
    Energy value of various feeds for the young pig.  J. Anim.  Sci.,
    24(2):555-558.

 8.  Firth, J.  A. and  B. C. Johnson. 1955. Sewage sludge as a feed in-
    gredient  for swine and poultry. Agr.  and Food Chem. 3(9) :795-796.

 9.  Gouwens,  D.  W.  1966.  Influence of dietary protein and  fiber on
    fecal amino  acid  excretion. Unpublished M.S.  Degree Thesis, Univ.
    of 111.  at Urbana-Champaign.

10.  Hackler,  L.  R.  1958.  Dried activated sewage sludge as  a nitrogen
    source  for ruminants.   Unpublished Ph.D. Thesis.  Univ. of 111. at
    Urbana-Champaign.

11.  Hackler,  L.  R. , A. L.  Neumann, E. E.  Hatfield, and B.  C. Johnson.
    1957. Dried  activated  sewage sludge as a nitrogen source for
    ruminants. Abstract in J. of Anim. Sci. 16(4):1090.

12.  Harmon,  B. G. ,  D.  E.  Becker,  A. H. Jensen, and D. H. Baker.  1969.
    Nutrient  composition of corn and soybean meal. J. of Anim. Sci.,
    28(4):459-464.

                                   253

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                              REFERENCES (cont'd.)

13.  Harmon, B. G., D. L. Day, A. H. Jensen, and D. H. Baker. 1972.
     Nutritive value of aerobically sustained swine excrement. J. Anim.
     Sci. 34(3):403-407.

14.  Harmon, B. G., D. L. Day, D. H. Baker, and A.  H. Jensen. 1973.
     Oxidation-ditch mixed liquor as a source of water and nutrients.
     Abstract in J. of Anim. Sci. 37(1):280.

15.  Harmon, B. G., D. L. Day, D. H. Baker, and A.  H. Jensen. 1973A.
     Nutritive value of aerobically or anaerobically processed swine
     waste. J. Anim. Sci. 37(2):510-513.

16.  Holmes, L. W. J. 1968. Concentration of proteinaceous solids from
     aerated swine manure.  Unpublished M.S. Thesis, Univ. of 111. at
     Urbana-Champaign.

17.  Holmes, L. W. J., D. L. Day, and J. T. Pfeffer. 1971.  Concen-
     tration of proteinaceous solids from oxidation ditch mixed liquor.
     Proc., International Symposium on livestock wastes, Amer. Soc. of
     Agri. Engin. PROC-271:351-354.

18.  Hurwitz, E. 1957.  The use of activated sludge as an adjuvant to
     animal feeds. Proc., Twelfth Industrial Waste Conference, Purdue
     Univ. Engineering Bulletin Ser. 94:395-414.

19.  Jones, D. D., D. L. Day, and A. C. Dale. 1971. Aerobic treatment
     of livestock wastes. Univ. of 111. Agri. Exp.  Sta. Bulletin 737 in
     cooperation with Purdue Univ.

20.  Liebmann, H. 1964. Parasites in sewage and the possibilities of
     their extinction. Proc., The Second International Conf., Advances
     in Water Pollution Research, Edited by J. K. Baars, Pergamon Press,
     Vol. 2:269-288.

21.  Nye, J. C., A. C. Dale, T. Wayne Perry, and E. J. Kirsch. 1972.
     Recovering protein from animal waste. Amer. Soc. of Agri. Engin.
     Paper 72-955.

22.  Orr, D. E., E. R. Miller, P. K. Ku, W. G. Bergen, D. E. Ullrey,
     and E. C. Miller.  Swine waste as a nutrient source for finishing
     pigs. Mich. State Univ. Report of Swine Research 232:AH-SW-7319.

23.  Pillai, S. C., M. I. Gurbaxani, and K. P. Menon. 1952. Influence
     of activated sludge on certain pathogenic bacteria. Indian Med.
     Gaz. 87:117-119-
                                   254

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                              REFERENCES (cont'd)

24.   Filial, S. C., E. G. Srinath, and M. L. Mathur. 1967. Activated
     sludge as a feed supplement for poultry. Water and Waste Treatment.
     May-June.

25.   Schendel, H. E. and B. C. Johnson. 1954. Activated sewage sludge
     as a source of vitamin B   for the pig. Agri. and Food Chem.
     2(9):23-24.

26.   Vetter, R. L. , R. D. Christensen, G. Frankl, and W. R. Masch. 1972.
     Feeding value of animal waste nutrients from a cattle confinement
     oxidation ditch system. Leaflet R170,  Anim. Sci. Dept., Iowa State
     Univ.

27.   Wax, J. E., B. G. Harmon, and G.  R. Schmidt. 1972. Effect of liquid
     feeding oxidation ditch mixed liquor on the palatability of pork.
     Abstract  in J. Anim. Sci. 35(5):1100.
                                    255

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          Grass Filtration for Final Treatment of Wastewater*
                                  by
             R. M. Butler, J. V. Husted and J. N. Walter**
                             INTRODUCTION

Grass filtration is one of three systems for land treatment of waste-
water1 »2»3.  xhe other land treatment systems, spray irrigation and
rapid infiltration, utilize the plant root zone and the soil profile
as a filter medium to renovate wastewater.  Grass filtration waste-
water treatment systems are designed to utilize the soil surface and
plant cover as the filter medium.  The treatment mechanisms in the
grass filtration system may include physical removal of particulate
matter from the water, chemical adsorption of ions in solution on the
surface of soil particles, uptake of nutrients in solution by plant
roots, and biological treatment by microorganisms.  In grass filtra-
tion systems, wastewater is applied along the top of a sloping site
and flows  through  the soil-plant filter with subsequent runoff.  The
runoff from the grass filtration systems can be returned to a stream,
held in a  pond or  reservoir for reuse, or used for ground water re-
charge.  The  quality of  the runoff water must be compatable with the
quality standards  of the receiving stream.  The runoff water quality
depends on the nature of  the wastewater, the characteristics of the
site,  the  type of  vegetation,  and the  operating methods of the system.

ADVANTAGES

Grass  filtration systems  require less  extensive wastewater piping
systems and less land area  than spray  irrigation systems.  If adequate
renovation can be  attained  by  grass filtration, savings in terms of
wastewater application  equipment and  land  can be obtained.   If treat-
ment by overland flow is adequate, land with  low infiltration capacities
can be used for wastewater  treatment.  An  additional  advantage of  the
  * Authorized for publication as paper no. 4635  in the journal series
    of the Pennsylvania Agricultural Experiment Station.
    This investigation was supported by funds provided by the United
    States Department of Interior, Office of Water Resources Research,
    as authorized under the Water Resources Research Act of 1964 through
    the Institute for Research on Land and Water Resources, The Pennsyl-
    vania State University and the Pennsylvania Agricultural Experiment
    Station.
 ** Department of Agricultural Engineering, The Pennsylvania State
    University, University Park, Pa.
                                    256

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grass filtration system is that the treated water remains on the soil
surface.  This facilitates sampling and monitoring of treatment effec-
tiveness, and the treated effluent is readily available for recycling
or reuse.

EXAMPLES

The grass filtration system has been successfully used to treat food
industry wastewater.  A comprehensive study of an overland flow system
for cannery wastewater treatment was conducted for the Campbell Soup
Company at Paris, Texas^.  The results of the study indicate 92 to 99
percent removal of volatile solids, 86 to 93 percent removal of nitro-
gen and 50 to 60 percent removal or phosphorus.  Preliminary tests of  a
grass filtration treatment system for beef feedlot runoff water have
been successful^.

A spray-runoff treatment system for raw municipal sewage is being test-
ed by scientists at the Robert S. Kerr Environmental Research Labora-
tory .  The experimental system produced an effluent that is of terti-
ary treatment quality without sludge production.

Only limited information is available on the feasibility of using grass
filtration for final treatment of municipal effluent.  In a study con-
ducted by Wilson and Lehman?, municipal sewage effluent from an oxida-
tion pond was applied to grassed plots 1000 feet long for final polish-
ing prior to artifical recharge.  During one trial, nitrogen and phos-
phate concentrations were reduced by approximately A and 6 percent,
respectively, during flow over the plot.  They concluded from these
tests that the quality of the effluent obtained from their systems was
not suitable for recharge.

PROJECT OBJECTIVES

The primary purpose of the research, summarized in this paper, was to
determine if grass filtration can be used to remove nitrate and phos-
phate from secondary treated municipal sewage effluent.  In addition,
the effects of various hydrologic parameters on the treatment process
were investigated.  Factors studied in field and laboratory tests
included; detention time, flow rate, flow distance, application fre-
quency, and seasonal effects.
                                   257

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                               PROCEDURE

Two sets of field tests and a laboratory study were conducted.  In the
field studies, the effects of hydrologic variables and application fre-
quency on nitrate and phosphate removal were investigated.  The effects
of detention time on nitrate removal were investigated in the labora-
tory.

HYDROLOGIC STUDY

Three plots 150 feet long by 20 feet wide were established on a 6 per-
cent slope in the Summer of 1972.  A stand of reed canarygrass was
established and maintained on the plot area.  Stations were installed
at 50-foot intervals on each plot to intercept the overland flow for
water quality samples and to monitor the flow rate.  At each  station
runoff was intercepted in 4-inch deep plastic lined trenches  and di-
rected to an "H.S." flume equipped with a water level recorder to give
a continuous record of flow rate.  Runoff water quality samples were
taken periodically from the outfall of the flume.  Three-inch diameter
aluminum pipes perforated with  3/4-inch diameter  holes spaced 6 inches
on center were used to apply  the water at  the top of each plot and to
redistribute  the  effluent below the monitoring stations 50 and 100 feet
down the plot.  Water meters  were used to monitor the amount  of water
applied  to each plot.

The  chemical  composition  of  the municipal  effluent illustrated in Table
1 is based on samples  collected during 1971  and reported  by Sopper and
Kardos8. The effluent is from the  treatment plant which  serves The
Pennsylvania  State University and the borough of  State College, Penn-
sylvania.  Secondary  treatment includes  standard  and highrate trickling
filters  and a modified activated sludge  process followed  by final set-
tling.

Secondary  treated municipal  effluent was applied  to  the plots once each
week for six  to eight  consecutive weeks  during  the Winter,  Spring, Sum-
mer, and Fall of  1973.  Three wastewater application rates  were  com-
pared.   The  same  application rate   was applied  to a plot  during  the
entire  study.   In the Winter of 1973,  10,  20, and 40  gpm  were applied
for  8 hours  each  week.   The  results of  the winter runs  indicated that
 the  infiltration  capacity of the soil was higher  than anticipated.
For  subsequent trials,  the  application  rates were increased to 15,  30
and  60 gpm and the  operating time  reduced to 6 hours.

Water samples were  taken from the  inflow and from the outfall of each
 flume at 1 1/2 to 2-hour intervals  during each run.   The nitrate and
 phosphate concentrations and the pH were determined for all samples.
 Total Kjeldahl nitrogen determinations were made on selected samples.
                                   255

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Table 1.  Chemical composition of the secondary treated
          municipal sewage effluent used in the study.
Constituent
PH

Nitrate-N
Organic-N
NH4-N
Phosphorus
Calcium
Magnesium
Sodium
Boron
Manganese
Minimum
7.4
mg/1
2.6
0.0
0.0
0.250
23.1
9.1
18.8
0.14
0.01
Maximum
8.9
mg/1
17.5
7.0
5.0
4.750
27.8
15.1
35.9
0.27
0.04
Average
8.1
mg/1
8.6
2.4
0.9
2.651
25.2
12.9
28.1
0.21
0.02
                          259

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APPLICATION FREQUENCY STUDY

Three parallel plots, each 20 feet wide by 150 feet long were installed
on a grassed slope.  The plots had an uneven slope of 6 to 8 percent,
with the greater slope occurring near the lower end of each plot.  Each
plot was seeded with a mixture of 3 parts of a commercial quick cover
seed mixture, 2 parts annual ryegrass, and 2 parts reed canarygrass.
The faster germinating ryegrass dominated the growth on each plot dur-
ing all runs.  Effluent was distributed at the head of each plot with
perforated pipes similar to those used in the hydraulic study.  Efflu-
ent collection and monitoring stations similar to those used on the
hydrologic study plots were installed at the bottom of each plot.

Three run sequences were conducted during the period from mid-June to
early November, 1973.  During the first run sequence from June 13 to
July 20, effluent was applied to each plot at an approximate rate of
100 gpm.  The second run sequence was a continuous flow study in which
effluent was applied 24 hours a day for 70 hours at a rate of 40-50
gpm.  This run was conducted from August 13-16 on the plot which had
received effluent 2 times per week in the first run sequence.  A third
run sequence, similar to the first, was conducted from September 17
through November 2 with an effluent inflow rate of 50 gpm.

Application frequencies during  the first and third run sequences were
2, 3, and 6 times per week with durations of 3, 2, and 1 hours per
application, respectively.  Each plot received approximately the same
total amount of effluent per week.  The same application frequency was
used on a plot during an entire run sequence.

During the first few weeks of operation, the grass crop was in the
development stage.  Grass height was maintained at 4 to 8 inches by
weekly or semi-weekly cuttings throughout the study.  A rotary mower
was used and clippings were retained on the plot.

Samples of inflow and outflow were taken for all runs.  During certain
runs, surface water samples were taken at five additional locations,
spaced 25 feet apart, down the plot.  Water quality analyses conducted
on all samples included nitrate, phosphate, and pH.  Total Kjeldahl
nitrogen determinations were made on selected samples.

LABORATORY STUDY

A laboratory study was conducted to determine the rate of nitrate re-
moval from secondary  treated sewage effluent on the surface of reed
canarygrass sod.  Three treatments were compared in the study:   grow-
ing sod, dead sod, and bare subsoil.  The sod and the subsoil were from
the grass filtration  field test site.  The dead sod was obtained by
drying a sample of growing sod  for several weeks.  Galvanized steel
containers 10 inches wide, 30 inches long and 6 inches deep were used.
To drain the containers, a 1-inch diameter perforated conduit was in-
serted across each end and a 1-inch deep bed of crushed limestone was

                                   260

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placed on the bottom.  Approximately 4 inches of sod or soil was placed
on top of the crushed stone.

The experiments were conducted in a controlled environment chamber with
a daytime temperature of 80°F and a nighttime temperature of 65°F.  The
chamber was lighted by fluorescent and incandescent lighting approxi-
mately 14 hours per day.  To simulate water  flowing down a sloping plot,
the containers were placed on a frame that oscillated with an amplitude
of 1 inch and a frequency of 15 cycles per minute.

Effluent was applied to the treatments until there was 1/2 inch of free
water on the surface.  Each day the effluent was drained from the con-
tainers and more effluent was applied.   Samples of the effluent were
taken periodically from the surface water layer during the day for
nitrate-nitrogen analysis.
                                 RESULTS

HYDROLOGIC STUDY

Nitrate and phosphate were  the  nutrients  studied in greatest  detail.
Nitrate removal is possible from two sources;  plant uptake  and  denitri-
fication.  Results from a detention time  study showed that  50-foot
plots with application  rates of 15, 30 and 60  gpm had detention times
of 15, 10 and  8 minutes,  respectively. These  flow-through  times were
too short for  any significant plant uptake of  nitrate during  a  run.

Tables 2 and 3 present  the  percentage of  nitrate removed from the
wastewater for two different methods of  calculation.  The values in
Table 2 come from a  material balance in which  the weight of the nitrate
applied was compared to the weight lost  through infiltration  and the
weight remaining in  the runoff  water.  The result is a value  for
nitrate removal that is corrected for dilution caused by melting snow
and rainfall.  Table 3  is simply a comparison  of inflow and outflow
nitrate concentrations.  A  comparison of  the two tables shows to what
extent the melting snow in  the  winter and the  rainfall in the spring
affect the reduction in nitrate concentration.  The amount  of precipi-
tation that occurred during the Summer and Fall runs was insignificant.

The amount of  nitrate removal obtained in this study was quite low
 compared  to those reported  for  other overland  flow studies.  Informa-
 tion  reported  by Law, et al9, provides a possible explanation for the
 low nitrate removal.  It was reported that a continuous flow for 5 days
 or more was required before reductions in nitrate levels were obtained.
 Since the  system was operated only once a week for a relatively short
 period of  time, it  is possible  that anaerobic  conditions were not
 available  for  dentrification.  In addition, the short detention times
 may not have  allowed adequate time for nitrate removal.  The labora-
 tory  study has shown that detention times of several hours are re-
 quired for a  high percentage of removal,   Another factor that makes

                                   267

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Table 2.  Effect of flow rate on the percent estimated nitrate loss
          determined by a nitrate balance during four seasons.
SEASON

Winter
Spring
Summer
Fall

Low

8
4
3
0
Flow Ratea
Medium
Percent
5
2
1
1

High

3
2
4
-1 (gain)
a Winter  rates for Low, Medium and High were 10, 20 and 40 gpm
respectively, other  seasons had  rates  of  15, 30 and 60 gpm.
 Table 3.   Effect of flow rate on the percent  change  in  nitrate
           concentration during four seasons.

SEASON
Winterb
Springb
Summer
Fall

Low
51
17
7
2
Flow Rate3
Medium
54
12
4
1

High
33
13
-1 (gain)
 a Winter rates for Low, Medium and High were 10, 20 and 40 gpm
 respectively, other seasons had rates of 15, 30 and 60 gpm.

 b High reductions in concentration for Winter and Spring resulted
 from melting snow and rainfall.
                                    262

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this study different from overland flow studies reporting higher ni-
trate removals was the nature of the wastewater.  The wastewater was
highly nitrified secondary municipal effluent and contained relatively
small amounts of organic matter.  Denitrifying bacteria require organic
carbon for a source of energy.

Phosphate removal occurs by plant uptake or by adsorption on the soil
particles.  Since the water was detained on the plots for such a short
period of time, plant removal of phosphate should not have been impor-
tant during a run.  Soil adsorption was possible because of the contact
of the water with the surface of the soil.  The amount of phosphate
removed was low with an average of about 17 percent.

A statistical analysis was performed to determine which parameters
significantly influenced nutrient removal.  Each increase in flow rate
resulted in a significant decrease in phosphate removal efficiency.
This indicated that contact with the soil surface is important in phos-
phate removal.  Nitrate removal was not significantly changed by chang-
ing the flow rate from 15 to  30 gpm, but switching from 30 to 60 gpm
resulted in a significant decrease in nitrate removals.  The effects
of flow rate on nitrate reduction present a further indication that the
time spent on the plots was not long enough at  the flow rates used in
the study.

The distance traveled by the  water also was included in the statistical
investigations.  Significant  decreases in  the concentrations of both
nitrate and phosphate occurred after each  50 feet of flow.  This is a
further indication  that the most important parameter for nutrient re-
moval is detention  time which depends on the flow rate and flow dis-
tance as well as the percent  slope and the characteristics of the
vegetation.

The analysis showed differences exist from season  to season.  These
differences were partly caused by  dilution from melting snow and rain-
fall which can vary greatly from year to year.   From  a one
year study, however, it is difficult to  assign  much  importance  to  the
seasonal effects.

Grass yield data  (Table 4) show  the  effects  of  the  different applica-
tion rates.  For  the first 50 feet  of  the  plot  receiving 60 gpm, the
yield was 2.66 T/A  (tons/acre) which was  lower  than all areas  sampled
except  the 50  to  100-foot  section  of the plot  receiving 15  gpm.  This
area received  runoff from  the first  50 feet  of  the  15  gpm plot which
was about 6 gpm.  The maximum yield was  4.06 T/A for the 50 to  100-foot
section of the 60 gpm plot.   The  first  50  feet  of  the  30 gpm plot  had  a
yield of  3.27  T/A.  The amount  of  infiltration  is  an important  factor
in determining grass yield.   The  first  50  feet  of  the  60 gpm plot  had  a
lower yield because approximately  15 inches  of  water entered the plot
during  each run.  The first  50  feet  of  the 60  gpm plot and  the  first  50
feet of the 30 gpm  plot each  had  12  inches of water entering the soil
and produced  the  largest grass  yields.

                                   253

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Table 4.  Grass yield for two harvests from 50-foot plot
          sections receiving effluent at three flow rates.
Flow
Rate
gpm
15
15
30
30
60
60
Distance
Ft.
0-50
50-100
0-50
50-100
0-50
50-100
Yield - Tons
Harvest
I
1.60
1.33
2.03
1.72
1.20
2.92
per acre
II
1.09
0.90
1.24
0.96
1.46
1.14
dry matter
Total
2.69
2.23
3.27
2.68
2.66
4.06
                             264

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APPLICATION FREQUENCY STUDY

Results of the application frequency study were analyzed by comparing
nutrient reduction and  flow rate relationships.  First, mean nitrate-N
and PO^-P concentrations were compared  for each of the 3 run sequences.
Second, the relationships between  the rate of  inflow and outflow were
examined for each run sequence.  Both nutrient reduction and  flow rate
relationships were examined  for effects  of application frequency.

First Run Sequence

Effluent was applied at 100 gpm to all  plots during the first run se-
quence.  Nitrate-N and PO^-P reduction  apparently had some dependence
on application frequency.  Comparison of mean  nutrient concentration
for inflow and outflow  indicated that greater  overall nutrient reduction
was achieved on the plot receiving effluent  2-times per week.  An 8.5
percent reduction in PO^-P was observed  on  the 2-times per week plot
while a 1.6 percent reduction occurred  on the  6-times per week plot.
The lowest PC^-P and nitrate-N reduction was observed on the plot re-
ceiving effluent 6-times per week.  Tables  5 and  6 summarize  the results
of the first run sequence.

Inflow - outflow effluent flow rate relationships for each plot were
similar during the first run sequence.   Outflow  rate was approximately
one-third of the inflow rate once  the entire plot had been wetted and a
steady rate of infiltration was approached.  This condition was achiev-
ed for most runs approximately 45  minutes after  inflow had started.
Thus,  two-thirds of the effluent applied was lost through infiltration.

Second Run Sequence - Continuous Flow Study

Results of data collected during the continuous  flow run indicate that
somewhat greater reductions  in nutrient concentration were achieved.
Comparison of mean nutrient  concentrations  of  inflow and outflow show
a 10.5 percent and 4.9  percent reduction for nitrate-N and PC^-P, re-
spectively.  Variation  of nutrient removal  with  time was minimal during
the continuous flow run.  Table  7  summarizes  data from  the continuous
flow run.

Inflow during  the run varied between 40 and 50 gpm  due  to  scheduled
irrigation valve changes  elsewhere in the effluent  distribution system.
Outflow during the entire  70-hour  run was approximately  one-third of
inflow which is an indication  of  the relatively  pervious soil with  an
infiltration capacity of  approximately  1 inch  per hour.

Third  Run Sequence

 Inflow was approximately  50  gpm  during  the  third or late summer run
 sequence.  Application  frequency  effects for nitrate were  similar to
 those  observed during  the  first  run sequence and are  shown  in Tables 8
 and 9.  The results  for phosphate  were  different.  There was  an increase

                                   265

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Table 5.  Effect of effluent application frequency on mean nitrate
          reduction during the early summer run sequence.

Samples in mean
Inflow (mg/1)
Outflow (mg/1)
% Reduction
Frequency
2
24
11.0
10.8
1.8
of Application
3
25
11.8
11.6
1.7
(per week)
6
35
10.1
10.1
0
Table  6.   Effect  of  effluent application  frequency on mean phosphate
           reduction  during the  early summer run sequence.
Frequency of Application

Samples in mean
Inflow (mg/1)
Outflow (mg/1)
Z Reduction
2
21
5.11
4.93
8.5
3
23
5.03
4.93
2.0
(per week)
6
35
5.05
4.97
1.6
                                   266

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Table 7.  Mean nutrient reduction during the 70-hour mid-summer
          continuous flow run.
                                         Nutrient
                             N03-N                  P04-P
Samples in mean              10                     10

Inflow (mg/1)                13.3                    4.67

Outflow (mg/1)               11.9                    4.44

% Reduction                  10.5                    4.9
                                   267

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Table 8.  Effect of effluent application frequency on mean nitrate
          reduction during the late summer run sequence.
Frequency of Application

Samples in Mean
Inflow (mg/1)
Outflow (mg/1)
% Reduction
2
34
11.3
11.0
2.7
3
45
12.2
12.1
0.8
(per week)
6
74
12.1
12.1
0
 Table  9.   Effect  of  effluent application  frequency on mean phosphate
           reduction  during the  late summer run sequence.
Frequency of Application

Samples in Mean
Inflow (mg/1)
Outflow (mg/1)
% Reduction
2
34
5.27
5.19
1.5
3
45
5.98
6.06
-1.3*
(per week)
6
74
6.22
6.27
-0.8*
* Minus denotes increase
                                  268

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in PO^-P concentration on the plots receiving effluent 3 and 6 times
per week.  This increase may have been caused by washout of soluble
phosphorus from the increasingly dense grass clipping layer at the soil
surface.

Inflow-outflow effluent flow rate relationships were more varied and
possibly frequency dependent during the third run sequence.  Outflow
rate was approximately one-tenth of inflow rate for the plot receiving
effluent 2 times per week, one-quarter of inflow rate for the 3-times
per week plot, and one-third for the 6-times per week plot.  The varia-
tions observed appeared to be the result of antecedent soil moisture
conditions since the 2-times per week plot responded with an outflow
rate nearly one-third of the inflow rate when run immediately following
an all-night rain.

LABORATORY TESTS

During the first two days of the experiment, there were only slight
decreases in the nitrate content of the effluent on the dead sod.  After
the second day, the daily results were relatively consistent.  The ni-
trate concentration of each wastewater sample was converted to a ratio
of nitrate concentration of the sample to the nitrate concentration of
the effluent applied that day.  The initial nitrate concentration ranged
from 12 to 14 mg/1 nitrate-N.  A set of curves for the data for the
third through ninth days of the experiment was obtained using an expo-
nential relationship and regression analysis (Figure 1).

The average reductions in nitrate concentration after 8 hours of incu-
bation were 95 and 72 percent for the growing sod and the dead sod,
respectively.  The difference in these values can probably be accounted
for by plant removal of nitrate.  The effluent incubated on the bare
subsoil increased in nitrate content during each day.  This result in-
dicates that there was not enough organic carbon in the effluent to
support denitrification.

The results of the laboratory experiment help explain the results of
the field experiments.  In the field, the flow-through time for the 150-
foot long plots was approximately 30 minutes.  Under laboratory condi-
tions, a 22 percent reduction in nitrate concentration was obtained on
the growing sod in this time.  The maximum reduction in nitrate content
obtained in the field was approximately 10 percent during the continuous
flow test.  The difference between this value and the laboratory value
for the same detention time is probably due to the fact that the labor-
atory test was for a batch system and the field tests were for a con-
tinuous flow system subject to temperature variations.  If under the
best field conditions, the removal rate is about one-half that obtained
in the laboratory, detention times of about 12 hours would be required
in the field to reduce the nitrate concentration from 12 to 1 mg/1
N03-N.  Additional laboratory tests are being conducted to determine
the effects of initial nitrate concentration of the effluent and tem-
perature on nitrate removal.

                                   269

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                                            BARE
                                            SUBSOIL
                                            GROWING
                                              SOD
                3456

                 TIME,  HOURS
8
Figure 1.  Nitrate removal from wastewater during
          incubation on three laboratory treatments.
                       270

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                              CONCLUSIONS

No significant reduction in phosphate or nitrate concentrations was
observed in secondary treated municipal sewage effluent after grass
filtration with the flow rates, plot slopes, and plot lengths used in
this study.  The results of the tests will be useful, however, in plan-
ning future experiments in which the application procedure and the plots
will be modified to improve nutrient removal.

The results of the field studies indicate that the removal of both
phosphate and nitrate increased as the application frequency was de-
creased from 6 times to 2 times per week.  The duration of application
was increased as the frequency was reduced so that approximately the
same depth of effluent was applied to each plot.  Comparison of samples
taken during each application period indicated only slight differences
in both nitrate and phosphate renovation during a run.  These results
suggest that the grass filtration system should be operated with runs
of long duration separated by several days of rest.  The field tests
also indicated that leaving grass clippings on the plot surface may
result in increased phosphate concentrations as phosphate is leached
from the decomposing grass into the wastewater.

The greatest problem to be overcome in designing a grass filtration sys-
tem to remove relatively high nitrate concentrations from municipal
sewage effluent is to obtain sufficiently long detention times.  In
laboratory tests, 5 to 6 hours were required to achieve a 90 percent
reduction in nitrate concentration from effluent with an initial con-
centration of 12 mg/1 N03~N.  Under field conditions, the detention
times would probably have to be 1 1/2 to 2 times the laboratory values
for a similar reduction in nitrate concentration.  Detention times of
this length could be obtained by restricting the flow rate over the
plots with barriers or by recycling the effluent.
                                   277

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                           References Cited

1.  Reed, S. C., et al, "Wastewater Management by Disposal on the Land",
    Special Report 171, Corps of Engineers, U. S. Army, Cold Regions
    Research and Engineering Laboratory, Hanover, New Hampshire, 1972,
    183 p.

2.  Thomas, R. E. and C. C. Harlan, Jr., "Experiences with Land Spread-
    ing of Municipal Effluents", in Proceedings of Conference on Land
    Disposal of Municipal Effluents and Sludges, EPA-902/9-73-001, pp.
    212-225, March 1973.

3.  Stevens, R. M., et al, Green Lands and Clean Streams, A Report for
    the Study of Federalism on The Beneficial Use of Waste Water through
    Land Treatment, Temple University, 1972, 330 p.

A.  Mather, J. R., Editor, "An Evaluation of Cannery Waste Disposal by
    Overland Flow Spray Irrigation", Publications in Climatology, Vol.
    XXII, No. 2, C. W. Thornthwaite Associates, Elmer, New Jersey, 1969,
    73 p.

5.  Thomas, R. E. and C. C. Harlin, Jr., "Experiences with Land Spread-
    ing of  Effluents", pp. 212-225.

6.  Thomas, R. E., "Spray-Runoff To Treat Raw Domestic Wastewater",
    presented at The International Conference on Land for Waste Manage-
    ment, Ottawa, Canada,  October 1973, 11 p.

7.  Wilson, L. G. and Lehman, G. S., "Grass Filtration of Sewage Efflu-
    ent for Quality Improvement Prior  to Artificial Recharge", presented
    at The  1966 Winter Meeting, American Society of Agricultural Engi-
    neers,  Chicago, Illinois, 1966.

8.  Sopper, W. E., and L.  T. Kardos, "Vegetation Responses  to  Irrigation
    with  Treated Municipal Wastewater", Recycling  Treated Municipal
    Wastewater and Sludge  through Forest and  Cropland, The  Pennsylvania
    State University Press,  1973, pp.  271-294.

9.  Law,  J. P., R. E.  Thomas, and L. H. Myers,  "Nutrient Removal  from
    Cannery Wastewater by  Spray Irrigation of Grasses", U.  S.  Dept.  of
    Interior, FWQA Water Pollution  Research Series,  16080,  73  p.,  cited
    by Reed,  S.  C., et al, Wastewater  Management by  Disposal on  the
    Land, Cold  Regions  Research and Engineering Laboratory,  Hanover,
    New  Hampshire, 1972, pg. 86.
                                   272

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             USE OF CATTLE  FEEDLOT  RUNOFF  IN  CROP PRODUCTION l

                                    by

              G.  W.  Wallingford,  L.  S.  Murphy,  W.  L.  Powers,
                      H.  L.  Manges,  and L.  A.  Schmid 2
                                 ABSTRACT


 Land  disposal of  beef-feed lot-lagoon  (run-off)  water was studied.
 Lagoon water was  applied during  the summers of  1970, 1971,  1972 and
 1973  by  furrow  irrigation to a si Ity  clay  loam  soil.  After four
 years the  five  treatments averaged 0, 7,  13, 22 and 37 cm/yr.  Corn
 (Zea  mays  L.) forage yield and plant  content of N, P, K, Ca, Mg, and
 Na were  measured.  Surface soil  samples and soil cores were taken
 from  the plots  after harvest each year.

 Electrical conductivity ranged from 1.6 to 7.6  (3.1 average) mmho/cm
 in the lagoon water applied at the study site and from 1.0 to 12.8
 mmho/cm  in samples taken from 12 Kansas feedlots.  Electrical conduc-
 tivities of extracts from saturated pastes of the surface soil samples
 were  increased  linearly by accumulative treatment all years.  The 1970,
 1971 and 1972 soil cores showed accumulations of NOa-N, P, K, and Na
 in the top 30 cm at all treatment rates.  Movement of NO^-N and Na
 down to  100 cm  was noted in 1971 in cores from  plots receiving 43 cm/yr.
Movement of NOs-N down to 240 cm was  recorded in 1972 in cores from
 plots that had  received 20 and 41 cm/yr.  Extractabie Ca and Mg in
the soil  cores  was not affected by treatment.    Corn forage yields were
a linear function of treatment in 1970 and a quadratic function in
 1971, 1972 and  1973.   The positive effect on yield was attributed to
 increased soil   fertility;  the relative decreases at the higher rates
were attributed to increased soil salinity.  Maximum yield and uptake
of N and  P were reached at the 13 cm/yr disposal rate in 1971 and 1972,
and at the 22 cm/yr rate in 1973.
Key Words:  Feed lot waste, feed lot lagoon, waste-water,
            forage production, irrigation, soil  salinity.
  Contribution No. 1427,  Department of Agronomy, and Contribution
  No.  200, Department of  Agricultural  Engineering, Kansas Agricultural
  Experiment Station, Manhattan, Kansas  66506

  Research Assistant, Agronomist, Associate Agronomist,  Assistant
  Agricultural Engineer,  and Associate Civil  Engineer,  Kansas State
  University,  Manhattan,  Kansas.  The  work reported here was partially
  supported by the Environmental Protection Agency, Water Quality
  Office,  under Grant Nos.  13040 DAT and S800923.

                                  273

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                             INTRODUCTION
The increasing size of beef cattle feed lots has been paralleled by an
increase in the magnitude and incidence of their waste disposal
problems.  The Kansas State Department of Health requires that runoff
detention  lagoons hold three inches of surface runoff from feeding
areas and that these  lagoons be emptied as soon as practical  to main-
tain retention capacity (3).  The most common method of disposal  has
been to apply  lagoon water to adjacent crop and pasture land.  High
transportation costs  dictate that maximum rates be applied to these
fields close to the source.

Care must  be taken so that the soil used as the disposal medium does
not become polluted.  Characterization of beef-feedlot runoff has
shown  it to vary widely in chemical composition, and generally to be
high  in total  salt content  (1,2,5,6,7,8,9).  After runoff arrives at
a  lagoon,  evaporation can further  increase salt concentrations.  When
such  lagoon water  is  applied to a  soil, some of the salts can be used
as plant nutrients to increase productivity but excess salts can
create soil salinity  and dispersion problems.

Only  limited  data  are available on effects of beef-feed lot-lagoon
water on soil  properties and plant growth.  Travis, et al. (10),
found  total salts  increased by 200$ and  infiltration declined to
zero  in  soil  columns  from four Kansas  soils after  inundation with
 lagoon water  with  an  electrical conductivity  (EC) of 13.4 mmhos/cm.
They  suggested that the higher proportion of monovalent cations Na  ,
K+ and NHt in such soil could have dispersed colloids and  led  to the
cessation  of  infiltration.

Satterwhite and Gilbertson  (8) found  in  a one-year  field study that
sprinkler  applications of  up to  30.5  cm  of  lagoon water  increased
soil  N,  P,  K,  S, and  Cl.  They compared  (in a greenhouse) the  effects
of tap water,  water taken  from a  lagoon  in  1969, and water from the
same  lagoon  in 1970 on plant growth.   Both  lagoon waters  increased
total  salts  in the soil more than  did tap water.  Growth of  nine
grass  species was  enhanced  by tap  water  and by  the  1970  lagoon water,
but was  inhibited  by  the  1969  lagoon  water.

We evaluated  how growth and composition  of  corn (Zea mays  L.)  forage
 and chemical  properties of  a Kansas  soil  were affected  by  furrow
 irrigation with beef-feed lot-lagoon  water.


                        MATERIALS  AND METHODS
 The study area was located 6 miles north of Pratt,  Kansas,  on a siIty
 clay loam soil with a cation exchange capacity of 19 meq/100g and a
 pH of 7.0.  Lagoon water was obtained from a nearby commercial  feed lot

                                   274

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and was applied  by  furrow  irrigation  in  increments of 5 and  10 cm
during the summers  of  1970,  1971,  1972 and  1973 to plots 9 m wide and
61 to 111 m  long.   Five treatments were  replicated four times'.   In  1970
and 1973 the  last  10 cm increment of  lagoon water was not applied to
the four plots receiving heaviest applications until after harvest.
Inflow and outflow  was measured on each  plot.  The average amounts
applied after four  years were calculated to be 0, 7, 13, 22 and 37 cm
per year for the four  replications.  The two replications that were
sampled for soil core  analysis had received an average of 0, 8, 17,
26 and 45 cm per year  by the fall of  1970, 0, 8, 15, 23 and 43 cm per
year by the fall of 1971, and 0, 7,  14,  20 and 41 cm per year by the
fall of 1972.  Samples of the lagoon water were collected during each
application and analyzed for Ca, Mg, Na, and K by standard atomic
absorption and flame photometric_procedures, for ammonium-nitrogen
(NI-U-N) and nitrate-nitrogen (N03-IM)  by  steam distillation procedures
(4), and for soluble salts by resistance measurements using a Wheat-
stone bridge.

To compare composition of the material being applied at the study site
with that found at  other feedlots, samples were taken from 12 locations
in Kansas during early July of 1970 and  1971, and were analyzed in the
manner described above.  The lagoons were sampled at four depths in
1970:  5, 50, 100, and  200 cm.  Chemical composition did not vary with
depth in 1970, so a single sample was taken at 15 cm from each lagoon
in 1971.

All plots received  a preplant irrigation in the spring of 1971 and
1972.   Additional well water was applied during the growing seasons
to the control plots and to plots receiving the lower lagoon-water
treatments so all plots received approximately equal volumes of liquid.
No chemical fertilizers were applied.

Corn silage yields  (mechanical  harvest) were recorded,  and samples
from each plot were analyzed for Ca, Mg, Na, and K by atomic absorp-
tion and flame photometry after a nitric-perchloric acid digestion.
Nitrogen and P were determined by steam distillation and colorimetric
procedures respectively after a sulfuric acid digestion.  The 1972
and 1973 forage was analyzed for nitrate-nitrogen by hot-water extrac-
tion and subsequent analysis by steam distillation.

Composite surface soil samples (0 to 15 cm) were taken  at 18 locations
in each plot after  harvest.  Extracts from a water-saturated paste
were analyzed for soluble salts by resistance measurements.   Cores
were taken after harvest to a depth of 2 m in 1970 and  3 m in 1971
and 1972 from plots in two replications.  The surface meter of each
core was divided into  10 cm increments and analyzed  for extractable
Ca, Mg,  Na, and K by atomic absorption and flame photometry.   For
the 1970 and 1971 cores the extraction procedure included an initial
extraction with methanol  to remove the cations not on exchange sites.
This was followed by extraction with 1.0 N ammonium acetate,  pH 7.0,

                                  275

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to remove the exchangeable cations.  The 1972 cores were extracted with
ammonium acetate only.  Except for Na, only insignificant concentra-
tions were found in the methanol extract.  The total cations extracted
by these two methods are referred to as extractable Ca, Mg, Na, or K.
The surface meter  increments also were analyzed for Bray P-1 extract-
able P.  The  lower depths were divided_into 20 cm  increments and, along
with the upper depths, analyzed for N03-N by steam distillation tech-
niques  (4).  The 1972 cores were analyzed for soluble salts to a depth
of one  meter by resistance measurements of extracts from water-satur-
ated pastes.


                        RESULTS AND DISCUSSION
Lagoon Water  Analyses,

Results  of  analyses  of  the  lagoon water applied are given  in Table 1.
Total salt  concentration,  indicated by the electrical conductivity^
was  high.   Concentrations of  the monovalent cations NH4, K  , and Na
were considered to be high.   The high and  low values of all measure-
ments show  how the composition  of the  lagoons of a single  feed lot can
vary.  This variation was  not accounted for  in our interpretation of
soil and plant measurements.

Table 2  includes the analysis of the  lagoon  samples taken  at the 12
Kansas  locations.  Wide variation  in composition of samples among
 lagoons  each  year was similar to variation of  lagoon water  applied.
There was little correlation  between the  1970 and  1971  data from the
same locations.  Lack of uniformity between  lagoons and between years
makes  it essential that analysis of  lagoon waste water  be  known before
disposal  to predict  how the material will  affect soil properties and
plant growth.

Effects  On  Soil Chemical  Properties

Electrical  conductivities  of  saturation extracts from  1970, 1971 and
 1972 surface  soil  samples  were  linearly  related to the  accumulative
cm of  lagoon  water applied  (Fig.  1).  The  EC values of  plots  receiving
an average  of 7 cm/yr were  not  much higher than those of the  control
 plots.   Plots receiving 13, 22, or  37 cm/yr  had EC values  generally
 higher than the control  plots.  Since the  EC measurement  is directly
 related  to  total water soluble  salt content  of  a soil,  the three
 heaviest treatments  provided  more  salts than could be  used by the
 corn plants or leached lower  into  the  profile.  High R2 values for
 all  three years (0.878, 0.875 and  0.914)  indicate  good  correlation
 between  the EC measurement and  accumulative  amounts  of  lagoon water
 applied.  This suggests that  EC measurements could be  used to estimate
 amounts  of  lagoon water that  have  been  applied  to  a  soil.


                                   275

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To  improve the graphical presentation of the soil-core analysis data,
measurements taken  from two plots  receiving the same treatment were
averaged.  Data points were further  reduced by combining data from
adjacent depth increments, so that  in Figs. 2, 3, 4, and 5 values are
plotted for depths  of  10, 30, 50 cm, etc., rather than at depths of
5,  15, 25, 35, 45,  55 cm, etc.

Fig. 2 gives N03-N  concentrations  in the 1970, 1971 and 1972 soil cores,
In  1970, all plots  that received lagoon water had higher NOs-N concen-
trations at the 10  and 30 cm depths than did control plots.  No NOs-N
accumulated deeper  than 30 cm in 1970.  Soil cores taken in 1971 showed
accumulations at  10, 30 and 50 cm depths at all dispersal  rates.  The
23 and 43 cm/yr rates caused particularly  high concentrations at 10
and 30 cm depths.   A N03-N peak was found  at 100 cm under the plots
that received 43 cm/yr.  In 1971 peaks were less well defined at 200
and 240 cm under the plots that had received lagoon water treatments
of  15 and 43 cm/yr, respectively.  By 1972 there was significant
movement of N03-N to the 240 cm depth under plots that had received
20 and 4_1 cm/yr.  Because the control plots received no additional  N,
their NOs-N levels  were lower than what would be expected in an irri- .
gated soil being managed for maximum corn  production.  The two highest
disposal rates were, therefore, the only treatments that caused signi-
ficant accumulations of NOa-N in the soil   profiles.

All disposal rates  caused extractable P to accumulate to a depth of
10 cm in the 1970,  1971 and 1972 soil cores (Fig. 3).  Movement to the
50 cm depth was found under plots receiving 41  cm/yr in 1972.  The
general  lack of P movement indicates that the soil's capacity to
immobilize the added P had not been exceeded except possibly at the
highest application rate.

Analyses of the 1970 cores for extractable Na,  K, Ca, and Mg, and
analyses of 1971 and 1972 cores for extractable Ca and Mg showed no
trends due to treatment.  Extractable K was increased to a depth of
10 cm in the 1971 cores and to 30 cm in the 1972 cores at all  disposal
rates (Fig. 4).  Movement below these depths was probably restricted
by exchange reactions with clay colloids.   Extractable Na accumulated
at all  depths in the 1971  and 1972 cores (Fig.  5).  Deeper movement
of Na than K reflects greater competitiveness of K for cation exchange
sites.   Soluble salts as indicated by the EC measurements of the 1972
soil cores had increased throughout the top meter under plots receiving
14, 20 and 41  cm/yr (Fig.  6).

Effect On Yield, Elemental  Uptake And NO^-N Content Of Corn Forage

Thirty-five cm of lagoon water had been applied to the plots receiving
heaviest treatments by the time the 1970 corn forage was harvested.
There was a general  linear trend in yield due to treatment in 1970
(Fig.  7).   That positive effect on yield in 1970 probably resulted
from increased soil  fertility (nutrients in the lagoon water).

                                  277

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Maximum yields were recorded at the 13 cm/yr rate in 1971 and 1972 and
at the 22 cm/yr rate in 1973.  Yields fell off at higher application
rates, which gave a quadratic relationship between accumulative appli-
cations and yield in 1971, 1972 and 1973  (Fig. 7).  The  initial  in-
crease in yield again can be attributed to improved soil fertility,
while the yield decline at the higher rates likely resulted from salt
buildup in the soil (Fig. 1).  Yield depression was less in 1973
probably because of higher rainfall that year.  The yield decline was
relative; all plots receiving lagoon water yielded more than control
plots.

Uptake of N, P, K, Ca, Mg, and Na was calculated from the yield and
elemental analyses of the corn forage.  Linear and quadratic coeffi-
cients and  intercepts from a regression analysis on uptake as affected
by the accumulative applications of lagoon water are presented in
Table 3.   In  1970 uptake of all elements  increased linearly with
applications as did the 1970 yield.  Uptake in 1971 and  1972 was a
quadratic  function of treatment as was yield.  Maximum uptake of most
elements occurred at about the same application rate that gave the
highest yield (Figs. 7, 8, 9 and 10).

The concentration of nitrate-nitrogen increased linearly with accumu-
lative treatment  in the 1972 and 1973 corn forage (Fig.  11).  Two of
the values measured were high enough to be considered dangerous to
livestock  if  ingested.
                       SUMMARY AND CONCLUSIONS
Feed Iot-1agoon water can contain large amounts of salts, particularly
the monovalent cations.  Considerable variations in composition of
feed lot-1agoon water is to be expected with varying rainfall, runoff,
and evaporation.  Continued applications of feed lot-1agoon water
significantly  increased salt content of soil we studied.  Increases
in the  electrical conductivity of the soil were  linearly related to
the amount of  lagoon water applied.  The heaviest lagoon-water treat-
ments contributed more salts than could be utilized by corn plants or
leached into the  lower portions of the soil profile.  The resultant
accumulations could have produced higher osmotic pressures in the soil
solution.  Nitrate-nitrogen accumulated in the soil  from the 22 and
37 cm/yr  lagoon water applications reflecting the relatively high
nitrogen  content of lagoon water.  Phosphorus also accumulated with
lagoon  water applications but accumulations were mostly restricted to
the surface 20 cm, reflecting lack of movement of P in the soil.
Maximum yields of corn forage occurred at an average application rate
of 13 cm/yr after the second and third year and at 22 cm/yr after the
fourth  year.  At higher rates, yields declined.  Maximum removal rates
of applied nutrients, an important consideration in maintaining viabil-
ity of  soil, were achieved at the same application rates that produced
maximum yields.
                                   278

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Disposal  of feed lot-lagoon water can produce excess accumulations of
salts if  soil  composition is not closely monitored.  Large amounts of
monovalent cations may contribute to undesirable conditions by restrict-
ing water infiltration by dispersion of soil colloids.   Yearly soil
analyses  of disposal areas are recommended.  Results of this investiga-
tion suggest that electrical conductivity measurements  can be used to
estimate  the amount of lagoon water previously applied  and to predict
effects of applications on soil chemical conditions and plant growth.
                           LITERATURE CITED
1.  Clark, R. N., and B. A. Stewart.  1972.  Amounts, Composition, and
    Management of Feedlot Runoff,  Tech. Report No.  12, Texas Agr. Exp.
    Station.

2.  Gilbertson, C. B., T. M. McCalla, J. R. Ellis, 0. E. Cross, and
    W. R. Woods.  1971.  Runoff, solid wastes, and nitrate movement
    on beef feedlots.  J. Water Poll. Control.  Fed. 106:5457.

3.  Kansas State Board of Health Regulations,  1970, Chapter 28,
    Article 18.  Kansas State Department of Health, Environmental
    Health Series, Topeka, Kansas.

4.  Keeney, D. R., and J. M. Bremner.   1966.   Determination and
    isotope-ratio analysis of different forms  of  nitrogen  in soils.
    Soil Sci. Soc. Amer. Proc. 30:583-587.

5.  Manges, H. L., L. A. Schmid, and L. S. Murphy.   1971.  Land
    disposal of cattle feed lot wastes.  Proc.  Inter. Sym.  Livestock
    Wastes.  Ohio State U. p. 62-65.

6.  McCalla, T. M., J. R. Ellis, and C. B. Gilbertson.  Chemical
    studies of solids, runoff, soil profile and groundwater from
    beef cattle feedlots of Mead, Nebraska.  Waste Management
    Research, Proceedings of  1972 Cornell  Agricultural  Waste
    Management Conference,  p. 211-223.  Graphic  Management Corp.,
    Washington, D.C.

7.  Miner, J. R., L.  R. Fina, J. W. Funk,  R.  I. Lipper, and G.  H.
    Larson.   1966.  Stormwater runoff from cattle feedlots.
    Management of Farm Animal Wastes.  Amer. Soc. Agri. Eng.,
    St. Joseph, Michigan.

8.  Satterwhite, M. G., and C. B. Gilbertson.  Grass response to
    applications of beef-cattle  feedlot runoff.   1972.  Waste
    Management Research, Proceedings of 1972 Cornell Agricultural
    Waste Management  Conference,  p. 465-480.  Graphics Management
    Corp., Washington, D.C.

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 9.   Swanson,  N.  P.,  L.  N.  Mielke,  J.  C.  Lorimor,  T.  M.  McCalla,
     and J.  R.  El Its.  Transport of pollutants from sloping  cattle
     feedlots  as  affected by rainfall  intensity,  duration, and
     recurrence.   1971.   Livestock  Waste  Management and  Pollution
     Abatement, Proceedings of the  International  Symposium on
     Livestock Wastes,   p.  51-55.  Amer.  Soc.  of  Agri.  Engr. St.
     Joseph, Michigan.

10.   Travis, D. 0., W.  L. Powers, L.  S. Murphy, and R.  I.  Lipper.
     1971.   Effect of feed lot lagoon water on  some physcial  and
     chemical  properties of soils.   Soil  Sci.  Soc. Amer. Proc.
     35:122-126.
                                  280

-------
No
Co
         Table 1.   Elemental  Composition And Electrical  Conductivity Of Beef-Feed lot-Lagoon Water

                          Applied To Research Plots During 1970, 1971 And 1972.


High
Low
Average
Electrical
Conductivity
nnrnno/cm
7.6
1.6
3.1
Na

660
112
295
K

1840
259
671
Ca

615
83
225
Mg

239
35
87
Nht-N
mg/ i
179
4
73
NO^-N

63
1
13
Total
Sol i ds

18134
1879
4771
COD

6445
593
1868
Volati le
Sol ids
at
7>
44.4
27.1
37.8

-------
 Table 2.  Elemental Composition And Electrical Conductivity Of Beef-Feed lot-Runoff Lagoon
   Samples Taken At 12 Locations In Kansas During July, 1970 and 1971.  The 1970 Data Are
    Averages Of Samples Taken At Four Depths: 5, 50, 100 and 200 cm.  The 1971 Data Are
                           From A Single Sample Taken At 15 cm.
Location
1
2
3
4
5
6
7
8
9
10
11
12
Electrical
Conductivity
1970 1971
mmho/cm
3.1
2.7
3.3
1.0
4.2
6.3
8.6
12.8
7.3
	 *
	 #
— *
3.0
2.7
4.1
4.8
3.9
4.3
12.6
4.2
	 #
10.6
1.4
6.0
Na
1970 1971

142
139
221
73
268
339
653
1690
834
	 *
	 #
— *

122
220
293
329
335
299
1320
403
__ _ *
1460
317
500
K
1970 1971

387
166
330
52
550
826
1100
2030
752
	 #
	 #
	 *

583
325
604
617
729
708
1920
458
	 #
2210
483
750
Ca
1970 1971
ppr
152
101
160
65
141
177
523
403
212
	 *
	 #
	 #

101
100
158
127
143
135
310
136
	 *
108
101
187
Mg
1970 1971

74
61
85
34
97
78
153
189
112
	 *
	 *
— ~*

78
70
111
124
81
89
171
82
	 *
235
64
170
Total 1 norganic
N (NH^-N + NOa-N
1970 1971

195
164
151
16
220
130
366
207
221
	 *
	 *
	 *

145
119
167
161
61
218
543
127
	 #
136
133
143
Lagoon dry at sampling time.

-------
    Table 3.  Corn Forage Yield, Electrical Conductivity Of Soil
 Saturation Extracts (EC), and Uptake By Corn Of  Indicated Elements
In 1970, 1971, 1972 and 1973 As Affected By Accumulative Applications
Of Beef-Feedlot-Lagoon Water (cm).  The Data Are Expressed As Factors
            Of A Regression Equation:  y = ax2 + bx + c.
Year
1970






1971







1972








1973

Measurement (y)
Yield (MT/ha)
N uptake (kg/ha)
P uptake (kg/ha)
K uptake (kg/ha)
Ca uptake (kg/ha)
Mg uptake (kg/ha)
Soi I EC (mmho/cm)
Yield (MT/ha)
N uptake (kg/ha)
P uptake (kg/ha)
K uptake (kg/ha)
Ca uptake (kg/ha)
Mg uptake (kg/ha)
Na uptake (kg/ha)
Soi I EC (mmho/cm)
Yield (MT/ha)
N uptake (kg/ha)
P uptake (kg/ha)
K uptake (kg/ha)
Ca uptake (kg/ha)
Mg uptake (kg/ha)
Na uptake (kg/ha)
Soi I EC (mmho/cm)
Forage Nitrate-
Nitrogen (ppm)
Yield (MT/ha)
Forage Nitrate-
Nitrogen (ppm)
R2
0.316
0.482
0.107
0.197
0.0
0.186
0.878
0.372
0.574
0.290
0.079
0.157
0.188
0.387
0.875
0.269
0.584
0.161
0.465
0.212
0.132
0.333
0.914
0.561
0.327
0.482
a
0.0
0.0
0.0
0.0
	
0.0
0.0
-0.00410*
-0.0221*
-0.00256*
-0.000190
-0.00280
-0.00139
-0.000212*
0.0
-0.00322*
-0.0177*
-0.00179
-0.0183*
-0.00237
-0.00117
	
	
	
-0.00106
	
b
0.498*
2.75*
0.388
1.60*
	
0.0890
0.0166*
0.425*
2.63*
0.264*
, 0.367
0.301
0.137
0.0261*
0.0191*
0.426*
2.91*
0.270
2.83*
0.350
0.170
0.00415*
0.0200*
5.29*
0.245*
2.26*
c
41.5
98.9
33.7
162
	
19.8
0.495
36.3
85.0
17.2
131
20.9
14.8
0.649
0.470
49.2
96.3
29.3
164
28.9
21.8
0.558
0.391
-8.86
33.8
75.7
 * Coefficient significant at the 0.05 level.
                               283

-------
NO
s
             0.0-
             3.2H
             2.11-
              1.6-
              0.0-1
                          FflU .19.70
10     20     3'0   .  I'D
 LflGOON WRTER. RCC.  CM

    FflLL  1972
                            £0
                      30     60     90    120    150
                       LRGOON WRTER.  RCC. CM
                                                  FfllL  19.71
22    11     66     88    1 0
 LRGOON WRTER. RCC. CM
           Fig.  1.
The electrical conductivity  (EC)  of saturated paste extracts from surface
(0-15 cm) soil samples  taken  in  the fall  of 1970, 1971 and 1972 as affected
by accumulative applications  of  beef-feedlot-lagoon water.

-------
                                FflLL  1970
                   £ia
Kg
Qa
                                  O—O  CONTROL
                                  «—•  8 CM/TERR
                                  «—«  17 CM/TEflR
                                       26 CM/TEflR
                                       15 CM/TEflR
                     20CH
                     60
                    2UO
                     300
             CONTROL
             7 CM/TERR
             1U CM/TERR
             20 CM/TERR
             Ul CM/TERR
   8     16    2<1     32
   NITR9TE-NITROGEN. PPM
                                                              FfiLL  1971
                             V     8     12    16    20
                                N1THHTE-N. PPM

                                 FflLL  1972
                                                               B—m CONTROL
                                                               «	» 8 CH/TEflR
                                                                   15 CM/TERR
                                                                   23 CM/TERR
                                                                   43 CM/TEflR
                                                                            300
                                                                30    US
                                                             NITRRTE-N.  PPM
                                                                                                      M>
                                                                                  r.
                                                    U !
                Fig.  2.
The  N03-N  content  of soil  cores  taken  in the  fall of  1970,  1971  and 1972 as
affected by depth  and average  yearly application  rate of  beef-feed lot-lagoon
water.

-------
                 FRLL  1970
      MO
    I
    u
    ! .
      80-
             CONTROL
              8 CH/TEflH
             17 CH/TEflR
             26 CM/TERR
             MS CM/TEflR
     100
             "5     I'B    3'7     36
                PHOSPHORUS. PPH

                 FflLL  1972
                                                              FflLL  19.71
                          'i,
                                                                           CONTROL
                                                                           8 CM/YEHR
                                                                           15 CM/TEflR
                                                                           23 CM/TEflR
                                                                           M3 CM/TEflR
22     M4     6'6     88
   PHOSPHORUS. PPM
                                                                                 1  0
      . o
      HII
    1
    •
    a
                      CONTROL
                      7 CM/TERR
                      1M CM/TEBR
                      ?0 CM/TERR
                      MI CM/TERR
     in
             25    50    75     100
                PHOSPHORUS. PPrl
                                    125
Fig.  3.
The  Bray P-1  extractable  P content of  soil cores taken in the fall  of  1970,
1971  and 1972 as affected by depth and  average  yearly  application  rate of
beef-feed lot-lagoon  water.

-------
   20
   'id
   100
              FflLL  19.70
   •ii
   100-1—
    150
Fig.  4,
CONTROL
8 CM/YEftR
17 CM/ TEBR
26 CM/YEflR
US CM/ YEHR
           90    180   270360
         EXTRflCTflBLE POTflSSIUM. PPM

               FHLL  1972
                                                              FflLL  1971
                                                          5
                                                             BO

                        U50
                                                            100
                                                                           B—Q CONTROL
                                                                           »	» 8 CM/TEflR
                                                                               15 CM/TEflR
                                                                               23 CM/TEflB
                                                                               M3 CM/TERR
260uoso
    P3TOSSIUM.  PPM
                                                                            800
                                                                                  1000
                     CONTROL
                     7 CM/TERR
                     14 CM/TERR
                     20 CM/TERR
                     Ml CM/TERR
300    1150    600
    POTRSS1UM.  PPM
                            750
                                  900
Extractable  K content of soil  cores  taken  in the  fall of  1970,  1971 and  1972 as
affected by  depth and average yearly application  rate of  beef-feed lot-lagoon
water.

-------
             FflLL  1970
                    CONTROL
                    8 CM/TEF>R
                    17 CM/TERR
                    26 CM/TEflR
                    
-------
                         FflLL   1972
         20-
         40-
         80-
        100-
C0NTROL
7 CM/TERR
14  CM/TERR
20  CM/TERR
41  CM/TERR
          0.0     0.5     1.0     1.5
                        E,  C,   MMHOS/CM
  2.0
2.5
Fig. 6.  The electrical conductivity of saturated paste extracts from soil  cores taken
       in the fall of 1972 as affected by depth and average yearly application rate
       of beef-feed lot-lagoon water.

-------
.   FflUL  1970
N)
10
o
8     16     24    32
 LftGOON HflTER. flCC. CM


    FflLL  1972
                      30     60     90     120
                       LRGOON WRTER. flCC. CM
                        150
                                                         60
                                                                     FflLL  19.71
                                          22  '   44'   66     88
                                           LflGOCN WflTER. flCC. CM

                                               FflLL  1973
35     70    F5?"    140
 LfiGOON WRTER. flCC.  CM
                         no
                                                                                          175
           Fig.  7.   Corn forage yield (metric tons/hectare,  corrected  to  30/t  dry matter)  in  1970,
                    1971,  1972 and 1973 as affected  by accumulative  applications of  beef-feedlot-

                    lagoon water.

-------
Kj
10
             280-
             250-





            cr200-!
            i

            CD


            iu'150-
            Q_

            ID
             1 DO-
              SO-
                          FflLL  1970
7     14     21     28
 LRGOCM WRTER. RCC.  CM

    FflLL  1972
                     lo   '  60  '   90   '  TaT"
                       LRGOON WflTER. flCC. CM
35
                         150
                                   200
                                                FflLL .19.71 .
22    44     66    88     110
 LRGOON WflTER.  RCC. CM
         Fig. 8.  Uptake of  N  by  1970,  1971  and 1972 corn forage as affected  by  accumulative

                  applications of  beef-feed lot-lagoon water.

-------
                  FflLL  1970
                      FflLL .19.71
              7      1'U     21   '  28
              LRGOON HRTER,  RCC. CM

                  FflLL  1972
35
22    44     66     88     110
                   LRCOON WRTER, RCC.  CM
DLT

0:45-
T:
0
GC
O_ J
0-15-
n.

X
xX
X



              30     60     90
              LRGOON WRTER.  RCC. CM
150
Fig.  9,   Uptake of P by 1970, 1971  and 1972 corn forage as affected by accumulative
         applications of beef-feed lot-lagoon water.

-------
Nl
<0
             300-
              100
             yoo-
                           FflLL  1970
7     14    21     28
 LfiGOON WflTER. flCC.  CM

    FflLL  1972
35
                      30     6'0     9'0    120    150
                       LRGOON WflTER.  flCC. CM
                                   2140-
                                                          180-
                                                        
-------
  1200'
   900-
Q_
O.
UJ
h—

-------
        IRRIGATION OF TREES AND CROPS WITH SEWAGE STABILIZATION

                  POND EFFLUENT IN SOUTHERN MICHIGAN

                                  by

       Jeffrey C. Sutherland9, John H. Cooleyb, Daniel  G.  Nearyc

                           and Dean H. Urie
INTRODUCTION

The "living filter" concept of sewage wastewater treatment, docu-
mented extensively at The Pennsylvania State University',  is being
tested in Michigan with systems designed both for research and prac-
tical  solutions to immediate needs.  Twenty to twenty-five rural
projects serving populations of under 1,000 to over 10,000 are now in
stages from preliminary to "in use".  All of them involve initial
treatment through the secondary level in facultative Qonds, with or
without pretreatment in anerobic or aeration ponds '  .

Soil and vegetation renovation of wastewater in Michigan is a natural
extension of the wide use of outdoor ponds for sewage treatment  in
rural  communities which began around I960.  Under orders to reduce
phosphorus  in Great Lakes tributaries (beginning in 1968), many
Michigan communities are choosing  irrigation over in-plant treatment
for reasons of economy.

The advantages of  irrigation over other  advanced treatment methods
are well known:  assimilation of dissolved carbon, phosphorus,
nitrogen, micronutrients and filterables usually can be assured  in
a  single treatment step.  No residues need be trucked away for dis-
posal.   Increased  farms yields are  realized, especially in drouthy
areas, and  new "ground water"  is occasionally a valuable water
supply asset.
aHead geologist, Williams &  Works,  Inc.,. Grand Rapids, Michigan.
bPrincipal  si Iviculturalist,  North  Central  Forest Experiment Station
  (U.S.D.A.),  Cadillac, Michigan.
cGraduate  research  assistant,  Forestry  Department, Michigan State
  University,  East Lansing, Michigan.
Principal  hydrologist,  North  Central Forest  Experiment Station
  (U.S.D.A.),  Cadillac, Michigan.

  Support of the studies  was  obtained  through  the United States  Forest
  Service  (U.S.D.A.),  The Michigan  State University  Institute of
  Water  Research, and  Williams  &  Works,  Inc.
                                   295

-------
irrigation of woodlands is much less common than irrigation of farm
crops.  Yet woodlands, in addition to being living filter systems,
have potentially great advantages: the Great Lakes region has a rela-
tively short growing season (5-1/2 months).  But year around irrigation
of woodlands has been successful'.  Public forest land often can be
acquired  inexpensively, or free, while costs for farm land reasonably
close to  service areas may be prohibitive.  The expenses of annual
planting  and frequent mowing would be avoided  in areas where there is
no established agriculture to benefit from crop yields.   Land too
rough to  farm or develop  is often  left in timber.  Such  land could be
used economically for irrigation.

Isolation of residences from irrigation spray  and, occasionally, from
visual contact with treatment sites,  has  been  necessary  in Michigan.
Woodlands can provide such isolation  with a border zone tens of acres
smaller than would be needed in open  farm areas.  Irrigation of mature
stands can  improve growth of understory vegetation, and thereby in-
crease food  and  shelter for ground birds, rabbits and deer4.  Woodlands
on  drouthy  soils can absorb relatively high quantities of water with-
out  fear  of  tree damage,  with attending increases in  rate of growth
being of  potential benefit.   In river lowlands and other wet areas,
drainage  improvement as part of wastewater treatment  design can make
or  keep  land suitable for tree  irrigation programs.   Irrigation and
management  of  immature ornamental  and shade trees to  be transplanted
to  park,  walkway and mall areas could be  a practical  adjunct of renew-
al  and beaut ificat ion programs.

Since all economic  indicators  suggest demand  for wood products will
grow faster  than supply,  increased markets for timber crops seem
assured.

Studies with young tree  stock  were started  in  lower Michigan to  learn
how various  species respond to  irrigation on  glacial  soils common to
the  Lake  States  region, and whether  there are  important  differences
among species as to nutrient  uptake.   Experimental  irrigation of es-
tablished tree stands was begun  to study  growth  response,  water quality
improvement  and  the effects upon  existing ground water chemistry.
Studies at  Middleville and Belding are the first  in Michigan  in which
sewage wastewater  is  being applied to trees  in nursery and  plantation
areas.  The  studies  in progress  at Belding involve  irrigation of orna-
mental and  shade trees.   Data  from one season  of  irrigation are avail-
able from Belding,  but one or more years  of  further  study  are  needed
 for meaningful  interpretations.   In  the  Spring of  1974,  irrigation  of
Christmas trees, pulpwood species and northern hardwood  trees  with
 pond stabilized  wastewater will  be started at Harbor  Springs  in the
 Little Traverse  Bay area.

The availability of  irrigation  facilities with adjacent  woodlands  and
 areas that  could be  planted  in  trees determined the  location  of  initial
experiments.  Such a  facility exists at  Middleville,  Michigan,  about
 32 kilometers  (20  miles)  southeast of Grand  Rapids.   The present  paper

                                    296

-------
discusses the results of two years of research at Middleville.

A sketch of the treatment area is shown in Figure 1.  The Middleville
system (2,200 population) consists of two 4.4 ha facultative ponds
and II ha of farm  land (corn) receiving chlorinated effluent.  Two
experimental tree  irrigation areas near the treatment site are receiv-
ing effluent from the south pond.  The Middleville treatment system
was designed as an irrigation agricultural program.

NUTRIENT QUALITY OF STABILIZATION POND EFFLUENT

The average nutrient concentrations irrigated are listed in Table I.
Total  phosphorus (total P) is less than 4 mg/I.   Total nitrogen (total
N) is a little greater than 7 mg/l of which approximately 4.5 mg/l is
organic nitrogen (Org-N).  The qualities changed between 1972 and 1973.
The nitrate nitrogen (NO-j-N): ammonia nitrogen (NH3-N) ratio of 0.2 in
1972 rose to 2.9 in 1973 and total P fell  by 40 percent in the same
period.  In 1972 the south pond received raw sewage directly.  In 1973
the south pond received partially stabilized water from the north pond
and no raw sewage.  This operation change accounts for the higher
NOj-N:NH3-N ratio.  The reduction in total P is not easily explained.
Retention of phosphorus in sedimented organisms is a speculative pos-
sibility.

CORN

Corn (hybrid no. 4444) is planted and harvested by a local  breeder
turkey farmer on 8.9 ha of the Middleville irrigation site.  In making
turkey feed, the corn is dried and mixed with soybeans and other grains.
The corn field is mostly in Plainfield sand (Figure I) which is natu-
rally drouthy, infertile and easily moved by wind.  Prior to the  1973
season, nitrogen rich fertilizer was added at II3 kg/ha rate.  Irriga-
tion in May through July was intermittent, but in August and September
a 24-hour schedule was kept, which resulted in average applications
of 68 mm/week.  Corn rows are spaced at 97 cm (38 in.) and individual
plants grow closely in a given row.

The results of irrigation were satisfying to the grower.  Corn reached
heights of 2 m and higher within the wetted perimeter; beyond the
spray limits no corn exceeded 0.5 m in height.  The yield on 8.9 ha
(22 acres) was 221 bu/ha (88.5 bu/acre) of dry shelled corn, or roughly
500 bu/ha (200 bu/acre) of ears.

Prior to the 1974 season, nitrogen fertilizer in twice the previous
amount will  be app i ied to the f ields.  Limi ng will be done in the wi n-
ter.   The corn stover was plowed-in (October) in order to help build
the soiI.

The estimated value of the 1973 corn crop is $5,000, of which \5% to
2Q% is consumed in planting, harvesting, trucking and drying.  Were

                                   297

-------
                 TYPICAL
               QUARTER CIRCLE
                  NURSERY
                   PLOT
                                          LEGEND

                                       GENERALIZED SOILS

                                       Bellefontaine Sandy Loom
                                       Fox Sandy Loam
                                       Ploinfield Sand
                                       * or Boyor Sandy  Loom
    -~l20m
\
          STABILIZATION
              POND /
               4.4 ha
               STABILIZATION
                  POND
                  4./4ho
  \
     \
              =f=
               1
       AGRICULTURAL
         IRRIGATION
           AREA   \
            II ha
 /THC
THORNAPPLE^
RIVER 500m
                PS
\ Bs
   \

  J
                                                 Middle ville
    RED  PINE
    IRRIGATION
      AREAS
 o°o    o
    o °
        o
°°o£i
               Figure  I.   Sewage Treatment Site
               and Experimental  Tree Study Areas,
                   MiddlevFIle,  Michigan
                             298

-------
                            Table  I
           Irrigation and Water Quality Data by Year,
               20-Year Old Red Pine Plantation,
                     Middlevi Ile, Michigan
           Parameter
1972
973
Total
Waste
Water
Appl led
at ...
25 mm/wk
50 mm/wk
88 mm/wk
Average NH-j-N
Concentra- TKN
tion NO,-N
of Effluent Total N
(mg/l) Total P
Average
Load ing
Rate
(kg/ha)
1 rrigation
Season
25 mm
N 50 mm
88 mm
25 mm
P 50 mm
88 mm
Began
Duration
420 mm
820 mm
1350 mm
2.1
6.9
0.4
7.3
3.9
30.7
59.9
98.5
16.4
32.0
52.6
July 7
15 weeks
460 mm
920 mm
1610 mm
0.7
5. 1
2.0
7.1
2.3
29.8
65.3
1 14.3
10.6
21.2
37.0
May 24
*I8 weej<^
•cept  for two  weeks shutdown  in July  for  repairs  due to
     ing damage.
                               299

-------
the community to manage such an operation, the net value of the crop
could pay for operation and maintenance of the ponds and irrigation
system.

TWENTY YEAR OLD RED PINE PLANTATION

A stand of red pine trees  (Pinus resinosa) established in very well
drained sandy soils was irrigated during the  1972 and 1973 growing
seasons.  Red pine is the most common plantation species in Michigan.

Information on red pine response to wastewater irrigation is available
from The Pennsylvania State University wastewater disposal studies5.
Following ten years of irrigation with secondary level effluent, red
pine receiving 2.5 cm per week exhibited  increased height and diameter
growth over those of unirrigated trees.  Trees receiving 5.0 cm per
week showed reduced growth.  The growth reduction occurring at the
higher  irrigation rate is  believed due to the combination of boron
toxicity and excessive soil moisture.  The red pine at Penn State grows
on  loam soil of the Hublersburg-Hagerstown complex.  Most red pine
stands  In Michigan are on  sandy textured  soils of glacial origin having
little or no clay in the sub-soil and consequent low moisture holding
capacity.

Study Site

The Middleville trees grow on  irregular  land  developed  in gravelly mo-
raine.  Soil is a typic hapludalf of the  Boyer  (Bellefontaine) series,
which is light colored, well drained sand and  loamy sand overlying cal-
careous sand and gravel.   Prior to red pine establishment, the  land
had been farmed and then abandoned.

The plantation has an average  spacing of  2.4  m.  The  average basal area
is  7.5  rrrVha and the average tree height  is  11.6 m.   The average DBH
(diameter at breast height)  is 18.1 cm.

Procedures

Twelve  circular areas of  7.5 m radius were  selected for  irrigation.
AlI trees were marked, pruned  to a height of  2.4 m  and measured for DBH.

The following four treatments  were randomly assigned  to  three areas
each:  (1) control - no  irrigation;  (2) 25 mm  of effluent per week;  (3)
51 mm of effluent per week;  (4) 88 mm of  effluent per week.

Water was applied during one 8-hour period  each week  by  a  single  rotary
aerial  sprinkler  installed  at  the center  of each area.   The amount of
water applied varied predictably with  distance  from the  sprinkler.   The
4.9 m radius was  selected  as the point  for  soil water quality  studies
since design rates of application were most nearly  met  here.
                                    300

-------
Samples of sewage pond effluent were withdrawn from a tap on  the  irri-
gation line during spray periods.   Soil  water samples were removed  from
60 cm and 120 cm depths using 51  mm diameter suction lysimeters.   Vac-
uum was drawn on the samplers prior to irrigation and they were allowed
to fill during the ensuing 7 day period.

All samples were preserved with IN HgCI  (I mg/100 ml).   Analyses  (total
Kjeldahl nitrogen — TKN, NH3~N,  N03~N and total P) were conducted  by
laboratories of the Institute of Water Research and Department of For-
estry at Michigan State University, using standard EPA methods^.

At the end of the growing seasons, needles from the second or third
whorls from the  leader were sampled from four trees in each area  for
nutrient analysis.  Average needle length and dry weight per fascicle
were determined  using 100 needle groups collected from the base of  the
current season's shoot.  The terminal bud of each branch used for nee-
dle samples was  also measured.  The DBH of each tree was recorded.
Tree heights were determined  in 1973 for both years using five repre-
sentative trees  per irrigation area.

Observations

The availability of percolating water at the 60 and  120 cm depths of
sampling was erratic.  However, sampling was usually successful at 60
cm under all three  irrigation  rates.  The samplers  placed  in control
plots captured so  little water that only occasional analyses could be
made.

The average total N and total  P concentrations were calculated from
mean monthly soil water data.  Total monthly phosphorus values averaged
0.04 mg/l and  less.  Total N values were  less than  I mg/l, of which
NO,-N was 25 to  40  percent  (Table  2).  Higher total  N concentrations in
the  1973 percolate  than  in  1972 relate to significantly  heavier  rain-
fall,  hence dilution, during the  1972 season.

The extent of  nutrient  renovation  of wastewater  was  determined by
weighting the  concentration values by the volume of  precipitation and
Irrigation  in  excess of the cumulative potential evapotransplration  .
Nitrogen removal through the  60 cm depth was 83  to  92 percent, and
phosphorus uptake was  in excess of 96 percent  (Table 3).  The  renova-
tion  figures show  increased percentages of applied  nitrogen  passing
through the soils at  heavier  rates of irrigation.   One  probable  reason
for this  is that heavier applications promote more  rapid  percolation,
allowing  less  time  for  removal reactions.   Levels of NH3-N  in  soil
water  were essentially  unchanged  due to  irrigation  treatment.  Nitrate-
N  concentrations were  significantly  higher  under the higher  rates of
irrigation.  Total  N  concentrations  were  also  significantly  higher
under  heavier  irrigation,  largely due to  the  higher N03-N.   Organic
forms  of  nitrogen  accounted  for approximately  half  of the  total  N.
Possibly the growth of  algae  in the  suction  soil  samplers  converted

                                   307

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                             Table 2
             Concentrations of N and P in soil water
                   at 60 cm depth (mg/l), 1973
                                 Nutrient Parameter!/
Irri
Rate



gat ion
(mm/ week)
25
51
88
NH3-N
0. 10 a
0.20 a
0. 14 a
TKN
0.36 a
0.64 a
0.55 a
N03-N
0. 1 1 a
0.34 a
0.39 a
Total N
0.47 a
0.98 a
0.94 a
Total
0.02
0.04
0.03
1 P
a
a
a
      not followed by the same letter are significantly different
at the 2.5$ level (paired student t test).
                               302

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                Table 3a.
Renovation of N and P in effluent (1972)

Parameters

Ground
Water
Recharge
(mm)
Total
Nitrogen
(kg/ha)
Total
Phosphorus
(kg/ha)


Rate

25 mm
51 mm
88 mm

25 mm
51 mm
88 mm
25 mm
5! mm
88 mm


Jul.

0
0
51

0
0
0.3
0 <
0 <
0. 1

Renovation of
Parameters

Ground
Water
Recharge
(mm)
Total
Nitrogen
(kg/ha)
Tota I
Phosphorus
(kg/ha)
Rate

25 mm
5 1 mm
88 mm

25 mm
51 mm
88 mm
25 mm
51 mm
88 mm
Jun.

47
74
1 12

0.5
1 .6
1.6
O.I
O.I
<0. 1

Aug.

145
272
460

0.6
2.6
2.3
CO. 1
:o. i
0. 1
Tab
N and
Jul .

0
10
86

0
O.I
1.3
0
<0. 1
<0. 1

Sept.

79
202
356

0.2
0.8
1.8
<0.l

-------
NO-T-N to organic forms during the 7-day collection period.

The few successful field control samples (data not given) contained
total P similar to the irrigation plot samples, indicating that no
phosphorus above background  levels penetrated to the subsoils.

The mean growth responses of red pine to the different irrigation rates
during the 1972 and  1973 growing seasons are presented in Tables 4 and
5.  Needle lengths in  1972 averaged 144.9 mm, with no significant dif-
ferences due to irrigation rate.  This was most likely the result of
the  late start  in the  irrigation schedule (July 15) since 90 percent
of red pine growth normally occurs in June and early July^.   In 1973
there were significant differences in needle growth which relate di-
rectly to the amount of water applied.  Needle lengths increased 12,
28, and 36 percent over control areas with increasing irrigation levels,
going from 121.0 mm  for the  unirrigated trees to  164.4 for trees receiv-
ing the greatest  irrigation.  The decrease in needle length in the
control area trees from  1972 to  1973  is attributed to low rainfall  in
June,  1973 (2.69 cm),  compared to that of June, 1972 (11.05 cm) which
is close to the 30 year mean rainfall for June of  10 cm.

Terminal bud  lengths exhibited no significant differences in  1972 which
could be related to  irrigation.   In  1973, trees receiving 25 mm had the
smallest terminal buds,  but  this  is unrelated to  the amount of irriga-
tion since no similar  reduction was evident  in the higher irrigation
rates.  Branch terminal  bud  length thus proves not to be a sensitive
biological indicator of  red  pine  response to  irrigation.  Measurement
of the terminal bud  of the main  stem  might be a better indication of
irrigation response, but  it  is  feasible only with  small trees.

The growth trends in dry  weight  per fascicle parallel those of needle
length.  No significant  variations from the overall mean of 71.59 mg
per fascicle were evident  in  1972.  The  1973 data  show that the needle
dry weight increased 8,  38,  and  56 percent over the controls with the
application of 25, 51, and 88 mm of effluent.  The significant differ-
ences occurred between the  low and intermediate application rates.
Increases in both needle  dry weight and  length point to greater photo-
synthetic capability in trees  irrigated at rates  of 51 and 88 mm per
week than those receiving  less  irrigation.

The anticipated result of  increase in total energy fixation would be
additional height and/or diameter growth.  Table  6 lists the mean DBH
increment from 1972 to 1973  as calculated by measuring the parameter
for all trees in each plot.  The average height and DBH data are in-
cluded for reference.  No significant increase in  DBH has yet appeared.
Any growth trends will show  up  in subsequent years as the 1973 needles
assume a larger role in photosynthesis.  Needles  normally remain alive
on trees for three years, contributing the most to photosynthesis in
the second and third years.
                                   304

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                              Table 4
        Mean Red  Pine Growth Parameters for Varying Sewage
      Effluent Irrigation Rates at Middleville, Michigan, 1972
Irrigation Rate
(mm/ week)
0.0
25
51
88
Needle Length
(mm)
142.6 al/
146.0 a
155.0 a
136.2 a
Termi na 1
Bud Length
(mm)
26.6 a
24.0 a
23.7 a
24.2 a
Dry Weight
Per Fascicle
(mg)
73.90 a
69. 17 a
81.12 a
62.18 a
j/Means not followed  by the  same  letter are significantly
  different at the 5%  level  (Tukey's  test).
                               Table 5
         Mean Red  Pine Growth Parameters for Varying Sewage
       Effluent Irrigation Rates at Middleville, Michigan,  1973
Irrigation Rate
(mm/ week)
0.0
25
51
88
Needle Length
(mm)
121.0 al/
136.4 b
154.9 c
164.4 c
	 . 	 	
Terminal
Bud Length
(mm)
28.3 a
24.5 b
27.9 a
30.2 a
Dry We ignt
Per Fascicle
(mg)
68.98 a
74.76 a
95.23 b
107.39 b
  I/ Means not  followed  by  the  same letter are significantly
 —'  •.. ,,  	_i_  _j_  a..	t^cf io,,Qi  fTnWf=\/'c; test).
different at the 5%  level  (Tukey's test)

                             305

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                                Table 6
        1973 Red Pine DBH  Increments and Representative Total DBH
         and Height for the Sewage Effluent  Irrigation Project
                       at Middleville, Michigan
     rrigation Rate        DBH  Increment        DBH        Height
        (mm/wk)                  (mm)             (cm)           (m)
            0.0                3.99 ai/          17.75         I 1.80

            25                 4.I I a            16.41         I I.83

            51                 4.67 a            18.39         12.77

            88                 4.09 a            16.46         12.77
    — ' Means not followed by the same  letter are significantly
       different at the 5%  level (Tukey's test).
The nutrient analysis of the foliage collected  in  1972 indicated only
one significant variation between  irrigated and unirrigated trees.
Only in the case of boron has a distinct  irrigation rate response oc-
curred.  Sopper and Kardos^ found  boron  levels of 23 and 33 micrograms/
gram (ug/g) in foliage from red pine irrigated at rates of 0 and 51 mm
per week, respectively.  They reported no statistical significance to
the observed differences.  Boron  levels  in the foliage from Middleville
specimens were 33, 28, 27 and 22 ug/g for the 88, 25, 51  and 0 mm rates,
respectively.  The boron content of trees receiving 88 mm of effluent
per week is significantly higher than that of the unirrigated trees,
and high enough that occurrence of boron toxicity conditions in the
red pine is a distinct possibility.  Should this condition continue
and toxicity symptoms result, red  pine on well drained soil may have
to be eliminated from consideration as suitable for receiving treated
sewage wastewater, at least at levels approaching 88 mm per week.

CUTTINGS AND SEEDLINGS

Cuttings and seedlings were planted in soi Is of the Boyer series devel-
oped on a moraine of gravelly till composition.  Some of the test plots
are positioned on slopes where erosion has removed much of the fine
material leaving a gravelly sand surface soil with little capacity to
retain moisture.  Others are on areas where the fine material has been
deposited, resulting in a mantle of fine  loamy sand which has a much
greater moisture retention capacity.  Still  others are on a temporary

                                   305

-------
haul road or fill material which has been covered with roughly a foot
of very stony loamy sand.  A compacted substratum severely restricts
percolation rate on these plots.

On the eroded slopes broad leaved weeds such as wild strawberry (Fragaria
vi rginiana) and hawkweed  (Aurantiacum spp.) were major components of
existing vegetation but on the undisturbed flat areas there was a very
luxuriant growth of forage grasses, mainly brome grass, and scattered
low brush, mainly hoptree (Ptelea tri folfata).  Clovers, fescues, and
brome grass seeded on the disturbed soils had become well established
before testing was begun.

Procedures
The tests included two hybrids of populus species, one an aspen type
hybrid (Populus canescens X I? gradi dentata) and one a cottonwood type
(Popu I us del toi des X P. nicjra); two species of  larch, Japanese (Larix
leptolepi s) and European (L. deci dua); a hydrophytic conifer, northern
white cedar (Thuja occidentaI is); and three indigenous hardwood species,
tulip poplar (L_i_r i odendrcm t u I i p i f era) which at this latitude grows
naturally only on fertile, protected sites, northern red oak (Quercus
bo re a Ms) which grows on more exposed slopes and ridges, and green ash
(Fraxinus pennsyIvanica var. Ianceolata) which occurs on a wide variety
of sites but reaches best development on moist bottomlands.  The two
populus hybrids were planted as rooted cuttings and the other species
as seedlings under 45.7 cm (18 inches) tall.

The cottonwood hybrid, green ash and white cedar were planted only on
the loamy sand with compact substratum; the other species were planted
on all  soil conditions described.  The plots were rototiI led thoroughly
before planting.

AI I  except the cottonwood hybrid and red oak were assigned at random to
quarter circle plots of twenty-five trees each  (Figure I).   The white
cedar and green ash were planted in 12 quarter-circle plots; and the
larches, tulip poplar and aspen hybrid were planted in 9 such plots.
The cottonwood hybrid and red oak were planted  in circles 8.8m from plot
centers.  The cottonwood was planted in six circumferential plots, and
the red oak in nine.

After the first year (1972), surviving European larch were moved to
circular rows 1.5 and 2.7 m from plot centers and cuttings from the
cottonwood hybrid were planted in the quarter circle originally occu-
pied by the larch.   Two cuttings were planted where each of the 25
larch had been.  One was cut in February and held in cold storage for
three months and the other was cut just before planting.

At intervals throughout the first growing season weeds were cut and
the soil tilled.  At the beginning of the second season (1973), dichlo-
benil  (2, 6-dichlorobenzonitri le) was applied at the rate of 168 kg/ha

                                  307

-------
to half of the area occupied by each species.  The other half was
mowed often enough to keep weeds and grass from smothering the trees.

Effluent was applied through Rainbird 24A-23°FP-TNT sprinklers.  A
single sprinkler was used for the low rate and two on a single riser
were used for the higher rate.  The amount applied varied widely over
any one plot due to wind drift and from year-to-year due to malfunc-
tions in the distribution system.  During the first year, the medial
total amounts were approximately 420 and 685 mm for the low and high
rates (46.6 and 76.2 mm/week, respectively).  Irrigation did not be-
gin until June 23, was interrupted for three weeks in July because of
low water in the ponds and was terminated on September 7 to allow trees
to harden off before the first frost.  During the second year 330 and
889 mm were applied in total (20.6 and 55.6 mm/week, respectively)
starting on May 17 and ending September 13.  A two-week interruption
in August due to equipment failure coincided with an extended hot, dry
spelI.

Observations

Variations  in weather during the planting period and differences in
effectiveness of weed control had a great deal of influence on survival
and growth during the first growing season.  Also, there was an even
distribution of rainfall through most of the season which probably
minimized the effects of irrigation.  Nevertheless, there were differ-
ences that seemed to be related to irrigation (Table 7).  Height growth
of the aspen hybrid was only slightly better in plots irrigated at the
low  level than in unirrigated plots, but  it was one third better In
plots irrigated at the higher level.  In contrast, the cottonwood hybrid
grew nearly 25 percent better at the lower  level but only slightly
better at the higher level.  Growth of other species was affected lit-
tle, If at all, by irrigation, but survival of Japanese larch, tulip
poplar, white cedar and red oak was  improved.  Survival of European
larch was poorest in plots that received the higher level of irrigation.

Growth during the second season provides a better indication of the
effects of Irrigation because most tree species were well established,
weed control was more uniform and there was an extended hot, dry
period (Table 8).

The rooting and survival of the new cottoqwood hybrid cuttings were
greater under lower level irrigation (15%) than in unirrigated areas
(4050 and under the higher  level (68%} (Table 9).  Possible development
of shallow root systems in the higher level areas followed by aggra-
vated moisture stress during the two weeks with neither rain nor irri-
gation could have caused the higher mortality.  By the end of the
season surviving cuttings in irrigated plots were more than twice as
tall as those in unirrigated plots.  The heavier irrigation increased
height growth little more than the lighter.  Survival and height growth
of stored and fresh cuttings were about the same.

                                   308

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                   Table 7
Survival and Growth of Cuttings and Seedlings
    During the First Growing Season (1972)

Tree
Species



Cottonwood
hybrid
Aspen hybrid
Green ash
European larch
Japanese larch
Tul ip pop lar
White cedar
Red oak

1 rrigati
on Level
None Lower Hi


Survi
(f>
98

100
95
76
68
73
79
86

Height
val Growth
(cm)
50

42
30
12
12
5
5
1


Survi va 1
<*>
100

100
96
80
99
77
87
99

Height
Growth
(cm)
62

45
31
3
9
4
5
0


Survi va
<*>
100

100
100
65
88
83
91
94

qher

Height
1 Growth
(cm)
52

56
27
10
II
4
5
1
                      309

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                           Table 8
        Survival and Growth of Cuttings and Seedlings
           During the Second Growing Season (1973)

Tree
Species
Cotton wood
hybrid \J
Aspen hybrid
Green ash 2j
European larch
Japanese larch
Tu 1 i p pop 1 a r
White cedar \J
Red oak

None
Survival
(*>
100
97
100
54
94
52
86
100
1 rrigat
ion Level
Lower Hi
Height
Growth
(cm)
27
33
21
18
27
5
2
-1
Survival
(*>
100
99
98
76
92
75
94
83
Height
Growth
(cm)
101
60
38
17
38
22
10
-1
Survi va
(*>
100
100
95
79
89
80
86
95

cjher
Height
1 Growth
(cm)
1 12
69
36
28
26
21
II
-2
     \J Difference in height growth between control and
        treated plots is highly significant (\% level)

     2/ Difference in height growth between control and
        treated piots is significant (5% level)
                           Table 9
  Survival  and Growth of Cottonwood Hybrid Cuttings (1973)
Irrigation
Survival _[_/
Average
Height ]J
  (cm)
None

Low I eve I

High level
   40.0

   74.7

   68.0
 33.3

 70.0

 74.9
     \J Differences between control and treated plots are
        significant (5% level)
                             370

-------
Contrary to first season results, irrigation improved survival  of  the
European larch that was replanted at the beginning of the second  sea-
son (Table 8).  The greatest improvement was in plots with compact
substratum.  Ninety percent of the larch on unirrigated plots died
compared with 75 and 32 percent with the lower and higher levels  of
irrigation, respectively.   A similar pattern of mortality occurred !n
tulip poplar.  Ninety-four percent of the unirrigated seedlings in
plots with compact substratum died, compared with 24 percent with  lower
level  irrigation and 20 percent with the higher level.   The only  unir-
rigated Japanese larch seedlings that died the second year had been
planted on the gravelly sand.  No red oak planted in unirrigated  plots
died even though there were some losses in irrigation plots.  A few
green ash died, ail in irrigated plots, and there was slightly heavier
mortality in white cedar but it was not related to irrigation treat-
ments.

During the two week period with neither rain nor irrigation some  mor-
tality occurred in all irrigated plots.  Most of the mortality among
European larch and tulip poplar occurred in this time interval.

The irrigation effects on  height growth of the two populus hybrids,
tulip poplar and white cedar, were dramatic.   Irrigated white cedar
seedlings grew five times  as much as those on unirrigated plots;  irri-
gated cottonwood hybrids and tulip poplar grew about four times as
much; and irrigated aspen  hybrids grew about twice as much.

Green ash grew rapidly in  all plots, but irrigation increased its
growth about 80 percent.  Irrigation had a similar effect on Japanese
larch growing on the  loamy sand, and increased growth about 35 per-
cent in that growing on the graveliy sand.  However, in the plots with
compact substratum the higher level of irrigation reduced growth  by
85 percent and the  lower level increased growth only slightly.  Irri-
gated European larch on loamy sand increased in height more than  five
times as much as in unirrigated plots but growth on gravelly sand was
inferior to that in unirrigated plots.  The poor growth response  of
larches under normally heavier irrigation in certain soils may result
from greater susceptibility to drought, as speculated above for the
cottonwood hybrid.

On the soil with compacted substratum, tips died back on many seedlings
that received no irrigation or low level irrigation, whereas most trees
grew a small amount where  irrigation was heaviest.  Many of the red
oak died back and height growth of the others was very siow.   Irriga-
tion had no discernible effect on growth of this species.

Di scussion
It is evident from the early results of this study that survival  and
initial growth of several tree species can be increased by irrigating
with stabilization pond effluent.  The long term effects of irrigation


                                  377

-------
are, of course, still to be explored.  There is a distinct possibility
that heavy irrigation, expeciallyon poorly drained soils, will  inhibit
development of the deep root system necessary to support large trees.
This could limit the size attainable in irrigated stands.  There is
also a possibility that insect or disease problems will  be aggravated
by irrigation.  Diseases of root systems seem most likely to be aggra-
vated because of higher soil moisture.  These are among the factors
that we hope to illuminate as we carry on additional  studies including
other tree species, other soil types and other kinds of distribution
systems.   In the near future we need to consider starting pilot tests
with some of the most promising species, such as the hybrid poplars,
so that we can find out more about the operational problems that may
be encountered when  irrigating stands on a commercial  scale.

SUMMARY

Corn in drouthy, infertile soil has shown excellent response to irri-
gation with stabilized sewage wastewater.  The net value of the crop
at Middleville after farming, trucking and milling costs are subtracted
could support most or all  of the sewage treatment site operation and
maintenance program.

The use of pond stabilized sewage wastewater for irrigation of hard-
wood and conifer plantings in southern Michigan has produced several
distinct results after two years of treatment.  Water quality monitored
beneath a twenty year old red pine plantation has indicated 83-92 per-
cent renovation of nitrogen, and 96 percent removal  of phosphorus.
Irrigation rates as  high as 88 mm per week have resulted in an in-
creased flow of applied nitrogen through the soil-plant system.
Nitrate nitrogen levels have been significantly greater under irri-
gation rates of 50 and 88 mm per week than under 25 mm per week, but
have remained below  1.0 mg/l.  Irrigated red pine has shown increases
in length and dry weight of needles by as much as 36 and 56 percent,
respectively, over that of unirrigated controls.  At the present time
no trends  in DBH increment or height growth have been observed.   But
with continued increases in needle length and dry weight, increases
in DBH or height are anticipated.  Nutrient analysis of the red pine
foliage has indicated elevated levels of boron which may lead to tox-
icity conditions in  future years.  No definitive statements on the
economic impact of wastewater irrigation of red pine are possible this
early in the project.  If red pine volume growth increases in future
years, however, partial recovery of the irrigation cost should result.

The use of wastewater for the irrigation of hardwood cuttings and
seedlings has produced considerable  increases in survival and height
growth.  A cottonwood hybrid, an aspen hybrid, and green ash exhibited
the most dramatic responses to irrigation.  These species grew an
average of 112, 69, and 36 cm, respectively, during 1973.  Tulip poplar
and white cedar, while producing less total growth, increased their
growth by four and five times over the range of treatments.  Irrigation

                                   372

-------
produced moderate increases in height growth in European and Japanese
larch, and little effect on red oak.

REFERENCES

I.  Parizek, R.R., Kardos, L.T., et_ aj_. Wastewater Renovation and
    Conservation.  The Pennsylvania State University Studies No. 23,
    1967,71 p.

2.  Williams, T.C.  Utilization of  Spray  Irrigation for Waste Disposal
    in Small Residential Developments.  Proceedings of the Symposium
    on Recycling Treated Municipal  Wastewater and Sludge Through
    Forest and Crop Land, Wm. E. Sopper and L.T. Kardos, eds.  The
    Penn State University Press,  1973, pp. 385-395.

3.  Urie, Dean H.  Opportunities and Plans for Sewage Renovation on
    Forest and Wildlands in Michigan.  Michigan Academician. IV (I):
    I  15-124,  1971.

4.  Wood, Gene W., Simpson, D.W., and R.L. Dressier.  Effects of Spray
    Irrigation of Forests with Chlorinated Sewage Effluent on Deer
    and Rabbits.  Proceedings of the Symposium on Recycling Treated
    Municipal Wastewater and Sludge Through Forest and Crop Land,
    Wm. E. Sopper and L.T. Kardos,  eds.  The Penn State University
    Press, 1973, pp. 311-323.

5.  Sopper, Wm. E., and Louis T. Kardos.  Vegetation Responses to
    Irrigation with Treated Municipal Wastewater.  Proceedings of the
    Symposium on Recycling Treated  Municipal Wastewater and Sludge
    Through Forest and Crop Land, Wm. E. Sopper and L.T. Kardos, eds.
    The Penn State University Press,  1973, pp. 271-294.

6.  Standard Methods for the Examination of Water and Wastewater, 13th
    Edition.  APHA, AWWA, WPCF.   1971.

7.  Thornthwaite, C.W., and J.R. Mather.  Instructions and Table for
    Computing Potential  Evapotranspiration and the Water Balance.
    J_0(3):  184-311, 1973.  DrexeI  Inst. of Technology, Publications
    i n CIimatology.

8.  Neary, D., Day, M. and G. Schneider.  Density-Growth Relationships
    in a Nine Year Old Red Pine Plantation.  Michigan Academician.
    V  (2)  : 219-232, 1972.
                                   313

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     USES OF POWER PLANT DISCHARGE WATER  IN GREENHOUSE  PRODUCTION^
                B.  J.  BondXw. K. Furlong,3/!.  D. Si
                     C. E.  Madewe 11,27 j.  3. Martini/
                              INTRODUCTION
AVAILABILITY OF WASTE HEAT FROM POWER PLANTS

By the  term "waste"  heat^we mean energy which  is  so degraded  in tem-
perature  that its economic uses are, historically, limited.   Usually
it has  been considered practical only to discharge this energy
directly  to the environment.   Typically, such  energy appears  in the
large quantities of  cooling water necessary to condense the steam  in
power generation facilities.   For example,  a 1000 mW fossil-fired
plant with a 20° F.  rise  through the condenser and a 7 percent stack
loss discharges slightly  over  1000  cubic feet  per second of warm
water.  Such cooling water generally is discharged in the range of 60
to 110  F., depending on  the temperature of the available inlet water,
the quantity circulated,  plant load, and the use  (if any) of  supple-
mentary cooling devices.   For  instance, at  the TVA Browns Ferry nuclear
plant,  condenser water discharge is calculated to vary from a low  of
70  F.  in January/to 110° F. in July and August,  for 100 percent once-
through cooling.* If cooling  towers, either evaporative or dry, are
used for  part or all of the heat rejection, then  the temperature of
the water increases  significantly.  For evaporative towers, condenser
water temperature would range  from  75 to 125°  F., depending upon the
time of year and location.  At Brogns Ferry, predicted condenser dis-
charge  temperatures  range from 110  F. in January to 12.U  F.  in July
and August,  if all of the heat were to be rejected by evaporative
cooling towers.   For dry  cooling towers, the water temperature would
be 20-M)  F.  higher  than  temperature with once-through cooling.

   ^Research sponsored  jointly by the TVA's  Divisions of Agricultural
Development  and  Power Resource  Planning and by the U.S. Atomic Energy
Commiesion under contract with  the  Union Carbide  Corporation.
    ^Division of Agricultural  Development, Tennessee Valley Authority,
Muscle  Shoals, Alabama
          Ridge National  laboratory, Oak Ridge, Tennessee
           example is for illustration only and is not meant to imply
that TVA will permit the  discharge of water as hot as 110  F. directly
to natural bodies of water.
                                  314

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The annual quantity of waste heat presently available in the U.S. is
staggering—on the order of 101  Btu, equivalent to 1.6 billion barrels
of fuel oil.  It is projected that by 1980 TVA's coal-fired and nuclear
power plants will be generating waste heat at an annual rate of lO1^
Btu.

TVA is concerned with developing all the resources in the Tennessee
Valley region.  Consequently, for several years we have been looking
at methods of using some of this tremendous quantity of waste heat as
a resource.  Several projects under way are:  catfish production in
raceways using condenser cooling water, soil heating to extend the
growing season of horticultural and field crops, and the use of con-
denser cooling water for environmental control in greenhouses.  Waste
heat will also be used in a system we are developing to biologically
recycle nutrients in livestock waste.' '^  Suggested uses and actual
projects by other organizations are numerous.%>?>®>>  it is doubtful
that proposed systems can use more than a small fraction of the energy
discharged from power plants.  Nevertheless, if we could find an
economic use for only 10 percent of the heat rejected from all gener-
ating stations projected to be built between now and the year 2000,
the utilization would be about the same as all the electrical energy
sold in 1972 (1.85 x KX mW-hr).

PILOT GREENHOUSE

The TVA waste heat research greenhouse at Muscle Shoals, Alabama, is
the result of the cooperative efforts of many individuals.  Engineers
at Oak Ridgp National Laboratory (ORNL) developed the basic environ-
mental control system for the greenhouse.  They, in turn, provided
technical assistance to TVA engineers (Division of Chemical Development
who designed the actual facility.  Funds for construction of the
greenhouse were provided by the Division of Power Resource Planning.
The Division of Agricultural Development coordinates the project and
operates the greenhouse.

The three major overall objectives of the research project are to test
the capabilities of the environmental control system, to determine the
effect of the resulting environment on production of horticultural
crops, and to evaluate the overall economics of the system.  Results
of engineering and horticultural tests and economic analyses will be
used to refine the system.  If the resulting system proves viable,
there are tentative plans to build a facility of approximately one acre
at Browns Ferry nuclear power plant in north Alabama where TVA has
reserved l80 acres inside the exclusion area for possible waste heat
use.

Specific engineering and horticultural objectives are:
Engineering Objectives
1.  To obtain controlled-environment data over the entire spectrum of
    yearly operating conditions,


                                   375

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2.  To determine the response of the greenhouse to changes initiated
    by a relatively sophisticated control system as well as changes
    from external perturbations,
J.  To study the effect of the presence of crops on the performance
    of the environmental control system (heating, cooling, and
    dehumidification),
k.  To have an empirical check for our analytical models for green-
    house design and to improve those models, and
5.  At a future date, to study alternate means of greenhouse environ-
    mental .conditioning.

Horticultural Objectives
1.  To obtain quantitative data on the yield of various crops,
2,  To study crop performance, including the incidence and control
    of disease, under high relative humidity (95-100 percent)
    conditions,
3.  To study hydroponics versus growth in rooting media, and
k.  To determine the effect of root media heating on crop performance.

                                DESIGN

SUMMARY

The greenhouse, shown schematically in Fig. 1, is a conventional
aluminum-framed glass-glazed  structure.  The simulation of waste heat
is from an electric water heater  (boiler).  Cooling is by evaporation
from  aspen fiber pads through which air is circulated by two prope Hep-
type  fans.  Air can be recirculated through an attic plenum or dis-
charged directly outdoors.  The air flow is controlled by automati-
cally-adjusted louvers.  Heating  is by  sensible  heat transfer from
warm  water flowing over the aspen fiber pads to  recirculated air at
saturation.  Dehumidification can be provided by a bank of fin-tube
heaters supplied with warm water.

FUNCTIONAL BEQUffiEMENTS

In view of the objectives stated  above, several  functional requirements
were  identified at the start  of the design and served as guides for
all subsequent design work.   These requirements  will be discussed
belov.

Flexibility

Because of the research nature of the greenhouse, operational
flexibility was paramount in  all  decisions pertaining to the heating,
cooling, control and structural components and systems.  This means
that  we tried to provide for  operation where a given variable  (e.g.
air flow rate) departed considerably from the traditional or expected
range of variation.  In all cases, flexibility was deemed more impor-
tant  than minimal cost.


                                  316

-------


Intake
Shutters
/ i
/
v|f
^^^
\
^2^^^^*H^
^^^




>^

v^
Aspen
Pad
V

1









V \ ~ 1


T r
Sump


i













x
w
^-













^-i
00
oo
00
00
00
00
00
oo
oo
00
oo
00
00
00
oo
oo
00
00
oo
00
00
oo
oo




Fin
^r-
Recirculation
Shutters
// V \ \ N 	




i
Tube


Heater
J
'




t

Pump
_/_
LV
^^L







-t















Water
Heater

1 Diverter Valve






\ \ \ \


A
T
^ V

8\
!U* x


"s-



Growing
Area
j ./
V
/ ^
Exhaust/
Shutters

Figure 1.  Waste Heat Research Greenhouse, Muscle Shoals, Alabama.

-------
 Major Variables

 The operating variables which bear on the engineering design are
 summarized below, along with the design values for the range of each
 variable.                                             /
 1.  External ambient dry bulb temperature:  16-91  F.
 2.  External ambient relative humidity:  40-100 percent
 3.  Air flow rate:  11,000-36,000 cfm
 k.  Air exchange rate in crop region:  1-2 volumes/min.
 5.  Warm water flow rate:  0-100 gpm
 6.  Makeup water flow rate:  0-2 gpm
 7.  Power required for warm water source:  0-l80 kW
 8.  Maximum relative humidity in crop region:  80-100 percent
 9.  Warm water inlet temperature:  70-124° F.
10.  Minimum air temperature over crops:  55-60  F.
11.  Maximum transmitted solar flux:  278 Btu/hr./sq.. ft.

 Humidity Reduction

 Humidity reduction was considered a third functional requirement.  This
 may turn out to be unnecessary, if humidity-resistant strains can be
 developed and disease control maintained.  For example, operation
 close to 100 percent relative humidity has been successful at the
 University of Arizona Puerto Penasco Project-'.  Nevertheless, because
 of the research nature of our greenhouse and in the interest of
 flexibility, provision was made in the design for reducing relative
 humidity below 100 percent when operating with recirculated air or
 in the presence of high ambient humidity.

 Data Sensing and Recording

 Greenhouse temperatures, humidity, water flow rates and temperatures,
 external ambient conditions, fan speed, and louver bank position were
 selected as the major data which required detection and continuous
 recording.

 Other Considerations.

 We recognize that use of warm water to heat the root zones (and, to
 whatever extent possible, the greenhouse air itself), constitutes a
 highly desirable future experiment.  In addition, we desired to use
 one side of the greenhouse for hydroponics while the other employed
 rooting media.  Another requirement was the provision for partial or

       i                            Q
     ^-^Phe ASHRAE design guidelines  (for air conditioning) indicate
 that 5^ hours per year will be below 17° F. and 150 hours will be
 above 9^  F. at the greenhouse location.
                                   318

-------
complete recirculation of air back to the pads under ambient conditins
where warm air must be conserved.  These considerations were identified
early so that the design would reflect any special requirements.

The detailed design proceeded after establishing the functional
requirements discussed above.  The sections which follow summarize
the detailed design.

COOLING

Evaporation was the only type of cooling considered for the greenhousa
In this sense, the greenhouse becomes a small horizontal cooling tower
when coupled to a power plant, regardless of the presence or absence
of crops.  The use of a bank of aspen pads approximately 23 ft. x 7 ft.
x 2 in. thick follows conventional greenhouse practice.  This instal-
lation is shown schematically in Fig. 1.  We expect to experiment with
other pad arrangements and materials in the future in an effort to
improve the cooling.  An important contribution to improved cooling is
increased air flow through the greenhouse.  This not only reduces the
air>4T through the house, due to the solar load, but also increases
the heat and mass transfer coefficients in the pads themselves.  The
latter vary directly with the face velocity according to'ORNL
experiments.    Our fans are capable of producing two air changes per
minute in the growing space.  This is equivalent to a face velocity
at the pads of about 250 ft./min.

HEATING AM) DEHUMDIFICATION

Due to the relatively low temperature water available, it was decided
that a contact type heat exchanger would be required to supply  the
major portion of heat to the greenhouse.  We chose to try to use the
simple, inexpensive pads as contact heat-exchange surfaces for  heating
the greenhouse with simulated waste warm water.  (Several other heating
methods are available, such as sprays, underground pipes, compact fin-
tube radiators, and even heat pumps.  These alternate methods will not
be discussed in this paper.)

Two precedents existed for the direct-contact heating—the experience
at Puerto Penasco by the University of Arizona researchers and  our own
experience at ORNL with a small test greenhouse.

One feature of the direct-contact mode of heating is that the air
quickly reaches saturation and remains near that condition as it is
recirculated back through the pads and the greenhouse.  To permit
dehumidification, we installed a bank of fin-tube heaters about 5 ft.
downstream of the pads  (Fig. l).  This heater bank is spread out to
cover the entire flow area.  Each tube is about 20 ft. long, headered
at each end to form two circuits of four passes each to provide even
temperature distribution.  The fin-tube bank can be operated with warm
water either in series or in parallel with the pads, as shown in Fig.L


                                  319

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It is designed to lower the relative humidity from 100 percent to 80
percent by providing TO kW.  One-inch copper tubing is used with
aluminum fins.  Air pressure drop across the surface is negligible.

Water at waste heat temperatures for the pads and fin-tube heater is
provided "by a standard commercial l80 kW hot water heater.

Heating of the plant root zones is accomplished by diverting warm
water from the system just downstream of the hot water heater.  This
water flows through coils of 3/U inch FVC pipe buried 8 in. below the
surface of two rooting media (soil and pine bark/vermiculite mix).
The water is discharged to the pump suction basin.

CONTROL AND INSTRUMENTATION SYSTEM

The control system is designed to maintain a  preset air  temperatureana.
a  preset relative humidity with a given fixed supply water temperature.

Air temperature  is controlled by changing fan speed and by modulating
the amount of air that is recirculated through the house.  The possible
air flow modes are:  once-through, and 25 percent, 50 percent, 75 per-
cent and 100 percent recirculation.  Control is achieved  by individual^
opening or closing each of four banks of louvers on the inlet, outlet,
and recirculation flow paths.  Five thermistors in the greenhouse
supply temperature signals; control can be from any one or combination
of the five.

Relative humidity control is effected by diverting warm water through
the fin-tube heater.  A diverter valve  (Fig. l) modulates the flow  in
response to a signal from two  gold-grid humidity detectors in the
growing  space.

Instrumentation  for the greenhouse consists of detectors  for flow,
temperature, and humidity signals plus appropriate readout and, in
some cases, recording equipment.

The warm water supply temperature is preset on the water  heater.  Water
temperature is measured by thermocouples at six locations:  upstream
of the water heater, downstream of the mixing valve, outlet of the
fin-tube heater, pad inlet and outlet, and makeup water inlet.  These
values are recorded on a multi-channel strip recorder.  Ambient air
temperature and  five internal  greenhouse temperatures are measured  by
thermistors and  similarly recorded.  Five portable recording hygrom-
eters have been  used initially for both wet and dry bulb  data, but
they will not be retained  for  long-term operation.

Relative humidity is measured by two separate gold-grid detectors
mounted  in aspirated cabinets at the entrance and exit of the growing
space.  These signals are  fed  to a controller which has a provision
for recording, but they are not recorded at present.  A visual
indication is provided on the  control panel, however.

                                  320

-------
Fan speed and louver position have visual  indicators on the control
panel and are also recorded on a strip-chart device.  The TVA weather
station at Muscle Shoals can provide hourly summaries of wind speed
and direction, dry bulb and dew point, barometric pressure, and, most
significantly, solar and total radiant flux.  We expect to utilize muh
of these data in lieu of duplicating the same measurements ourselves.

                 INITIAL ENGINEERING TESTS AND RESULTS

One design error had to be rectified:  the attic barrier, originally
4-mil polyethylene, was replaced with corrugated fiber glass-rein-
forced acrylic panels.  Over a period of about 8 months, the poly-
ethylene had become severely embrittled and weakened by the combinatai
of solar radiation and heat in the attic.  Also, an exhaust fan may be
installed for the attic, to operate when the main fans are in the
once-through mode (summer cooling).

TESTS

In general, our engineering tests sought to measure:
1.  Air flow rate, both fast and slow speed, with once-through flow
    and with partial and full recirculation.  These data were taken
    with a hot-wire anemometer using a 12-point grid set up to
    represent equal flow areas per point.
2.  Fan performance, such as speed, head,  flow and power requirement,
3«  Pressure drops, especially for the pads, fin-tube heater, and atti^
h.  Air temperature and humidity at various locations and under various
    operating conditions,
5.  Control system performance, such as the maintenance of preset
    temperature or humidity,
6.  Water temperature as controlled by the water heater, and
T.  Pad and fin-tube performance, as measured by water temperatures
    and air enthalpies.

RESULTS

Our engineering tests are not complete as  yet, but a summary of
qualitative results is presented below.  (Items 1, 2, 3 and 8 apply to
an empty greenhouse.  The others apply to-a house with a mature
cucumber crop-)
1.  Air flow decreases as the fraction recirculated increases, with
    80 percent of the once-through flow obtained under full recircu-
    lation.  This applies to either fan speed.
2.  There is considerable variation in air velocity with location in
    the plane perpendicular to flow.  Flow decreases as elevation
    increases, with a change as much as 50 percent in some locations.
3.  The water heater, with its 10-step controller, maintains water
    temperature within j£° F. of the setpoint.             o
h.  The greenhouse air temperature ranged  between 65 and 68  F. for a
    clear night where the ambient reached  a 19  F. while operating
    with water at 7*4-  F.  The fin-tube heater supplied about 20-30

                                 327

-------
    percent of the heat under these conditions.
5.  Data taken over sevenQdays in early December, with ambients
    ranging from 19 to 35  F. indicated air temperatures 7 to 11° P.
    lower than vater inlet temperatures to the pad.  (The fin-tube
    heater was supplying about 20-30 percent of the total heat during
    this period.)
6.  The coldest night tQ date, 13  F., produced a 63  F. house
    temperature with T^ F. water.  The fin-tube heater supplied about
    20-30 percent of the heat.
7.  We have calculated that 95-100 kW of heat was used when the ambient
    temperature reached 19  F. This is in good agreement with the
    design value of 111 kW at 16° C,
8.  Greenhouse cooling was checked at two extremes of water tempera-
    ture, 6£>  F. and 119  F.  In the former case, the water was
    recirculated and the pad inlet and outlet temperatures were the
    same as the ambient wet-bulb 66° F.  The switch to high-tempera-
    ture water brought the pad outlet up to 73  F*  In both cases,
    the ambient air was cooled, by 6° F. with 66  F. supply water and
    by U  F. with the 119  F. water.  Further cooling data must await
    spring and summer operation.
9.  The heating system is highly buffered against changes in outside
    temperature as evidenced in Fig. 2.  A change of 31  F. outside
    resulted in only a 5° F. change inside.

                INITIAL HORTICULTURAL TESTS AND RESULTS

We are testing tomatoes, cucumbers and lettuce in the greenhouse.
These three crops were chosen because they occupy about 93 percent of
the greenhouse area devoted to vegetable production in the United
States.  In one experiment, rooting media are being tested:  heated and
unheated soil (Ochlockonee fine sandy loam), heated and unheated pine
bark/vermiculite mix, and wheat straw bales (cucumbers only).  The
pine bark/vermiculite mix is a variation of the peat moss/vermiculite
mix conanonly used for greenhouse tomatoes.  The straw bale culture is
a technique commonly used in Europe and England for greenhouse
cucumber production.  All media treatments received fertilizer
according to the schedule in Table 1.
                                  322

-------
    70
 1
•I  66
&  64

 o»


    62
 o>
 o>
    60
       10
Water  Temperature, 73°F

   Pad flow 2.5 gpm/ft

   Fin tube flow 40 gpm
       20              30

          Outside Temperature
40
so
                Figure 2. Response of greenhouse temperature to outside temperature changes.

-------
           Table 1.  Fertilizer Schedule*/for Rooting Media
           (NH4)2HP04      mpO^      TO^Ca(N03)2       MNO"
                               grams/100  liter

1
2--3
4-10
11-12
13-17
134
67
67
134
134
134
67
67
0
0
0
0
134
268
268
0
0
268
268
268
0
0
134
134
268
In a  separate experiment, two hydroponic  systems are being  compared.
One system consists of gravel-filled troughs which are periodically
pumped  full of nutrient  solution and then allowed to drain.   They are
filled  four times per day during clear weather and fewer times during
cloudy  periods.  The other  system consists of troughs of coarse  sand
to which  nutrient solution  is  surface applied daily.  Both systems
use the nutrient solution shown in Table 2.

        Table 2.  Nutrient Solution Used  in the Hydroponic  Systems
Element      Concentration
                 ppm
N
P
K
Ca
Mg
S
Mn
Cu
Mo
Zn
Fe
01
B
135 (increased to 200 after first fruit
62
156
165 (increased to 230 after first fruit
49
64
0.62
0.05
0.03
0.09
5.00
0.86
0.44
set)


set)









                                  324

-------
Femfrance cucumbers were transplanted into the greenhouse on October
10 at a population density of one plant per  8 ft.  of greenhouse area.
Femfrance is a long, seedless cucumber developed especially for
greenhouse production.  Yields for the entire harvest period are
shown in Table 3-

There was no significant effect of media heating on yield.  This is
because soil and mix temperatures have been  at an acceptable levelQto
date.  For examgle, on December 3 with 8h° F. water, the mix was 7^  F«
unheated and 82  F. heated.  Respective values for soil were jk  F. and
ar F.

There was no significant effect of media on  total or grade no. 2 yield.
However, yields of grade no. 1 cucumbers in  bale culture were  less than
yields in soil or artificial mix.  Also, the unheated soil was
superior to artificial mix in number of grade no. 1 cucumbers  produced.

Hydroponic yields cannot be statistically  compared to the other yields.
Significantly fewer grade no. 2 cucumbers were produced in the sand
culture as compared to the gravel culture.   Type of hydroponic system
had no effect in the other two yield categories.

Grand Rapids leaf lettuce and Bibb  lettuce were  transplanted into  the
greenhouse on November 15.  The hydroponic  systems and the heated  and
unheated soil and mix used were identical  to those used for cucumber
production.  In late January, cucumbers were replanted and the
lettuce replaced with tomatoes.

During November, the greenhouse was  operated without  humidity  control
for  IT days; i.e., the fin-tube heater was  inoperative.   During this
period of high humidity  (95 to  100  percent relative humidity), there
was an outbreak of powdery mildew  (Erysiphe  cichoracearum).  A
reduction in humidity to about  85 percent  by activating the fin-tube
heater and a change from zineb  fungicide  to benlate  checked the fungal
outbreak.  It is probable that  simply  the  use of benlate  would have
had  the  same result.

Since Femfrance cucumbers are different  in appearance and taste from
cucumbers available in  stores  in  the Muscle  Shoals  area,  a consumer
acceptance test was conducted at  a  local supermarket.  Results are
given in Table U.  The Femfrance  cucumbers were  well accepted  and  sold
for  about twice the price of  the  field variety.
                                   325

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                                Table _3_
Cucumber Yield, Nov. 8-Jan. 30.
Cu
Soil

(1)

(2)



(3)




Total yield: Ibs. /plant
Cucumbers/plant
Grade no. 1 yield:
Ibs. /plant
Cucumbers/plant
Average weight, Ibs.
Grade no. 2 yield:
Ibs. /plant
Cucumbers/plant
Average weight, Ibs.
Unheated
19 .k
16.1
/
15. 60?
12. 5a
1.25

3.4
3.2
1.10
Heated
18.2


15. 5a
12.3ab
1.26

2.6
2.U
1.10
Pine Bark
Verraiculite Mix
Unheated
18. U
lU.o

13. 6a
9.9c
1.37

3.6
2.9
1.26
Heated
19.1
1U.7

13. 9a
10.3bc
1.35

U.7
3-9
1.21
Straw
Bales
1U.1
11.7

8.6b
6.6d
1.30

k.2
3-7
1.13
Hydroponic
grave 1
21.2
18.0

16.8
13-7
1.23

3-9a
3-7a
1.0
sand
22.3
19-5

20.1
17-3
1.16

2.2b
2.2b
1.0
   ^Values followed by the same letter are not significantly different at the 5 percent level of
     probability.   Hydroponic yields cannot be statistically compared with yields from other treatments.

-------
                Table U.  Market Test, Nov. jO-Jan. i8
Price


Femfrance
Field Variety
Range
- _ _
.25-..U9
.15-. 29
Mean
$ - - -
.38
.20
Number
Sold

U06
1013
                         ECONOMIC IMPLICATIONS

Results of an initial economic evaluation stress the need for refine-
ments in greenhouse design to make it show greater economic potential.
One example would be to recirculate the air through an adjacent house
and thus, eliminate the fiber glass ceiling presently used to form
the recirculation attic.  Also, if power plants go to closed-loop
cooling, the higher temperature water available will lower capital and
operating cost.  For example, lower water flow rates or smaller heat
exchangers would be required.  Also, cost of disease control would be
reduced if lower humidity could be maintained with the warmer water.
Of course, many factors which would improve production and sales in a
conventional greenhouse would also be important in improving the
economic potential of waste heat greenhouses.  These factors would
include improved crop varieties, batter and cheaper disease and insect
control, improved labor efficiency, and the increasing demand for off-
season, greenhouse grown products.

                             IN CONCLUSION

We are pleased with the engineering system because it has performed
as designed to date.  However, we do plan modifications to the
present house to test variation on the central theme of heating and
cooling with a single contacting device  (pads)'   Similarly,
horticultural production has been satisfactory to date but we will
continue screening crops, varieties, and rooting media to increase
production.

An important future question which we  intend  to examine is that of the
interface between a large greenhouse installation and a power plant.
Of interest are  such things as variation of water temperature wi^Ii
load, plant shutdown, water quality and activity  monitoring, greenhouse
revenue and rate structure, legal liability to commercial lessees, etc.
In addition, many questions remain regarding  crop marketing and
operational economics.

We believe that  this pilot facility, although still  in the early
stages of operation, will continue to  provide us  with good engineering
guidelines for the design of a  larger  demonstration  facility to be


                                   327

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located at an operating power plant, and we 3ook forward to initiating
conceptual design work on such an installation.
                                   328

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                             References
1.  Williams, G. G., "TVA Programs-Waste  Heat Utilization in
    Greenhouses and Other Agriculturally  Related Projects."
    Proceedings of the National Conference  on Waste Heat Utilization,
    October 27-29,  1971, Gatlinburg,  Tennessee.  CONF-TllOJl
    (May 1972).

2.  Bond, B0 J., C. E. Madewell, J. B.  Martin, Jr., and D. A. Mays.
    "TVA Projects—Beneficial Uses  of Waste Heat."  Proceedings of
    the National Conference  on Complete WateReuse, Washington, B.C.,
    April 2J-27, 1973-

3.  Madewell, C. E., J.  B. Martin,  and  B. G. Isom,  "Environmental and
    Economic Aspects of  Recycling Livestock Wastes—Algae Production
    Using Waste Products," paper presented at  the Association of
    Southern Agricultural Workers meeting January 31-February 3, 1971,
    Jacksonville, Florida.

h.  Yarosh, M0 M.,  e_t al.  "Agricultural  and Aquacultural Uses of
    Waste Heat," USAEC REPORT ORNL-1+797 (July 1972).

5.  Yarosh, M. M.,  and B.  L. Nicholas (Oak Ridge National Laboratory),
    "Waste Heat and How  It Might Be Used,"  "The Potential  for
    Agricultural and Aquacultural Uses  of Waste  Heat,"  and  "Factors
    That Need to be Considered in Utilizing Waste Heat,"  (Published
    in AWARE Magazine) Reprints available from:  AWARE Magazine,
    615 North Sherman Avenue, Madison,  Wisconsin  5570^-  Price
    each.

6.  Beall,  S. E.,  and G. Samuels, "The  Use of Warm Water  for Heating
    and Cooling Plant and  Animal Enclosures," USAEC Report  ORNL-TM-
    3381  (June  1971).

7.  Symposium:  Beneficial Uses for Thermal Discharges, Journal  of
    Environmental Quality 2:178-228, Apr.-June 1973-

8.  American  Society of  Heating, Refrigeration,  and Air Conditioning
    Engineers,  "Handbook of Fundamentals,"  1967 ed.,  Chapter 22.

9.  Jensen, M.  H.,  "The  Use of Waste Heat  in Agriculture,"  Proceedings
    of the  National Conference on Waste Heat Utilization,  October
    27-29,  1971,  Gatlinburg, Tennessee, CONF-711031 (May 1972).

10.  Furlong,  W. K., M.  I.  Lundin, L. V. Wilson, and M. M.  Yarosh,
     "Activities Report—Beneficial Uses of Waste Heat Covering ORNL
    Activities  Through December 31, 1972,  in the Joint AEC(ORNL)-TVA
     Program,"  ORNL-TM-la95  (June 1973)-
                                  329

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PART 5    NONTECHNICA L RES TRAI NTS
         SESSION CHAIRMAN

        JOHN H. MARSH, P.E.
PRESIDENT, ENGINEERING ENTERPRISES
        NORMAN, OKLAHOMA

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            LEGAL CONSTRAINTS ON THE USE OF WASTEWATER

                        FOR FOOD AND FIBER

                                 by

              William R. Walker and William E.  Cox*


                           INTRODUCTION

The world demand for increased production of food and fiber places
an ever increasing strain on the available water supply.   The problem
is accentuated by the current energy crisis, which is not likely to
be of short term duration.  Prior estimates of water demands may
well be grossly underestimated when one contemplates the consumptive
use of water in energy production (e.g., coal-gasification) and for
irrigated agriculture.  Effective management of our wastewater re-
sources moves from something which is highly desirable to something
which is absolutely essential.

The recycling of municipal sludges and effluents on land has been
used more extensively in other parts of the world than it has in the
continental United States; therefore, this alternative for waste dis-
posal has not had an in-depth evaluation  in the United States
relative to other methods.   Increased interest in recent years has
stimulated additional research into the health aspects of this method
of disposal (e.g., survival  of viruses) and the direct effect on food
and fiber production.   No  assessment of this waste management tech-
nique can be complete,  however, without some discussion of the
institutional processes by or through which this waste management
scheme functions.  Although  these institutional considerations in-
clude such diverse matters as the form and powers of water organiza-
tions, financial arrangements, public attitudes, and political
traditions, this paper  will  concentrate on legal aspects as manifest
in legislative enactments  and the common  law.  It will first examine
direct government control  over this disposal technique by the federal
and state levels of government.  Second,  since relatively large areas
of land are an indispensable component in this method of wastewater
treatment, governmental regulation of land use must be examined along
with the corollary common  law rights of other property owners in the
same area.  Third, both statutory and common law rights related to
water must be fully analyzed in  terms of  this method of disposal
since land application  of wastewater alters the flow  regimens of both
surface and ground water.

  *Director and Research Associate, respectively, Virginia Water
   Resources Research Center, Virginia Polytechnic  Institute and
   State University, Blacksburg, Va.

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                   DIRECT GOVERNMENT CONTROLS

FEDERAL

The federal government has historically left the jurisdiction of
water pollution control in the province of state and local govern-
ment, viewing it essentially as a public health matter to be dealt
with under the police powers.  The first comprehensive federal   -,
control effort was the passage of the Water Pollution Control Act
in 1948.  This legislation relegated the federal government to a
"back-up" position with primary responsibility for pollution control
to remain in the hands of the states.  The Federal Water Pollution
Control Act of 1956^ contained the first authorization of federal
grants on a large scale to assist states and municipalities in plan-
ning and building facilities for treatment of wastewaters.  The Act
contained prohibitions and omissions that were not encouraging to
the land treatment method.  For example, the cost of land for sewage
treatment, including use of land as an integral part of wastewater
processing, was not eligible for grant assistance, and no provisions
were made for grants in connection with recycling and reclamation of
wastewater.

On October 18, 1972, the Federal Water Pollution Control Act Amend-
ments of 19723 became law.  This Act is without question the most
comprehensive and complex legislation that has ever been enacted to
clean up the nation's waters.  It establishes a national policy that
a major effort be made to develop technology necessary to eliminate
discharge of pollutants into the navigable waters, waters of  the
contiguous zone, and the ocean, and several specific provisions of
the legislation give impetus to consideration of land disposal as a
serious alternative for alleviating the water pollution problem.

One of  the basic encouragements is the requirement that publicly-
owned  treatment plants achieve secondary treatment results  by 1977\
including  removal of all floatable solids  and 85 percent  of suspended
solids.  In addition to increased costs in general, this  requirement
will  result in greater quantities of sludge to  be disposed  of, a task
made  more  difficult in some  cases by restrictions on ocean  disposal
of sludge  contained in the  legislation.  Thus an  incentive  for land
application of wastewater is produced since this  technique  has the
capability of meeting  the secondary  treatment  requirement  and hand-
ling  a  part of the sludge disposal problem as well.

Land  treatment is also facilitated by  provisions  of the 1972 Amend-
ments  which permit the use  of  federal  grants  for acquisition of  land
that  will  be an  integral  part  of the treatment  process or is used
for  ultimate disposal  of  residues  resulting  from  treatment opera-
tions5.  Grants cannot be used to  acquire  land  upon which  a
conventional  treatment plant is  located.
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Provisions of the Act and regulations adopted for its implementation
show a definite intent that land treatment be considered as an alter-
native form of waste treatment.  For example, the legislative
provision requiring the Administrator of the Environmental  Protection
Agency (EPA) to encourage the development of revenue producing waste
treatment facilities makes specific reference to recycling potential
sewage pollutants through the production of agriculture, silviculture,
or aquaculture productso.  Another relevant provision is included in
the proposed regulations containing cost effectiveness analysis
guidelines.  This provision requires the consideration of all feasible
alternatives, specifically including systems using land or subsurface
disposal techniques7.

Although this most recent manifestation of federal water quality
policy suggests and even encourages the use of land disposal where it
is most cost effective, primary responsibility still resides with the
states subject to the Administrator's approval of the state's permit
program^.

STATE

Land application of wastewater is generally subject to state control
as are other methods of waste disposal.  Acceptance of this treatment
concept varies among the states.  A 1972 study by Temple University
indicates that 14 states had a favorable orientation toward land
treatment and 11 were negative in their outlook, with the remaining
25 either neutral or not subject to classification on the basis of
available information^.

Control over land treatment facilities has largely been provided
within the discretion of the state regulatory agencies, with few
formal regulations having been adopted.  A survey reported in a 1973
EPA publication conducted by the American Public Works Association
with state health and water pollution control agencies indicates that
most state agencies have no set policies on this phase of wastewater
handling or attendant environmental impacts, do not impose specific
conditions on installations, seldom inspect existing systems, and
seldom require monitoring procedures and the filing of official
reports on operation.  Only five states indicated that official regu-
lations governing irrigation with wastewater were in effect: Arkansas,
Arizona, Colorado, New Mexico, and Texas.  Only four states, Arizona,
Arkansas, New Mexico, and Texas, indicated that they have rules
governing the type of crops approved for wastewater irrigated lands.
Permitted crops range from "forage only" to "all types."  Texas regu-
lations demonstrate concern over consumption of raw crops from waste-
contact areas, with a prohibition in effect against wastewater
irrigation of those crops to be consumed in the raw state.  The few
states which invoke crop restrictions also specify the quality of
effluent to be applied to the land10.
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Other states in addition to those listed as having regulations  govern-
ing wastewater irrigation have controls with regard to land treatment
in general.  For example, Californial! and Idahol2 have regulations
of this type.  These controls would appear to apply to any proposal
for wastewater irrigation.

                         LAND USE CONTROLS

Policy concerning land use control is currently undergoing consider-
able re-evaluation as indicated by efforts to enact federal land
use planning legislation and the establishment of land use control
mechanisms at the state level.  Provisions of these new laws and
regulatory procedures will likely impose direct controls over land
application of wastewater, but until these programs are implemented,
the traditional controls imposed by local zoning ordinances and
private control measures will provide the most significant con-
straints.

The applicability of zoning ordinances to land treatment facilities
is a complicated matter because of dependence on such factors as the
provisions of enabling legislation and the terms of the particular
zoning ordinance involved in a given situation.  Two different
situations may arise in which the applicability of zoning would be
significant.  The first concerns the extent to which a governing body
is subject to its own zoning restrictions, and the second involves
application of zoning restrictions of a second governmental entity
where facilities are to be constructed on land lying outside the
jurisdiction of the owner.

With regard to the question of whether a governmental body is subject
to its own zoning restrictions, the answer is frequently provided by
provisions in zoning ordinances which exempt governmental uses from
their provisions.  For example, The Supreme Court of Pennsylvania in
a 1952 easel3 upheld the right of a municipality to construct a sew-
age treatment plant in a residential district since the zoning ordi-
nance specifically permitted "municipal use."  Where no specific
exception  is made for governmental use, the decision of whether the
restrictions apply is sometimes based on a distinction between
proprietary and governmental functions.  Proprietary functions are
generally  held to be subject to zoning restrictions while govern-
mental functions are not!4.  In general, government functions are
those imposed upon a municipal corporation by the state as a part of
the sovereignty of the state to be exercised by the municipality for
the benefit of the general public, both within and without the
corporate  limits.  On the other hand, proprietary functions are
those exercised with respect to a municipality's private rights as
a corporate body for the advantage of the  inhabitants of the city
and of the city itself 15.
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Of course, the key question which arises is whether operation of sew-
age treatment facilities is proprietary or governmental  in nature,  a
determination which may have varying outcomes.  In a 1935 New York
casel6, a municipality was enjoined from erecting a garbage disposal
plant in a restricted district on the grounds that it was performing
a proprietary act and therefore bound equally with all other persons
by the terms of its own ordinance which prohibited disposal plants.
An opposing viewpoint is expressed in a 1969 Vermont easel? which
holds the construction of a sewage disposal plant to be a govern-
mental function and therefore exempt from local zoning ordinances.

Variation in outcome also characterizes attempts by one governmental
body to apply zoning restrictions to sewage treatment facilities to
be operated within its jurisdiction by a second political entity.  An
example of a case of this type where zoning restrictions were upheld
is given by a 1962 decision of the Supreme Court of Missoun'18,  An
injunction was awarded to a county for prohibition of the construc-
tion of a sewage disposal plant by a city in a residential district
of the county.  The opposite result was reached in a 1962 Arizona
decisionl9 in which the state supreme court held that the operation
of a municipal sewage disposal plant was exempt from zoning regula-
tions of another municipality in which the property involved was
located.  After recognizing a division of authority as to whether
sewage disposal is a proprietary or governmental function, the court
took note of the fact that the weight of recent authority appears to
support the theory of a governmental function.  This trend indicates
that municipalities are not likely to be inhibited by internal or
external zoning in the location of land disposal facilities in an
increasing number of localities.

In addition to formalized land use controls,  land treatment facili-
ties may also be subject to private control measures where such
operations have a detrimental impact on surrounding property.  Of
interest here is the system of civil law which defines private rights
and provides a mechanism for accountability where the activities of
one party injure or infringe upon the rights  of others.  This law is
embodied in the accumulated decisions of the  courts and  is enforced
by means of litigation, which may take  the form of an action  for
damages or suit for injunction.

The concept of private nuisance is likely  to  be a central  element in
any legal conflict arising out of situations  where land  treatment
facilities create conditions which unreasonably interfere  with the
use or enjoyment of other property.  Land  treatment facilities are
not likely to be declared a nuisance simply  because of the dis-
pleasure of an adjacent landowner; substantial  interference with a
legally protected  interest is required.  In  determining  whether  a
given  facility does constitute a nuisance,  the court  will  weigh  the
gravity of the harm produced against the social utility  of the
activity  in question.  Since  the utility of  waste  treatment  to  the

                                  334

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general public  cannot  be  questioned,  suits  based on nothing more than
a general objection to the  establishment  of such facilities almost
invariably have been unsuccessful.  Another effect of the relation-
ship of such operations to  the  public interest is the refusal by the
courts in most  cases to grant injunctions that would prohibit con-
struction or force cessation of operations21.   Although cases exist
where sewage disposal  plants have been enjoined22, the more common
remedy imposed  by the  courts is  an award  of compensate damages.

A successful suit for  damages must be based on a showing of injury in
excess of slight discomfort or  annoyance, but it is not necessary that
the property in question  be made totally  unusable or that an imminent
danger to health exist in order for a legal  course of action to arise.
The concept of  nuisance encompasses activities that create conditions
offensive to persons or ordinary sensibilities, including unpleasant
odors, loud noises, and disease-causing insects.  The courts have
generally recognized the  right  to an action for damages where these
conditions are  caused  by  the operation of sewage disposal facili-
ties".  Damages which can  be collected include compensation for
depreciation of property  values and an award for personal dis-
comfort24.

If nuisance conditions are  created by a waste treatment facility, the
right to recover damages  is not likely to be nullified by the claim
that the facility has  been  properly designed and operated.  A 1942
Iowa court decision25  addresses this issue  with regard to a conven-
tional sewage treatment plant.  The owner of the plant maintained
that liability should  not be imposed because the plant involved was
of the most modern type, had been designed  by a competent engineer,
and had been given approval by a state agency.  The court held that
these factors did not  relieve liability arising from the detrimental
impact on other property.   To the extent that proper design and opera-
tion of facilities actually prevent adverse effects, the likelihood
of legal  action is reduced, but compliance  with such procedures is no
shield from accountability  for injury produced.

In addition to the landowner whose use exists at the time of estab-
lishment of land treatment  facilities, the  owner who initiates a use
of near-by property subsequent to installation and operation of
treatment facilities also is likely to possess the right to maintain
a legal action for compensable damages in connection with property
injury.  "Coming to the nuisance" is generally no bar to an action
for recovery.  This principle has been applied to a variety of indus-
trial  or commercial  activities which were originally established in
undeveloped regions but were later encompassed by expanding urbani-
zation26.  It has been  indicated that public facilities are also
subject to such action.  For example, the court in a 1951  Florida
decision2' which denied a request for an injunction, noted that an
adequate  remedy for damages existed for actual injury to property
                                  335

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produced by a garbage disposal plant which had been encompassed by
urban growth.
                 CONSTRAINTS IMPOSED BY WATER RIGHTS

The third basic category of potential legal constraints on land appli-
cation of wastewater consists of adverse effects on private water
rights that may result from alteration of water quality and natural
flow patterns.  Although land treatment is intended to reduce overall
water pollution, any degradation of ground water quality may subject
the operator to legal action by ground water users.  In addition,
ground water recharge may produce damage to others where the water
table is artificially raised.  With regard to surface waters, the
principal adverse effect of land application is the reduction in
streamflow which may occur below the previous point of discharge and
result in injury to water users located there.  Thus it is necessary
to consider several aspects of private water rights which may con-
strain use of land treatment.

GROUND WATER

Perhaps the most obvious legal constraint on land application of
wastewater imposed by ground water law concerns protection of quality.
Although it is generally believed that the majority of pollutants in
wastewater will not survive oxidation in the soil, it is likely that
certain residuals and trace contaminants will find their way to the
ground water.   In some cases, the end result of the oxidation process
itself may be a form of contaminant.

The right of the landowner to uncontaminated ground water has not
always been recognized.  The courts in some cases have been reluctant
to impose liability where ground water pollution has resulted from
lawful uses of land on the basis that such injury could not have been
anticipated",  in other cases, however, the right to uncontaminated
water has been upheld and liability imposed on those responsible for
its pollution.  One of the common methods of protecting ground water
quality has been to declare activities which cause its contamination
to be a nuisance29.  in some cases the right to pure ground water has
been viewed as absolute, with liability imposed on the party respon-
sible for its degradation without regard to the reasonableness of his
activity or the care with which his operation is conducted^,  it is
interesting to note that acceptance of the strict liability concept
in the law is increasing31, a fact which generally enhances the right
of an injured party to recover from those responsible.

One of the most difficult aspects of ground water pollution cases is
the problem of establishing a cause-and-effect relationship between
the polluted water supply and the alleged source of the pollution.
Since direct evidence as to the source is often impossible to obtain,

                                  336

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the courts have often accepted indirect proof based on circumstantial
evidence in such cases.  It is difficult to generalize with  regard to
the type of evidence which will be sufficient in a given case,  but
factors that have been considered include the proximity of the
alleged source, the existence of other possible sources, the time re-
lationship between the alleged pollution-causing activity and the
injury, and the capability of the suspected source to pollute3^  Be-
cause of the fact that ground water contamination suits are  often
initiated and decided with relatively imprecise data, it would  appear
desirable that provisions for monitoring ground water quality include
data collection prior to operation of the site as a treatment facility
such that background data will be available for comparative  purposes
in the event that complaints of quality degradation arise.

In addition to the protection of quality, water law will also protect
the landowner from interferences with ground water levels which pro-
duce injury.  Underdrains are a method of mitigating both the quali-
tative and quantitative effects on ground water, but to date they
have not been extensively used.  A survey by the American Public
Works Association indicated that only 4.9% of the 122 disposal  fields
examined had underdrains.  Designers of the systems relied on evapora-
tion, plant transpiration, and ground water recharge to take up the
flow33.  it must be recognized that under certain circumstances the
increased subsurface flow may reach adjoining lands and so raise the
ground water level that agricultural or residential development of
the property is adversely affected.

Liability for artificially raising ground water levels has been im-
posed by the courts.  For example, there have been several cases
concerning damage to adjoining property as the result of  the con-
struction of reservoirs from which there was subsurface leakage.  In
an Ohio case34, the plaintiff proved to the satisfaction  of the court
that a nearby impoundment had caused water to flow, ooze, percolate,
or seep into the land of the plaintiff, thereby rendering it sour,
wet, or swampy, and permanently unsuitable for use as farm land or
for subdivision and sale.  The plaintiff based its claim  for damages
on the concept  that defendant had committed a "trespass"—a physical
invasion of the plaintiff's property which destroyed  the  Tatter's
right  to the enjoyment thereof.  The court held that  liability was
absolute without regard to whether the defendant  had  used due care
in the construction and maintenance of the dam and reservoir.

An earlier  Florida case35 had  a very similar  fact situation.  The
court  found in  favor of the  landowner  injured by  the  subsurface water
on the basis  that  damming a  stream and  causing  an increase  in  ground
water  levels  such  that injury  was produced was  unreasonable.   This
case also  addressed  the question  of whether  a defendant can  escape
liability  when  the  dam was  built  under authority  granted  by  the
United States  Government.   An analogy  could  exist to the  case  of land
disposal  since the federal  government  is  likely to provide  much of

                                   337

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the funds for the project and in so doing gives its implied consent
to the project.  The court in the reservoir case held that authority
to erect the dam did not justify an unreasonable use of the property
as it affects the rights of others.

SURFACE WATER

Rights to water in its natural state are considered and dealt with as
real property in almost all jurisdictions.  Water differs, however,
from other real property in that it is the use of the water and not
the water itself which is the subject of property rights.   This qual-
ification or condition is found in each of the two major water
doctrines prevalent in the United States—riparian and appropriative.
The riparian doctrine recognizes a property right in the water only
when the use being made is reasonable in terms of others having an
equal right.  Under the appropriative doctrine, a property right does
not arise until the water has been put to beneficial use.   So strong
is this requirement that it is often said that beneficial  use shall
be the basis, the measure, and the limit of the right to the use of
water.  There is general agreement that "beneficial use" and "reason-
able use" both carry economic as well as legal connotations.

The application of municipal sludges and effluents to land actually
constitutes an "add on" after water has been applied to beneficial
use by a local unit of government.  Although this recycling has most
of the attributes of irrigation, the rate of application and other
parameters are defined not in terms of optimum irrigation but of
obtaining improved water quality.  Improved water quality may well
be accepted as a national goal or objective, but unresolved is the
question of whether it can or should be obtained at the expense of
property rights in water as established by state law.  The question
must be asked as to whether this recycling of wastewater is a
beneficial use or a reasonable one under our current water law
doctrines.

The general rule in those states following the appropriative doctrine
is that a water use once established will remain substantially un-
changed with regard to its effects on other appropriators.  The right
in the use of water includes the right to change the place, nature,
and means of use as well as the point of diversion, but the right to
change is qualified in that it may not be exercised to the detriment
of other appropriators, including those with junior rights36.  Thus a
conflict arises where a proposed modification in use involves a
change from non-consumptive use to consumptive use which will deplete
streamflow.   Land application of municipal wastewater would appear
to fall into this category of change since irrigation consumes more
water than does traditional municipal use alone.  In fact, the
appropriator located immediately downstream from the prior point of
waste discharge may be denied all use of the previously existing
return flow.

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of junior appropriates will necessarily be protected.   A 1972
Colorado casej/ concerning a change in point of effluent discharge
is relevant to this issue.  In this case, the court draws a distinc-
tion between a change in the point of diversion and a change in the
point of effluent return.  The court acknowledges that an appropri-
ator may not change the point of diversion except upon conditions
which eliminate injury to other appropriates, but goes on to say
that changes in points of return of wastewater are not governed by
the same rules.  It is held that there is no vested right in down-
stream appropriates to the maintenance of the same point of return
of irrigation wastewater, and in the absence of bad faith or arbi-
trary or unreasonable conduct, the same rule applies to sewage
effluent from a municipality or sanitation district.  The court does
acknowledge that there may be instances in which a change of the
point of return may be enjoined, but the fact situation of this case
is not one of them.  The case has a strong dissent which argues that
the language of the court in previous cases is not limited to situa-
tions involving a change in point of diversion but that the court has
set forth guidelines applicable to any change which an appropriator
might make in the mode or manner of use of his water right.  Never-
theless, the majority holding in the case represents a viewpoint
that may permit land application operations to function without
accountability to those whose water rights are adversely affected
thereby.

The riparian doctrine is based on the simple premise that he who owns
land traversed by or bordering a flowing stream may make a reasonable
use of the waters thereof so long as such use is reasonable in the
light of others having a similar right.  After making a reasonable
and proper use of the water for his own purposes, a riparian generally
must return all the surplus water to its natural channel before it
reaches the land of lower riparian owners^°.   Because of the amount
of land involved in land disposal, it would seem unlikely that land
to be used for disposal  would be so situated that the return flows
would naturally reach the stream of origin at a point above the land
of all lower riparians.   Therefore, land application is likely to be
in violation of the rights of other water users on the stream.

The situation is further complicated where the return flows from land
application ultimately find their way to a stream different from
their origin.  Interbasin transfers are generally not recognized
under the riparian doctrine.  Of course, a cause of action in cases
of interbasin transfers  or other violations of riparian rights does
not arise in the absence of actual injury to the rights of a riparian
landowner39.  Therefore, legal  obstacles to land treatment operations
may not exist where a water surplus exists.  In the absence of a
surplus, the water rights of landowners on the stream may become a

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significant constraint.  If these water rights are given full  protec-
tion by the courts, land application may be excluded in some riparian
situations without special  provisions for acquiring the water rights
of those affected or returning the effluent after land treatment to
a point above the lower riparian owners.

The riparian doctrine is basically concerned with the interests of
landowners who hold property adjacent to a stream by protecting the
quantity and quality of the flow.  This procedure automatically
results in a substantial measure of protection to the natural  water
course itself, its scenic attributes, and many instream uses such as
recreation and fish and wildlife habitat.  More liberal interpreta-
tion by the courts on such questions as "standing to sue" and "class
actions" greatly expand the potential for liability where the recyc-
ling of effluents on lands significantly modify the regimen of the
stream.  It thus becomes evident that surface water problems must be
acknowledged and effectively dealt with in the design of land
disposal systems to be located in the East.
                     SUMMARY AND CONCLUSION
In conclusion, it is obvious that consideration of potential legal
constraints must be an integral part of the planning and design of
systems for applying wastewater to land for the production of food
and fiber.  Because of the nature of these constraints, they cannot
be treated as final design considerations but must be kept in mind
from the initiation of planning.  Ultimate feasibility of a given
installation may well be dependent on these factors, and basic
aspects of the physical design may be controlled thereby.  Failure
to give complete and timely recognition to these constraints is
likely to produce problems during the operational stage and result
in inefficiency and frustration in the application of the concept.
Only when these constraints are fully incorporated into the total
design framework can the potential of this technique be effectively
realized.
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                            Footnotes


 1.   Water Pollution Control Act of 1948, 62 Stat.  1155  (1948).

 2.   Federal  Water Pollution Control  Act of 1956,  70  Stat. 498  (1956).

 3.   Federal  Water Pollution Control  Act Amendments of 1972, 86 Stat.
     866 (1972).

 4.   J_dL, sec.  301(b)(l)(B).

 5.   I^., sec.  201(g)l, 212(2)(A).

 6.   Id.., sec.  201(d)(l)

 7.   40 CFR Part  35, Appendix A(e)(l).

 8.   Federal  Water Pollution Control  Act Amendments of 1972, 86 Stat.
     866 (1972),  sec. 402(b).

 9.   Center for the Study of Federalism, Green Land—Clean Streams:
     The Beneficial  Use of Waste Water Through Land Treatment.  Phila-
     delphia, Temple University, 1972,  p. 226.

10.   Environmental  Protection Agency, Survey of Facilities Using Land_
     Application  of Wastewater,  Washington, Environmental Protection""
     Agency,  1973,  pY 99.

11.   Cali forni a Administrati ye Cocte,  Title 23, Waters, Chapter  3, Sub-
     chapter  15.

12.   Idaho Board  of Environmental and Community Services, "Rules and
     Regulations  for the Establishment of Standards of Water Quality
     and for  Wastewater Treatment Requirements for Waters of the State
     of Idaho," sec. XI,  1973.

13.   Lees v.  Sampson^ Land Co...92A. 2d 692 (Pa. 1952).

14.   101 C.J.S. Zoning  sec. 135.

15.   James A. Ballentine, Ballentine's  Law Dictionary, Rochester, N.Y.,
     The Lawyers  Co-operative Publishing Company,  1969,  p. 530, 1012.

16.   O'Brien  v. Town of Greenburgh, 268 N.Y.S. 173  (N.Y. 1933),
     aff'd. 195 N.E. 210(N.Y. 1935).
                                  341

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17.  Kedroff v. Town of Springfield, 256A.  2d 457 (Vt.  1969).

18.  St. Louis County v. City of Manchester, 360 S.W.  2d  638  (Mo.1962);
     see also Jefferson County v. City of Birmingham,  55  So  2d 196
     TATa. 195TT:

19.  City of Scottsdale v. Municipal Cout of Tempe.  368 P.  2d  637
     (Ariz. 1962); see also City of Des Plalnes v. Metropolitan
     Sanitary District of Greater Chicago.  ?68 N.E.  2d  42F (111. 1971);
     People Ex. Rel. Scott v. North Shore Sanitary Dist.  270  N.E.  2d
     133 (111. 1971).'

20.  See, e.g. Bader v. Iowa Metropolitan Sewer Co.. 178  N.W.  2d 305
     TTowa 1970TrWard v. Hampden Township. 271 A. 2d  895 (Pa. 1970).

21.  See, e.g.. Fields Sewerage Co. v. Bishop, 30 S.W.  2d 412  (Tex.
     1930).

22.  See, e.g.. City of Marl in v. Criswell. 293 S.W. 910  (Tex.  1927).

23.  See, e.g.. City of Temple v. Mitchell. 180 S.W. 2d 959 (Tex.
     T9?4).

24-  See, e.g.. Aguayo v. Village of Chama. 449 P. 2d  331 (N.M. 1969).

25.  Ryan v. City of Emmetsburg,  4 N.W. 2d 435 (Iowa  1942).

26.  See, e.g., Hulbert v. California Portland Cement  Co.,  118 P.  928
         . 1911); Mitchell v. Mines, 9 N.W. 2d 547  (Mich. 1943).
27.  State ex. rel.  Knight v. City of Miami. 53 So.  2d 636 (Fla.  1951).

28.  See, e.g.. North East Coal Co. v. Hayes, 51 S.W.  2d 960  (Ky.
     1932); Rose v.  Socony Vacuum Corp. 173 Atl. 627 (R.I. 1934).

29.  See, e.g., Love v. Nashville Agricultural and Normal  Institute,
     304  (Tenn. 1922); Swift and Co. v. Peoples Coal and Oil^Co,.,   186
     Atl. 629  (Conn. 1936).

30.  See, e.g.. Berger v. Minneapolis Gaslight Co.,  62 N.W. 336 (Minn.
     1895); Berry v. Shell Petroleum Co., 33 P. 2d 953 (Kan.  1934).

31   W. Prosser, Handbook of the Law of Torts, St. Paul, Minn., West
     Publishing Co., 1971, p. 509.

32.  See, e.g., Hall v. Galey. 271 P. 319 (Kan. 1928); Bumbarger v.
     Walker, 164 A.  2d 144 (Pa. Super. Ct. 1960); Jackson v.  U.S.
     Pipeline  Co., 141 A. 165 (Pa. 1937).

33.  Environmental Protection Agency, op. cit, p. 73.

                                   342

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34.   City of Borbenton v.  Miksch. 190 N.E.  387  (Ohio 1934).

35.   Cason v. Florida Power_£o.. 76 So.  534 (Fla.  1917).

36.   Robert Emmet Clark, Ed., Waters and  Water  Rights, Vol. 5, Indian-
     apolis, The Allen Smith Company, 1972, p.  428.

37.   Metropolitan Denver Sewage Disposal  Dist.  No.  1 v.  Farmers Reser-
     voir and Irrigation Co..  499 P. 2d  1190 (ColoT 197277™^

38.   93 C.J.S. Waters sec. 62.

39.   See, e.g., Virginia Hot Springs Co.  v. Hoover, 130  S.E. 408 (Va.
     1925); Strattpn v. Mt. Hermon Bo.ys'  School,  103 N.E. 87 (Mass.
     1913).
                                  343

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         SOCIAL, POLITICAL, REGULATORY AND MARKETING PROBLEMS
                OF MARINE WASTE-FOOD RECYCLING SYSTEMS*
                            John E. Huguenin
                             Judy T. Kildow
INTRODUCTION

    The coastal areas of the United States are receiving ever mount-
ing public attention due to their increasingly apparent importance to
the national well being.  The pressures emanate from the increasing
demands on the coastal waters and exploitation of their contained re-
sources combined with the increasing risk of destroying some of these
very resources by pollution due to the presence and activities of
large adjacent populations.  Thus,at times,coastal zone resource ex-
ploitation and resource conservation appear to be conflicting ob-
jectives.  But need they be conflicting?

    It has become customary to consider any and all of the waste pro-
ducts of our society as pollutants,rather than resources, and to view
their discharge into the envirpnment as a form of pollution.  However,
the nature and ecological effects of man's wastes are so highly vari-
able that their common designation and implied common impact on the
ecosystem may be a concept both simplistic and misleading.

    There are at least some marine waste-food recycle concepts that
from a scientific and technical viewpoint seem very promising (see
Table 1).  There is also currently considerable effort being expended
both nationally and internationally in the science and technology of
using societies' wastes as a resource in marine aquaculture.  This
does not mean that there are not many serious obstacles to be over-
come before some of the proposed systems can be considered ready for
large scale operational use.  Others, on the other hand, are already
being used on a large scale both intentionally and unintentionally in
sea food production.  Agricultural and fishery processing wastes are
commonly used in formulated diets for animals both terrestrial and
aquatic, possibly without full understanding of some of the potential
 This presentation results from the activities of an interdisciplinary
 study group funded by the M.I.T. Sea Grant Program.
 John Huguenin is a Research Associate  in  the Biology Department of
 the Woods Hole Oceanographic Institution, Woods Hole, Mass.
 Dr. Judy Kildow is an Assistant Professor of Ocean Policy in the
 Ocean Engineering Dept.  of the Massachusetts Institute of Technology,
 Cambridge, Mass.
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                 Table 1.  Uses of,"Wastes" in Marine Aquaculture
         Waste
      Possible Uses
  Possible Benefits
      Comments
Thermal effluents from
power plants
Secondarily treated
domestic sewage
Agricultural and  fishery
processing wastes

Compacted solid wastes
 Sewage  sludge
Provide water temperature
control capability
As fertilizer to grow
marine plants
As components in formu-
lation of feeds

As construction material
for artificial reefs
As fertilizer or as input
for production of protein
to be used in animal feeds
Up to 500% (depending
on circumstances) in-
crease in yearly pro-
duction due to water
temperature control

Large "free" nutrient
source at base of
marine food chain and
also providing a form
of tertiary sewage
treatment

Reduced aquaculture
feed costs

Providing habitats to
enable denser concen-
trations of marine
animals

Reduced aquaculture
feed costs
Greal deal of current
research activity and
commercial interest
Current research
promising - many
possible variations
and applications
Already common
practice

Small scale research
in progress - Not a
preferred material
for reef construction

Small scale research
in progress

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risks involved.  As an example, the over 600 state and federal fish
hatcheries in the U.S. alone utilize more  than 41 million pounds of
fish food yearly, with almost 23 million pounds of this being organic
wastes  (Hinshaw, 1973).  The potential for the beneficial use of
power plant waste heat is widely recognized and is one of the aspects
of waste utilization currently receiving the most attention  (Mathur
and Stewart, 1970^; Yarosh, 19733).  The use of algae in processing
domestic sewage is accepted practice in many parts of the United
States.  Current efforts are in progress to extend these practices to
produce useful food organisms in a marine  environment (Ryther,
Dunstan, Tenore and Huguenin, 1972^; Allen, Conversano and Colwell,
1972^). The use of sewage ponds to grow aquatic food animals is not at
all a new concept (Allen, 1970^). Sewage products which are dumped in
large quantities into our coastal waters are certainly, at least in
part, responsible for the high sea food yields from some of  our
estuaries (Ryther, 1971').  However, this  increased productivity due
to fertilization with wastes is often unintentional, generally not
recognized and does not lend itself to management.  The risks are
nevertheless present.

    Time and research and development effort can be expected to make
some of these concepts both technically feasible and economically
attractive.  It is not clear, however, whether even strong technical
and economic justifications are sufficient to assure the application
and exploitation of new developments by our society.  Apparently not,
for there are other considerations and constraints.  These obstacles
must be systematically and rationally analyzed before considerable re-
sources are expended to develop technically sound systems only to find
them useless due to critical legal, socio-political or psychological
factors.

ADVOCATES AND BASIS FOR OPPOSITION

    Marine aquaculture, in general, is already constrained by a host
of institutional factors.  Previous efforts dealing with these seem to
have concentrated on land and water rights (Henry, 1971°; Kane, 1970*;
Shields, 197010).  However, there are many other problems upon which
effective opposition might focus (see Table 2).  The use of  resources
currently considered wastes in the production of marine foods may in-
tensify institutional scrutiny of such operations, due to both the
nature  of the wastes and the increased visibility of the projects.
This is further complicated by the fact that marine aquaculture does
not have any formally organized interest groups to lobby for its in-
terests and for its rights in the heavily  contested coastal  zone.  In
contrast there exist many well established and powerful groups which
could form effective opposition if they were to perceive marine aqua-
culture as a threat to their interests in  coastal and estuarine areas.
There is no lack of vulnerable points for  opponents to exploit.  How-
ever, this is counterbalanced by the growing awareness of shortages


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  Table 2.  Potential Sources of Opposition to Aquac.ult.ure








'Illegal use of chemicals by aquaculturists.



•Local and state conservation and game regulations.



•Competition for sites from alternate users.



•Industrial fears of more pressures to reduce water pollution.



•Secondary products flooding small markets.



• Countermeasures to prevent poaching and vandalism.




•Pollution potential of large cultures.



•Alterations to coastal areas.



•Transplants of organisms both planned and unintentional.



•Characteristics of occasional disasters.



•Presence in cultures of nuisance diseases and parasites.




•Concerns over potential hazards to public health.
                            347

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and the need to effectively manage our coastal resources.  The fact
that there are no organized advocates at present is a partial advan-
tage in that attitudes of potential political oppositions are still
flexible and there are no commitments to past mistakes.  Waste food
systems in particular have unique possibilities for a broad appeal to
many different elements of our society not possible with straight
commercial aquaculture.  Many of the potential opposition groups may,
if care is taken, become strong supporters.  This is due to the multi-
objective possibilities of such systems which combine pollution con-
trol, growth of food and the use of otherwise wasted "resources".
This provides both a strength and a weakness.  Making judgements in
areas where objectives conflict may prove troublesome.  In addition,
there is the danger that with more research, what now appear to be
generally compatible objectives may prove to be incompatible.  The
broad appeal may also lead to a premature "push" for acceptance re-
sulting in opposition based on scientific and technical issues which
could easily be diffused with more thorough research.  Many of these
potential issues involve areas in which so little research has been
done that it is not even known with certainty whether or not sub-
stantial problems in fact exist.  This is especially true for marine
waste-food recycle concepts, due to the many potential and unresolved
questions particularly in the areas of public health and quality
assurance.  Combining two such critical human activities as food pro-
duction and the removal of wastes can very easily produce opposition
based on fear resulting from uncertainty.

PREREQUISITES FOR ACCEPTANCE

    There are several prerequisites before societal acceptance of
waste-food or water reuse systems for general use is a realistic
possibility.  First, all legitimate public health concerns must be
adequately assessed and resolved (Shuval and Gruener, 1973^).  Two
examples where sewage products are involved stand out.  One is the
problem of enteric virus (Ramas, 197012; Metcalf, Vaughn and Stiles,
197013)t and the other is the possible presence of known carcinogenic
chemicals in the input wastes.  There are also unknowns involving the
transmittal and concentration of various "pollutants" through complex
food chains which include man, and the long term effects of these
processes.  Systems must be developed to reduce or circumvent the
high risk areas, and adequate public health safeguards must be pro-
vided.  If this is not done, there is very little chance for societal
acceptance unless the perceived needs are so imperative and pressing
that high risks are warranted.  The inverse is also true.  Definite
markets must either exist or be created for even low risk waste-food
systems to receive societal and regulatory acceptance.  These markets
may be  for direct domestic use or more indirect, such as  for the
benefit derived from exports or even for non-material advantages,
such as for environmental enhancement.  But these needs must be per-
ceived, and believed by at least a portion of our society for any
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hope of favorable actions.  The very existence of this conference
indicates that, to some degree, this is  already  fact.

    Given a latent but generally  perceived  societal need, potential
markets, and promising waste  food system possibilities, at least
some favorable public policy  decisions  seem a prerequisite for the
establishment of any full  scale demonstration projects.  At a mini-
mum, it would require some indications  that if the systems meet some
specified performance levels  the  outputs would be approved for public
use.   It is important to  remember that  most systems  at  this point are
not completely proven, and that uncertainties also exist about both
performance at full  scale  and over economic viability.  These un-
certainities  can remain a  basis  for skepticism and opposition in
spite  of successful  research  efforts. The economics,  in particular,
of many proposed aquaculture  systems are open to question, because
almost all  the published  research has  been done  in very small labora-
tory experiments and the  little data that exists for large cultures
is  generally  proprietary  information.   In addition,  pilot plant
facilities  are usually  designed to research a spectrum of possible
concept variations,  operating conditions and management approaches.
As  a  consequence,  they  are usually much smaller than the ultimate
systems,  since  the flexibility required for their continually changing
experiments and  from their physical plants would be cost  prohibitive
at  full  scale.   Therefore the operations and economics of  such re-
 search facilities, while very useful, have  limitations when attempts
 are made  to directly apply these experiences to large systems de-
 signed for routine use under closely defined operating conditions.
Thus,  these uncertainties can be fully  removed only with the design,
 construction and operation of full scale demonstration systems,  which
 need a favorable public policy environment  to justify the tremendous
 efforts and expenses involved.   This applied development stage may
 be the weak link in aquacultural development (Trimble  1972^) due
 to the current lack of government  support  and to the high risks and
 insufficient incentives for  private industry.   However, it must be
 emphasized that small scale  laboratory testing  and  sub-scale pilot
 plant facilities are an essential  prerequisite  for providing the
 needed design information for full scale demonstrations.  There are,
 however, pressures  to speed  the  development of  promising waste-food
 systems to large scale applications.   Prematurely committed demon-
 stration projects that fail, due to inadequate  preparation  pose a
 real  danger in that they  may discredit basically sound solutions.

              ermining  "safe" levels of contaminants  in foods poses

              s
                                           ..
             to all is that the       S          roducts found on the
                                    349

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future systems.  Also, while not necessarily easy, it is probably
not as restrictive to the aquaculturist as it may at first appear.
It must be remembered that the aquaculture output is a managed pro-
duct and inherently susceptible to much improved quality control
comparable to "wild" products, which in many cases have not even been
carefully monitored or analyzed.  When analyzed, the latter may be
found to have surprisingly high levels of some "pollutant" whose
presence may not even be related to any activities of man (e.g.,
mercury in swordfish and other fish).  Requiring completely "zero
additions" of any pollutant, such as is done for "known" carcinogenic
chemicals in applying the Delaney amendment to the Food, Drug and
Cosmetic Act of 1938, while allowing "non harmful" quantities of the
same substances if found "naturally", is certainly an unrealistic
double standard which could easily preclude the hope and potential
promised to mankind by aquaculture, whether it uses wastes in the
process or not.

CHOICES

    What sort of policy options do the marine waste-food advocates
have?  It is clear that the policies adopted by advocates as well as
their decisions in the fields of marketing and system design will
affect the types and severity of the opposition.  A major option is
in  the choice of the initial outputs which are somewhat independent
of  the long term objectives of the projects and seem a flexible
variable based on what is currently known about aquaculture food webs.
Thus the initial outputs may not be marine foods for human consumption
but rather products for education, research, bait or only as food
additives.  It seems probable that societal acceptance will be in-
versely proportional to the intimacy of the contact with the product
and its waste input (Bruvold and Ward, 197215).  Thus, the probability
of successful societal adaption might be increased by initially con-
centrating on other than direct food outputs.  Early systems should,
as much as possible, hedge against uncertainties by maintaining the
capability of producing a spectrum of different products.  Another
option, assuming a direct food output is desired, is whether to pro-
duce relatively small quantities of high quality, high value "luxury"
sea foods or go for large quantity "protein".  Production oriented
aquaculture operations in both developed and less developed countries
have consistently and without any notable exceptions attempted to
produce "premium" foods.  In the less developed countries this has
often been primarily for export although local food shortages may
exist.  In addition, experience has shown that cultured products al-
most invariably receive premium prices over the same product from
natural sources.  Thus, if economic criteria alone are used, any
large scale aquaculture operation is almost certain to involve high
unit value products.  However, waste food systems are much more like-
ly, due to their association with public utilities, potential health
hazards and involvement with current public concerns, to have decision
criteria that are not based solely on marketplace economics.

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    Another major policy choice  to be made  by waste food advocates is
whether to go for a high degree  of publicity, presumably favorable,
or adopt a policy of secrecy.  Obviously, this decision in many cases
may not be completely within  the powers  of  this  group.  Aquaculture,
in general, has been receiving a great deal of publicity, almost all
very favorable and also, by and  large, unrealistically optimistic.
On the other hand, actual  commercial aquaculture ventures have gener-
ally been carried out with a  very minimum of publicity.  This may be
due both to the proprietary nature of the operations and their vul-
nerability to opposition because of some of their practices, as well
as to the generally unfavorable  legal/political  environment.  In
addition, successful aquaculturists have generally been well inte-
grated with their rural power structures, have usually conducted their
business completely on private land and  have operated on a scale small
enough not to form a focus for opposition.   In contrast, well organ-
ized efforts to set up large  commercial  aquaculture operations needing
substantial capital and specialized expertise have often met failure,
due in part to their planned  scale of operations and association with
"outside interests".
    A noticeable exception to this low profile policy have been opera-
tions carried out in cooperation with power plants.  However, most of
these operations have been experimentally oriented rather than direct
commercial attempts.  In the  few commercial cases, publicity has been
due to the power company's desires for favorable public relations in-
volving their thermal discharges rather  than any change of policy on
the part of aquaculturists.   In  addition, while  these few operations
have not been opposed, they have been relatively small in scale with
little impact and they have been surrounded with an aura of research
and experimentation.  Thus, reactions to these operations are not
necessarily indicative of  the reception  to  be experienced by large
scale operational systems. It may not be reasonable to assume that
the past history of secret or semi-secret aquaculture operations is
a realistic possibility for future large aquaculture systems, es-
pecially if wastes are used.  A  policy of secrecy  for the planning
and development of a large commercial system might be realistic only
if it were known with confidence that the use of wastes so lowered
the production costs and that the associated technical risks and
probabilities of unpleasant external effects were  so low that the
risks involved with presenting society with a "fait accompli" would
be justified.

CONSUMER ACCEPTANCE

    Assuming that all other problems were solved,  there is still the
question of consumer acceptance  of waste-grown sea foods.  There are
several important variables involved.  Probably  the most critical  is
the degree and circumstances  surrounding the differentiation of
waste grown marine foods from other sources of the same products, as
may be required by regulatory agencies.  This is a problem mostly  in
the fields of packaging, labelling and advertisement.  It is

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 anticipated,  but not yet proven,  that the products  will look,  taste
 and smell the same as their managed counterparts  which are not grown
 with "waste"  inputs.  This is  obviously a critical  assumption.  If
 little or no  differentiation is  required, there is  in all  likelihood
 no marketing  problem.  Past rulings of the Food and Drug Administra-
 tion and the  Federal Trade Commission, suggest assuming the worst
 case conditions.

     Assuming  that distinct labelling will be  required,  to  what extent
 does a consumer's feelings of  repugnance for  wastes,  which are known
 to be acquired rather than innate or physiological, carry  over to
 food products grown with such  materials?  Unfortunately, there does
 not appear to be any available research directly  dealing with  this
 interesting question.  However,  if the fishing aspects  of  the  Santee
 Project (Merrel et al., 196716)  and the common use  of wastes in agri-
 culture are any indication,  this  associative  link may either not
 always occur, or can be nullified, even when  the  physical  facts are
 clearly known.   There is also  considerable literature on public
 attitudes towards the closely  allied concept  of using reclaimed water
 (Bruvold and  Ward,  197017;  Bruvold, 197118; Bruvold,  1972a19;
 Bruvold, 1972b20;  Gallop Poll, 197321).  Small scale  surveying at
 M.I.T.  (to be published)  has shown good correlation of  this data with
 consumer attitudes  towards  seafoods grown with waste  inputs.   Even
 studies on attitudes toward  irradiated fish (Yankelovich,  196622)  and
 on fluoridation (Sapolsky,  19682^) show surprising  similarities.
 However, while  it is relatively  certain that  a large  segment of the
 public would  not oppose the use  of wastes in  marine aquaculture,
 there is little information on the numbers or degree  of  activism of
 either strong supporters  or strong opponents.   This is a critical
 void,  since small vocal minorities have often swayed  public opinion
 in similar situations as  demonstrated  by the  history  of  local
 fluoridation  decisions  (Sapolsky,  19682^).

     It  must be  remembered  that the fact that  aquacultural  products
 are  cultured  and managed, presumably under adequate controls,  is a
 strong  selling  point and  one exploited  by all  present commercial
 aquaculture operations.   The available  literature indicates that
 those  individuals who approve  of  the use of potentially  objectionable
 inputs  do  so  under  the  assumption  that  such systems,  if  established,
would have  adequate  controls.  These feelings  of assured quality may
be strong enough for some consumers  to  make waste-food products pre-
 ferred  over sea  foods  of  "unknown" quality from other sources.   In
all  studies,  the major  sources of  consumer opposition have been un-
certainties and  intuitive feelings  concerning uncleanliness, impuri-
 ties and lack of public health safeguards.  Thus, the public's  con-
 fidence  in  the  safeguards and  quality control  of the output products
may well be the  single most important factor in consumer acceptance
of waste grown  foods.  Obviously one incident  of bad publicity in-
volving  the quality  of  the products  could  be extremely damaging.
                                 352

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    These problems highlight the importance of deficiencies and in-
adequacies in monitoring and quality control technology.  While very
sensitive analytic methods have been developed for detecting most
pollutants and contaminants, these methods, in many cases, are not
adequate for long term continuous or quick reaction monitoring in a
biologically active seawater environment,  as would be required for
applications in operational systems.   Research and development effort
is especially needed to apply and modify current  laboratory methods
and equipment to produce reliable and  practical monitoring systems.

CONCLUSION

    In spite of the many problems  (see Table 3),  the future for
marine waste-food recycling systems is very promising.  In fact, a
good case could be made that such systems  are already operating un-
intentionally on a large scale  in some regions of our coastal zone.
While at the present time  the recycling  of wastes into  our food
supplies is neither planned nor managed, this is  sure to change.  At
some point, the increasing demands  on  the  coastal zone  for both waste
disposal and food production can be safely met  only by  acquiring some
degree of control over the systems  involved.  Only in managed situa-
tions can the risks be controlled and  production  increased.  Our real
choice is whether this is  to be done methodically or haphazardly.  Due
to the required long lead  times for planning, research  and development,
the time for choice may be now.

ACKNOWLEDGMENTS

    The authors wish to thank Drs.  John  Ryther, Leah Smith and
William Kerfoot for reviewing  the draft  manuscript.
                                  353

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         Table 3.   Problem Areas Requiring More Effort
.  Resolving potential public health hazards.

.  Establishment of realistic "acceptable" levels for "pollutants".

.  Better understanding of food chain dynamics.

.  Applying existing technology to monitoring  and control
  problems.

.  Support for promising "pilot plant" and "demonstration"
  projects.

.  Economic evaluations and comparisons with alternative
  approaches.

.  Market testing of outputs for quality and acceptance.

.  Development of public and private institutional infrastructure.
                             354

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     1970.

18.  Bruvold, William  H.   Affective  response  toward uses  of re-
     claimed water.  J.  of Applied Psychology 55:28-33, 1971.

19.  Bruvold, William  H.   Public  knowledge  and opinion  concerning
     new water  sources in California.   Water  Resources  Research  JJ
     (5): 1145-1150, 1972a.

20.  Bruvold, William  H.   Consistency  among attitudes,  beliefs  and
     behaviour.   J.  of Social  Psychology  86_: 127-134,  1972b.

21.  Gallup  Poll  Release.  Americans  Concerned that Water Resources
     Will Become  Permanently Low  in  20 Years.   Princeton, N.J.,
     American Institute  of Public Opinion,  Wed., May  16,  1973.

22.  Yankelovich,  D.   Cost benefit  study  of selected  products in
     Atomic  Energy Commission's  low-dose  food irradiation program,
     prepared for the  A.B.C.  by Daniel Yankelovich,  Inc., N.Y.,N.Y.,
     Report  No. NYO-3666-1,  1966, 226  p.

23.  Sapolsky,  Harvey  M.   Science, Voters and the  Fluoridation
     Controversy.   Science 162:427-433, 1968.
                                   356

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                RECYCLING FOR A PURPOSE-BUT FOR WHAT
                   PURPOSE?  A SOCIOLOGIST'S VIEW

                                by

                      Lewis H. Irving, Ph.D.*


     With the political and social unrest persisting in today's world,
the public is beginning to question the very existence of many of the
social institutions.  One constantly hears statements such as:  "There
is no need to be concerned with the population problems, because as
the population grows so will our level of technology and everything
will be O.K.—besides, it's my right to have as many children as I
want"; or "There is no real energy crisis, it's just the government
and big oil companies trying to drive the price of gasoline up, the
little man out and the customers off their backs.  There is enough
energy for everyone."  In essence, the people do not know which line
of rhetoric to accept and when a true social issue is presented they
view it with a jaundice eye.  A key question which the physical scien-
tific community must address is "How do we determine the existing
public opinion toward a topic, and once known, how do we alter those
opinions to facilitate a social acceptance of the problems in order
to allow a joining of efforts on the parts of the scientific community
and the public community to abate the issue?"

     Turner and Killian have suggested that

     "At tijnes...problems arise for which tradition provides no
     clear-cut solution.  An issue is created and a public arises.
     Public opinion is formed and reformed, often with startling
     shifts, until the issue disappears.  The major norms of a
     society as embodied in its institutions persist for long
     periods in spite of varying degrees of dissatisfaction with
     them.  But periodically social movements develop whicb^cul-
     minate an institutional change and cultural revision."
                      WHAT IS PUBLIC  OPINION?


     In order to evaluate the attitudes  of the public, one must first
understand that public opinion  is  the "...effective  expression of the
public.... /realizing that; the public...is a  dispersed group of people
     "Assistant Professor  of  Sociology,  Central  State University,

                            Lewis M.  Killian.   Collective Behavior.
      J. VAJ. i Aw ± y * ^t*^f^* * *• * • *•*•                  _        A
Engelwood Cliffs,  Prentice-Hall,  Inc.,   1957,   p.  J.

                                   357

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Interested in and divided about an issue, engaged in discussion of the
issue, with a view to registering a collective opinion which is ex- 2
pressed to affect the course of action of some group or individual."
Of course there are many definitions of public opinion but for purposes
of this paper the above suffices.

     Consequently, one may interrelate public opinion and individual
opinion understanding that the feeling, attitudes and actions of the
group are attributable to the phenomena of the individual.   "Public
opinion then becomes a form of group thinking, and the process bears
more than an analagous relation to the individual's 'complete act of
thought.'"1*

     In order then to understand the attitudes of the group, one must
measure, in some  form or another, the attitudes of the individual
toward the question under consideration.  Peter Kelvin explained that

      "...an attitude has at least two components:  there is the
      'object1 of  the attitude or, rather, what the individual
     knows or believes about it; and there is his feeling towards
      it, which  is the basis for how he evaluates it.  If we...
      look at the  meaning we give 'attitude1 in everyday language,
     we  find a  third component, by implication related to behav-
      iour.'^

To examine the  three components of an attitude from another perspective,
"Feelings are often referred to as the affective components, thoughts
as the cognitive  component, and predispositions to act as the behav-
ioral  component."°

      To  the  layman the behavioral and cognitive components of attitudes
appear to be  the  most obvious factors for measurement; however, the
outward  displays  of personality do not necessary coexist with the
inward thought  patterns and vice versa.  To explain this in another
fashion:   when a  person discriminates either positively or negatively
toward another, it does not mean there is a coexisting positive or
negative prejudice acting  in conjunction with the former.  Therefore,
"Prom the  standpoint of research technique, observable behavior is
in fact  very  difficult to measure, and the actual behavior observed
      2Ibid.  p. 219.
      ^Lunberg, George A.   Public Opinion from a Behavioristic View-
 point.  American Journal of Sociology.   3£: 387-405,  November 1930.
      ^Clark, Carroll D.   The Concept of the  Public.  Southwestern
 Social Science Quarterly.  13.:311-320,   March 1933-
      t>Kelvin, Peter.The Bases of Social Behaviour—An Approach in
 Terms of Order and Value.  Great Britain, Holt,  Rinehart  and Winston,
 1970,  p. 40.
      6secord, P. F. and C. F. Backman.   Social Psychology.   New York,
 McGraw-Hill,  1964,  p.  97-
                                   355

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may have relatively little to do with basic beliefs and feelings,"?
Consequently, the focus of attitude research is toward the affective
component.  In essence, attitude measurement is the measurement of
feelings and opinions directed toward an object or issue.

     This phenomena gives rise to one of the major difficulties
prevailing between the physical sciences and the social sciences.  How
does one measure an ambiguous concept that has no mass or physical com-
ponent?  To do this the social scientist has had to essentially rely
on a method of ordering or scaling.


       ATTITUDE MEASUREMENT FOR THE FACILITATION OP CHANGE


     Predictivity must necessarily be based upon extrapolative condi-
tions and the facilitation of attitude changes toward ecological
phenomena must be accomplished by congruence between theory and prac-
tice.  In order to accommodate this task the community must be pre-
conditioned to accept the feasible technological solutions to perceived
problems.  The principle of attitude measurement or scaling "...is a
form of planned collection of data for the purpose of description or
prediction as a guide to action or for the purpose of analyzing the
relationship between certain variables	"°  If one is to understand
the level of thinking of an individual or public toward a topic (e.g.,
how a public feels toward recycled products), one must develop an
instrument of measurement appropriate for the specific task.  Once
this instrument has been developed, the data collected and analyzed,
the social researcher will then be able to formatively design a tech-
nique which will be useful in redirecting the public opinion toward a
more positive understanding of the concept.

     To change the attitudes of a conmunity or society is not an ef-
fortless task, as Thorstein Veblen warned, "Institutions are products
of the past process, are adapted to past circumstances, and are there-
fore never in full accord with the requirements of the present."9
Lewis A. Coser further examined Veblen's theory as

     "...a new technology erodes vested ideas, overcomes vested
     interests, and reshapes institutions-in accord with its
     own needs.  But this process may take considerable time,
     and in that time lag—when, for example, an industrial society
     v
     'Kelvin,  p. 40.
     ^Oppenheim, A. N.  Questionnaire Design and Attitude Measurement.
New York, Basic Books, Inc.,1966,p. 1.
     9Veblen, Thorstein.  The Theory of the Leisure Class.  New York,
Modern Library,  1937,  p. 191.


                                  359

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     is still governed by legal and moral rules dating from
     the handicraft era—society suffers from the waste that is
     brought about by the lack of correspondence between its
     institutions and its technology."10

     The change of attitudes of a social system ".. .implies a change
in the individual's order of values, associated with changes in his
beliefs and behaviour; and in essence any change in the way in which
the individual orders his environment naturally affects the predictions
which he makes about it and the way in which he adjusts to it and ma-
nipulates it."11  The ability of the individual to change to the new
technologies will be in concordance with the instrumentation used to
provide the special knowledge, the efficiency with which the communica-
tion or learning devices are utilized and the effectiveness or its
organization.

     To further extend one's understanding of this phenomena, Turner
and Killian suggest that "Certain fundamental conditions will determine
the effectiveness of attempts to manipulate any type of collectivity."12
These fundamental conditions are:

     "(1)  All effective influence obviously depends first of all
     on gaining access to the group to be influenced....

      (2)  .. .the receptiveness of the mass toward the proposed
     course of action or thought. In the mass, the receptiveness
     /meaning interests, motivations and understandings; is that
     of the individual, since individuals must decide and act	

      (3)  ...the possibility of carrying out the proposed
     action....

     fAnd finally;

      (4)   ...the inclination and opportunity to act, the recipient
     still makes some evaluation of the appeal itself and of the
     assumed source of the appeal."13
       Coser, Lewis A.  Masters of Sociological Thought—Ideas in
Historical and Social Context.  New York,  Harcourt Brace Jovanovich,
Inc.,  1971,  pp. 272-273.
     llKelvin,  p. 59.
     12Turner and Killian,  p. 2?7.
     ISlbid.  pp. 277-295.
                                  360

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                              SUMMARY
     In order to achieve a social change of attitude toward a new
philosophy of life the social scientist will have to be familiar with
the current thinking and trends being developed in the physical scien-
tific coirmunity.  The social scientist will have to evaluate the cur-
rent level of thinking of the community, design a program to institute
social change and implement these changes in order to bring the social
acceptability of the community in line with the degree of current
technical know-how.  This will require a formidable amount of lead
time, which heretofore has not been a luxury afforded the social
scientist.  In short, the apathetic attitude demonstrated by the
general public and many decision-makers cannot be dispelled unless
social and physical sciences merge toward a collective approach to a
solution.
                       SELECTED BIBLIOGRAPHY
Clark, Carroll D.  The Concept of the Public.  Southwestern Social
     Sciences Barterly.  13.: 311-320,  March 1933-

Coser, Lewis A.  Masters of Sociological Thought—Ideas in Historical
     and Social Context.  New York.  Harcourt Brace Jovanovich, Inc.,
     1971-

Kelvin, Peter.  The Bases of Social Behaviour—An •Approach:'-in Terms of
     Order and Value.Great Britain,Holt, Rinehart and Winston,
     1970.

Lunberg, George A.  Public Opinion from a Behavioristic Viewpoint.
     American Journal of Sociology.  36.:387-405,  November 1930.

Oppenheim, A. N.  Questionnaire  Design and  Attitude Measurement.  New
     York,  Basic Books, Inc.,1966.~

Secord, P. F. and C. F. Backman. Social Psychology.  New York,
     McGraw-Hill,   1964,  p. 97-

Turner, Ralph H. and Lewis M.  Klllian.  Collective Behavior.  Englewood
     Cliffs,  Prentice Hall, Inc.,   1957-

Veblen, Thorstein.  The Theory of the  Leisure  Glass.  New York.   Modern
     Library,   1937-
                                   361

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PART 6   NEW OR INTEGRATED SYSTEMS
         SESSION CHAIRMAN

       GEORGE H. ALLEN, Ph.D.
       PROFESSOR OF FISHERIES
    HUMBOLDT STA TE UNIVERSITY
        ARCATA,  CALIFORNIA

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               THE MICHIGAN STATE UNIVERSITY
             WATER QUALITY MANAGEMENT PROGRAM

                            by

        T. G. Bahr,b R. C. Ball,C and H. A. Tannerd

                       INTRODUCTION

One of the Nation's greatest environmental questions is
what to do with vast quantities of municipal wastewater.
Conventional methods of wastewater treatment can con-
tribute importantly to wastewater clean up but the dis-
charge of even these wastes has created eutrophication
of lakes and degradation of waterways through the nu-
trient materials that they contain even after advanced
treatment.  We are also becoming aware of perhaps an
even more important problem.  Many products used by man
contain elements that represent scarce and diminishing
resources or compounds that have a high energy demand in
their manufacture.  In an economy faced with both materi-
al and energy shortages it seems foolish to manufacture
products at high cost to both of these, use them once
and then discard them.  But this is exactly how we handle
plant nutrients.  We mine our increasingly limited sup-
plies of phosphorus and infuse our scarce fossil energy
into the production of nitrogen fertilizers in order to
sustain the food production that feeds the millions in
our cities only to waste these elements into the nearest
stream.  This gross mismanagement coupled with require-
ments for high water and air quality and the difficulties
of removing nutrients to levels acceptable to discharge
into natural waters has prompted an extensive search for
alternate methods.

It is well known that nutrients present in wastewater can
be turned into the production of harvestable food and fiber
o
 This program  is supported by grants from the Ford Founda-
 tion,  Rockefeller Foundation, Kresge Foundation, the U.S.
 Environmental Protection Agency, Office of Water Resources
 Research,  and the State of Michigan.

 Director,  Institute of Water Research, Michigan State
 University
/-•
 Associate  Director, Institute of Water Research, Michigan
 State  University

 Director of Natural Resources and Assistant Director for
 Water  Quality Management, Institute of Water Research,
 Michigan State University

                             362

-------
if applied in appropriate amounts to the land or to the
water.  The quantity and schedule of application of waste-
water to agricultural lands or to aquaculture systems to
achieve an optimal level of food or fiber production
and, at the same time, achieving a high level of water
renovation is not well understood at this time.  The
number and depth of research studies in this area has been
very limited and there are many important questions vital
to human health, energy and resource conservation that
have not been answered.  We believe that maximum efficiency
of research on these questions can be obtained from an
integrated study of recycling systems incorporating the
facets of both terrestrial and aquatic ecosystems.  The
plans, estimates and designs for functioning reuse and
recycling systems cannot be based on concepts but must
be reduced to reasonably accurate estimates based on
experience gained with working model systems.  Such sys-
tems will require a coordinated multi-disciplinary effort
of considerable magnitude.

                DEVELOPMENT OF THE PROJECT

Michigan is a state with vast water resources, bordering
on four of the five Great Lakes.  Our interests for the
future are all closely tied to the discharge of wastes in-
to these bodies of water.  Almost without exception every
pollutant that leaves a sewage disposal plant or runs off
from our fields or urban areas will reach one of the Great
Lakes.  This can be seen in Figure 1 by examining the
position of Michigan's major tributaries in relation to the
bordering Great Lakes.  Recent estimates by staff of the
Institute of Water Research at Michigan State University
place phosphorus  (P)  losses to Lakes Michigan and Erie from
Michigan's lower pennensula at approximately 35 million
pounds per year.  The greater Detroit area  in southeastern
Michigan alone contributes nearly  20 million pounds per
year, nearly all of which  is from municipal or  industrial
sources  (Bahr1).  Michigan State University has a long
history of research on pollutants  and nutrients in  streams
 (Ball and Bahr2) and  a concern on  the part  of many  of the
staff for the well being of our water resources.  The
wastewater recycling  project to be described in this paper
was designed and built as  a direct result of these  con-
cerns .

The development  and administration of the project is a
major part of the water programs of Michigan State  Univer-
sity's  Institute of Water  Research located  on  the main
campus.  Prototype plans received  financial  support and
approval to  continue  from  the Michigan  State University
Board of Trustees in  1966.  Since  then  we have  been helped

                            353

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Figure 1
Prinicpal rivers in the State of Michigan and
location of the Red Cedar River watershed,
site of the Michigan State University waste-
water recycling program.

                            364

-------
and encouraged by many faculty, citizens and public of-
ficials in both state and federal agencies.  Funding was
eventually obtained from the Ford, Kresge, and Rockefeller
Foundations and later from the U.S. Environmental Protec-
tion Agency and from the State of Michigan through its
Clean Water Bonding Program.  The project construction
was completed during the Fall of 1973.  Cost of construc-
tion, exclusive of land acquisition, totaled approximately
$2.5 million.

                    PROJECT DESCRIPTION

The physical facility for the Michigan State University
wastewater recycling project consists of four basic ele-
ments: (1) a conventional activated sludge sewage treat-
ment plant; (2) a 4.5 mile transmission line; (3) a lake
system; and (4) a land irrigation system.  This system is
schematically shown in Figure 2.  The following is a more
detailed discussion of the facility.

SEWAGE TREATMENT PLANT AND TRANSMISSION LINE

The East Lansing Sewage Treatment Plant services the City
of East Lansing, Michigan State University with its 50,000
students and employees, and the adjoining community and
represents the first element of our system.  It is present-
ly undergoing modification and enlargement to a capacity
of 15,000,000 gallons per day.  As an integral part of
this modification the Michigan State University program
will develop and operate a parallel unit within this plant
that will have a capacity of 2,000,000 gallons per day.
Our portion will differ from the total plant in that the
effluent being received will not have pre-treatment for the
partial chemical removal of phosphorus.  The effluent
(secondary or primary) from this subunit can be directed
to a pumping station with a present capacity of 2,000,000
gallons per day and provisions for additional pumps to
increase its capacity to 6,000,000 gallons per day.  Flow
will be transmitted through the second element of the sys-
tem, a 21 inch concrete-asbestos pipeline which traverses
4.5 miles to the southern border, of the Michigan State
University campus and on to the 500 acre research site.
Here it discharges into the first of four man-made lakes.

LAKE SYSTEM

The flow from the first lake is then by gravity through
each of the other lakes and to a control building and pump
house servicing the adjacent spray irrigation site.  The
lakes have a total surface area of 40 acres with the maxi-
mum depth of 8 feet at each outlet structure and a mean

                            365

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                PRIMARY and SECONDARY
                    TREATMENT
                    ,	-^v
                  X      4   J
                   \-~^~^^rJ
                                       CONTROL
                                       STRUCTURE
         LAKE SYSTEM-186 ACRES
         INCLUDING 40 ACRES OF LAKES
       22 INDEPENDENT SPRAY
        UNITS (REMOTELY
        CONTROLLED AND
          PROGRAMMABLE
           GROUNDWATER
            and EVAPO-
           TRANSPIRATION
STREAM
          TERRESTRIAL SYSTEM - 314 ACRES
Figure  2.  Schematic of the Michigan State University
          wastewater recycling system.
                         366

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depth of 6 feet.  This depth was chosen to maintain the
entire bottom within the euphotic zone in order to en-
courage the growth of rooted aquatic plants.   The bottom
was also contoured in a very uniform manner to ease their
mechanical harvesting.  Each lake has a collection basin
at the outlet so that water may be lowered to collect
fish or other aquatic fauna in a very small area.  This
area is serviced by a ramp to allow access by both boats
and trucks.  Control of discharge and lake level is af-
forded by both sliding gate valves and slash boards.  The
interlake transfer system and connection to the irriga-
tion site is so designed that effluent can be taken di-
rectly from the pipeline or water can be intercepted at
the discharge of any of the lakes in the system or mixed
from any combination of lakes (see Figure 2).  This fea-
ture will afford researchers with a wide range of water
qualities to be applied and tested on the irrigation site.

MARSH SYSTEM

Marsh systems with their high rates of internal nutrient
cycling may prove to be an extremely efficient system for
the uptake and conversion of wastewater nutrients into use-
able products.  To test the possibilities and feasibility
of this idea we constructed three, one acre marshes which
are fed from the discharge of Lake 2 in the system.  Re-
turn water from the marshes enters Lake 3.  The basins of
the marshes were constructed in  a terrace design that re-
sulted in three zones of depths  of 18, 24 and 36 inches.
It is estimated that this will allow us to create in these
basins biota quite comparable with natural marshes  of this
area.

In the construction of the lake  basins and marshes  par-
ticular attention was given to the sealing of the basins
to prevent loss of water through the bottom  soils.  They
were sealed with native clay and percolation tests  indi-
cated a low permiability of 0.07 inches per day.

LAND IRRIGATION SYSTEM

The last  element of the system  is a  terrestrial  site  lo-
cated on  350 acres immediately  south of the  lake  system.
As with much of the soils  of the glaciated parts  of Michigan
and the Great  Lakes basin  there  is a large array  of soil
types ranging  from heavy clay to light sandy soils.   Within
the terrestrial site  are forested areas,  a pine  plantation,
cultivated fields and fields that have allowed  to regress
into old-field plant  associations.   The  land is  gently
rolling and  the entire  area  is  drained by the Fenton  out-
let which enters a tributary of  the  Red Cedar River which

                             367

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in turn flows into the Grand River, a tributary of Lake
Michigan  (Figure 1).  The irrigation site is bounded on
three sides by an 800 foot buffer zone where we will not
spray effluent at the present time but it is available
for overland flow irrigation and research not requiring
spraying  of effluent.

Within the terrestrial area is a 145 acre spray irrigation
site.  Twenty-two individual valves, remotely controlled
and programmable from the control building, direct the
flow of water to the sets of surface irrigation pipe.
Four, 700 foot tapered aluminum laterals extending north
or south  from a valve comprise a set.  Water supply to
each set  is carried by a 21 inch underground pipe and the
system is designed for winter operation.  Spraying is by
conventional Buckner 8600 sprinkler heads.

Within and surrounding both the lake and land sites we
have drilled approximately 60 wells to monitor the level
and quality of subsurface water.  Depths vary from just a
few feet  into the glacial drift to over 200 feet, within
the underlying sandstone aquifer.  Approximately 1/3 of
these wells will be equipped with automatic monitoring
and sampling equipment as part of a major effort to charac-
terize the subsurface hydrology in the region.  All wells
are cased, sealed and fully protected from external con-
tamination.  Surface water flow for the entire watershed
in study  will be measured by means of a network of weirs.
Many of these are now installed.  Climatological stations
at selected points in the area provide the additional
information to generate a detailed look at the entire
hydrological cycle for the area.

TOTAL SYSTEM FLEXIBILITY

The physical facility as it was designed affords maximum
flexibility in the development of research involving an
integrated land and lake management system for the res-
toration  of water quality and the recycling of nutrients.
We have the potential for hydraulically stressing any area
of the terrestrial system with effluent far in excess of
its capacity of handling it to the extreme of no water
application above that of natural rainfall.  Within this
range we  have the capability of spraying at a variety of
qualities as selected by the investigator, and on any
temporal  pattern that lends itself to research in the
development of a particular management strategy.  Table 1
gives the estimated loading rates of various chemical
parameters if secondary effluent were directly applied to
the irrigation site.
                            368

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   Table 1.  Estimated soil loadings per acre on application of one  inch of East
             Lansing Sewage Treatment Plant secondary effluent.
Ui
0\
<0
Concentration in
Secondary Effluent Grams per
Chemical Parameter Og/1) Acre inch
Organic Nitrogen
Nitrate Nitrogen
Nitrite Nitrogen
Ammonia Nitrogen
Soluble Phosphorus
Total Phosphorus
Total Carbon
Total Organic Carbon
Dissolved Organic Carbon
Suspended Solids
Volatile Solids
Chlorides
Iron*
Manganese
Zinc
Nickel
Copper
Mercury
2.2
3.07
0.25
9.70
1.1
4.9
150
30
20
63
25
261
0.81
0.09
0.19
0.11
0.06
0.00005
227
317
26
1261
143
637
15,450
3,090
2,060
6,489
2,575
26,883
83
9
20
11
6
0.005
Pounds per Pounds per Acre
Acre Inch Per Year **
0.5
0.7
0.06
2.77
0.3
1.4
34
6.8
4.5
14.3
5.7
59.1
0.18
0.02
0.04
0.025
0.013
0.00001
36
50
4.1
200
23
101
2,445
489
326
1,027
408
4,254
13.2
1.5
3.1
1.8
1.0
0.0008
    *Iron  is being  added  for  chemical phosphorus  removal  at  the  East  Lansing  Sewage
    Treatment  Plant.
    **At a rate of  two  inches per week between March  and  November  (36 weeks).

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Within the capabilities of the system we can chlorinate
the effluent at several points including the point at
which it leaves the sewage treatment plant, at the point
where it enters the first of the lake series and at the
point where it enters the pump and distribution center
leading to the spray irrigation site.  We can also chlor-
inate it before entrance into any natural surface outlet
stream.

                       RESEARCH PLAN

AQUATIC STUDIES

The lakes will serve a multiplicity of purposes chief
among which is the removal of nitrogen, phosphorus and
other trace constituents of the wastewater.  The mechanism
planned to accomplish this is to incorporate these materi-
als into the biological systems of the lake.  The attenua-
tion of influent material will come about by several mech-
anisms:  (1) direct sedimentation to the lake bottoms;  (2)
incorporation  into the plankton populations followed by
precipitation  or coprecipitation with the algae;  (3) sec-
ondary incorporation into the animal populations of the
lake including crayfish, tadpoles, minnows, aquatic in-
sects, and food or forage fish; (4) aquatic macrophytes.
These higher plants will constitute a major mechanism  for
concentrating  and removal of dissolved materials from
inflowing effluent.

During the fall of 1973 ten species of aquatic macrophytes
were introduced into the lake system.  Freshly cut material
was transported to the campus lakes from nearby natural
areas.  Potamogeton foliosus was given general distribution
over the entire extent of the lakes and localized clones
of the following macrophytes were also established: P_.
pectinatus, P. crispus, Elodea canadensis, E_. Nuttallii,
Myriophyllum spicatum, Najas flexilis, Ranunculus sp.  and
Vallisneria americana.  Care was taken to exclude problem
species such as Ceratophyllum demersum.  Based on the  pre-
liminary studies of McNabb and Tierney^ we can expect  net
yields approximating 2100 gm/m^ ash-free dry weight over
a six month growing period.  Of this weight about 1.51
is P and 5% is N.

As the water flows from lake 1 through the sequence to lake
2, it can be diverted to 3 one acre marshes adjoining  lake
2.  The purpose of the marshes is to test the efficacy of
a marsh community in removing nutrients from wastewater.
There is some  evidence that they are unusually proficient
in denitrification processes.  The research strategy on

                             370

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these is to plant and develop marsh communities adaptable
to the uptake of high levels of nutrients, then pass
effluent through these three marshes at a rate compatable
with the maximum uptake of nutrient materials.  The con-
trol of the marshes is individual and the flow can be reg-
ulated at different levels.  Cognate research on the util-
ization of these marshes by waterfowl and wildlife will
be undertaken in detail.  It is anticipated that they will
produce a harvestable crop of wildlife as well as serve
for basic research.

TERRESTRIAL STUDIES

Forest

Within the terrestrial system is an area supporting a
mixed hardwood forest typical of most woodlots in southern
Michigan.  Here, the survival and growth of the trees will
be documented under different spray application regimes
and the penetration, uptake of nutrients, and loss from
the system will be measured by a variety of techniques.
Partially covered by the spray irrigation system is a pine
plantation which will serve as contrast for the deciduous
forest system.  Plans for additional research include the
planting of trees that can be used for intensive cropping
on a short term rotation.  It is presumed that these will
be used for pulp wood and their complete removal and
utilization will serve to take the accumulated nitrogen
and phosphorus that they have incorporated during their
growth completely out of the system.

Incorporated as a by-product of the forest research will
be a study not only of the nutrients removed from the
system but also a study of the return and internal cycling
of these materials in the form of leaf fall to the forest
soils.  This offers the possibility of the selection and
management of tree types for maximum binding and holding
of those nutrients that we desire to either remove from
the system or have tied up in such a manner that they
will not enter the potable water aquifer.

Old Field - Plant Ecosystems

Preliminary work over the past several years has indicated
that the natural diversity of an old-field community may
be one of the most effective and efficient units for cap-
turing nutrients from the wastewaters sprayed on the land.
There is also evidence from these studies that spraying
them with nutrient rich effluent will appreciably change
the composition of the plant community.  The efficiency

                             371

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of these plant communities in trapping nutrients and the
role of grazers, especially insects, in transporting nu-
trient material out of the study system will be a topic
of continuing research.

Cultivated Crops

In the demonstration phase of the Michigan State Univer-
sity program we are not only striving to clean up the
wastewaters of a city, but to also make use of those
materials in the wastewater that have been mined and fab-
ricated at considerable costs of natural resources and
energy.  It is self evident that the transformation of
these resources back into useable food or fiber is an
essential ingredient in this demonstration.  Thus, we
will be carrying out in-depth research on those crops
that can be used in this way.

Many crops now cultivated for either direct or indirect
human use can be benefited by the irrigation with nu-
trient rich waters.  But in addition to testing the
efficiency of these known plant crops we will undertake
a wide spread search for plants that meet the rather
unique conditions that we have set up in our recycling
program.  Over the centuries crops have been selected for
many characteristics among which are the ability to live
with a minimum of water and grow with a minimum of input
of nutrients.   There is now a need to reverse this par-
ticular selection and choose plants that will thrive in
an excess of water and in rich concentrations of those
nutrients that promote growth.  With these crops, when
they are found or developed from the gene pool of known
and possible crops of the world, we will again have to
select for palitability and other factors desirable in
plants to be used for direct or indirect food production.

It is recognized that certain plants take up heavy metals
and other materials that may be found in wastewater ef-
fluents and that these levels may reach the point where
they are unacceptable for either human or domestic animal
food.  Before such judgements can be made it will be neces-
sary to demonstrate such uptake and consider the possibil-
ities of either rejecting these forms as food or seeking
new uses for them.  Since one of the goals of such a pro-
ject is to keep heavy metals and other potentially haz-
ardous materials out of our streams and lakes it appears
desirable to concentrate them in plants produced in aquatic
or terrestrial systems.  The problem of their utilization
would then become the focus of additional research.  In
this manner we at least have the hazardous materials "in
hand."
                            372

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Soil Research

Any system such as the one proposed at Michigan State
University will be dependent to an important degree on
the: (1) selection of a site with soils compatable with
the concept of total waste management and recycling; and
(2) management of the soil complex in such a manner as to
accomplish the mission of the project over a long period
(approaching steady state); and (3) a subsurface geology
that both serves as a filter and does not present a barrier
to water movement downward from the water application site.

Detailed studies will be carried out on both the short
and long range effects on soil texture and composition of
heavy applicatons of wastewater.  This will involve studies
of the retention of heavy metals in the soil mantel and
development of strategies that will allow the heavy appli-
cation of nitrogen in its several forms and yet allow the
transformation and uptake of the nitrogen into forms that
will either be held in the crops or be driven off to the
atmosphere in the form of gaseous nitrogen.

The site selected has a great variety of soils characteris-
tic of the Great Lakes drainage basin thus affording the
opportunity for in-depth research on wastewater application
to diverse soil types.

                      MONITORING PLAN

CHEMICAL AND PHYSICAL

As both a service and as a basic research effort  there is  a
need for continuous monitoring of a  large array of  chemical
and physical parameters connected with both the operation
of the wastewater treatment plant and with the lake and  land
facets of the recycling system.  As  a service to  all inves-
tigators working on the total project, there will be avail-
able data on 52 chemical parameters  representing  collec-
tions starting with the raw sewage entering the sewage
treatment plant, the  attenuation and removal of materials
as they move through  primary and secondary treatment and
as they enter the pipeline  leading to the  lake system.
The chemical parameters of  interest  will be checked as
they move through the lake  system from one lake to  the next
and into the pump station.  Similar  and  further tests will
be made through the stream  outfall system  and to  the sev-
eral areas of the spray irrigation site.  A similar series
of analyses will be made from the  test well system.
These will identify movement of chemical materials  into
the soil mantel and down into the  subsoil  and ground water
aquifer if such movement does take place.

                            373

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Most chemical parameters will be serviced by the Insti-
tute of Water Research's chemical laboratory under the
direction of an analytical chemist and his staff.  Within
the limitations of modern technology we will monitor the
chemical parameters through automated equipment both for
the savings in time and cost and for uniformity of results.
There will be a constant check on the quality of the chem-
ical work through the splitting of samples to outside
laboratories and duplication of analyses within the labora-
tory.  This program will also be closely coordinated with
standardization methodologies set forth in the International
Joint Commission's program on pollution of the Great Lakes.

MICROBIOLOGICAL

One of the most widespread of all criticism of recycling
of wastewater has been in the arena of public health
concern.  There appears to be general acceptance that the
bacterial content of such water can be controlled and re-
duced to completely acceptable levels through chlorination
at some stage in the recycling process.  However, there
have been expressed doubts about the safety of using re-
cycled sewage effluent, regardless of the chemical and
biological stages that it has passed through.  We know that
such recharges into the ground aquifers are widespread in
areas where sewage disposal plants empty their wastes
into streams which in turn recharge ground aquifers.  How-
ever, in designing a recycling system it is essential
that all concerns of this type be answered fully and that
the final product of such a system be completely safe from
a health standpoint.

One of the major problems in identification of the health
hazards from a recycling system has been the high cost and
inadequate technology in identifying many of the more im-
portant viruses in dilute solutions such as one would find
in the recharge to a ground water aquifer.  To address our-
selves to this problem we are establishing a fully equipped
laboratory capable of both routine and research analytical
efforts in determination of the total microbiological
problems involved in this project.  Paralleling the chem-
ical monitoring program, the microbiological samples for
both bacteria and virus will be taken at every major trans-
fer point within the system.  This will include identifi-
cation from the raw sewage through the treatment plant,
through the pipeline to the lake system and to all aspects
of the spray irrigation site.  This will also include a
detailed consideration of the potential for dispersent by
aerosols in the spray irrigation process.
                            374

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                          SUMMARY

The total recycling concept that we are preparing to test
is based upon:

(1) The fact that our natural resources are limited in
supply and will, within the foreseeable future, become
critical both to industry and agriculture.  This is partic-
ularly true of phosphorus and is well demonstrated by the
fact that the world is now competing for a relatively
limited supply of available phosphorus to feed a vastly
increasing world population.

(2) That materials, such as nitrogen, found in abundance
in our wastewaters will be in short supply immediately
and are of concern as well because of the high levels of
energy that they require to manufacture them.

(3) These two essential nutrients are very great contrib-
utors to the pollution of our streams, our inland lakes,
and the Great Lakes.

Thus, we believe that for the conservation of our es-
sential natural resources and for the reduction in the use
of energy and the protection of our water resources of
the country, it is essential that we both remove these
nutrients from the waters and also make use of them in
the production of food materials.  The mechanism for doing
this is in part understood and in part there is a serious
lack of scientific knowledge upon which to base the re-
cycling and reuse of materials contained in wastewater.

Recently, in an unusual joint release, the Water Pollution
Control Federation and the American Water Works Associa-
tion stated their support for a massive research effort
to develop needed technology and evaluate the potential
health problems related to recycling of wastewaters to
domestic water supplies.  Noting that "sound management
of our total available water resources must include con-
sideration of the potential use of properly treated waste-
waters as part of drinking water supplies," the joint
resolution pointed out the lack of adequate scientific
information about possible acute and long term effects on
man's health for such reuse, and also noted that the
essential fail-safe technology to permit such direct re-
use has not yet been demonstrated.  The resolution em-
phasized the need for prompt action based on the fact
that "ever greater amounts of treated wastewaters are be-
ing discharged to the waterways of the nation and consti-
tute an increasing proportion of many existing water
supplies."  Although indirect recycling has occurred for

                             375

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many years with no obvious indications of damage to the
public health, almost no effort has been devoted to the
complex and long term evaluation and quantification of
possible hazards related to the ingestion of recycled
wastewater or  its by-products.  Furthermore, the nature
of hazardous materials in wastewaters will require many
years of research to develop monitoring and treatment
processes essential to their removal or control.

Since this is  a problem with applications throughout
the entire United States, the federal government can play
a major role in providing the kind of funds and coordina-
tion needed to implement the research, monitoring, and
pilot programs that must be developed to forward the pro-
gress in this  effort.  Because of the interrelated nature
of the many facets of a program such as this, it may well
fall to those  universities with a large and diverse staff
to undertake an overall evaluation (systems analysis) of
the process and to develop demonstration and educational
programs that  must accompany such an effort.  This is what
is proposed in the Michigan State University program.

                     LITERATURE CITED

1.  Bahr, T. G. (ed.).  Ecological Assessments for Waste-
    water Management in Southeastern Michigan.  Technical
    Report No. 29, Institute of Water Research, Michigan
    State University.  327 pp.  1972.

2.  Ball, R. C. and T. G. Bahr.  Ecology of the Red Cedar
    River.  In: Whitton, B. A. and M. Owens (eds.) "River
    Ecology,Tr~Blackwell Scientific Publications Ltd.,
    London (In press).

3.  McNabb, C. D., Jr. and D. P. Tierney.  Growth and
    Mineral Accumulation of Submersed Vascular Hydro-
    phytes in  Pleioeutrophic Environs.  Technical Report
    No. 26, Institute of Water Research, Michigan State
    University.  33 pp.  1972.
                            376

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           EXPERIENCES WITH A MARINE AQUACULTURE-TERTIARY

                       SEWAGE TREATMENT COMPLEX
                                           *
                           John E. Huguenin

                            John H. Ryther
                             INTRODUCTION

     Treated secondary sewage effluent has been shown to be a good
complete fertilizer for the growth of microscopic marine plants
(phytoplankton), which form the base of the marine food chain (Dunstan
and Menzel, 19711).  From this somewhat surprising fact has emerged a
concept for a combined aquaculture-tertiary sewage treatment system
(Ryther, Dunstan, Tenore and Huguenin, 19722), which has been under
active investigation at the Woods Hole Oceanographic Institution since
late 1969.  While the optimal operating points for the two objectives
of nutrient removal and maximizing aquacultural output are not coinci-
dent, they are close enough to make a combined system feasible.

     The essence of the concept is a treated sewage-marine phytoplank-
ton-bivalve mollusk  (oysters,  clams, mussels,  scallops)  food chain.
However, in reality it  includes  a  much more complex  food  web with
current  research involving  the  utilization of  flounder,  lobster,
abalone,  bait  worms  and  seaweeds  as  secondary  components in  such  a
system.   This  is due not only  to  a desire to hedge  against uncertain-
ties, but is also based on  experimental results which  indicate  that  multi-
species  aquaculture  systems  are more efficient and  productive  than
those with only one  species  (Tenore,  Goldman,  and Clarner, 1973 ).
Thus, efforts  are  leading to the  development not of a  single  system
but  rather to  a spectrum of  technically  feasible systems.  Efforts are
also underway  to investigate the  legal,  political,  regulatory, market-
ing  and  public health  aspects  of  such a. concept.  In addition,  once
 *John Huguenin is a Research Associate (engineer) in the Biology Dept.
  of the Woods Hole Oceanographic Institution, Woods Hole, Mass.
  Dr.  John Ryther is a Senior Scientist in the Biology Dept.  of the
  Woods Hole Oceanographic Institution, Woods Hole, Mass.
                                   377

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established, multi-species systems have value as research tools in
their own right, since complex natural processes involving different
trophic levels can be investigated under controlled conditions.
While a lot of work remains  to be done, from experience to date both
indoors and outside (Dunstan and Tenore, 1972^; Tenore and Dunstan,
1973a^), it is clear that such systems can be made to efficiently re-
move nutrients (Goldman, Tenore, Ryther and Corwin, 19746; Goldman,
Tenore  and Stanley, 1974O and produce rapid growth of marine  organ-
isms with high survival.

     Encouraging results and the need for better seawater research
facilities to more realistically evaluate the concept for practical
applications led to the design and construction of the Environmental
Systems Laboratoty (ESL) situated in Woods Hole, Massachusetts.  The
primary intent was to provide a flexible experimental facility to en-
able research over the complete spectrum of possible development for
such systems.

     Unfortunately, the ultimate application of the concept  in New
England would require the use of waste heat from power plants  to en-
able the animals to feed during the winter.  Due to this complication,
these same ideas may prove simpler to implement on a large scale in
areas of the country with more seasonally uniform water temperatures.
Small scale experimentation  has started at a site in Florida to
evaluate this concept in a semi-tropical environment, and a  larger
scale facility in that area  is now in the planning stage.  For the
same reason, the U.S. Pacific Coast is also of interest.

DESIGN  PROBLEMS

     In terms of system design, the nature of the nutrient source
(i.e.,  secondary sewage or not) is for most purposes irrelevant.  The
major design problems are those that would be common to any  other re-
search  oriented marine aquaculture system of equal complexity. The
main design constraint was clearly uncertainty over system require-
ments.  This was due to both the continually changing research ex-
periments and the evolution  and refinement of the concept itself.
Since  quantitative requirements could only be defined with confidence
 for the relatively near term, at most about two years, designing  for
 system flexibility and amenability to modifications became very im-
portant.

     An interesting fact is  that much of the necessary biological in-
formation needed for  the development of aquaculture systems  is not
available.  This problem exists for several reasons, even for species
that have been relatively well studied.  In spite of the tremendous
amount  of information available on the biology of some marine animals,
the data  from  recognized experts  can cover  such a range of values as
to be useless.  As an example, the published respiration rate of
oysters varied by a factor  of more than twenty.  On top of this


                                   378

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uncertainty was  the  fact  that within  the  components of our  system
there are many other sources and  sinks  for dissolved oxygen.  Also
surprisingly, important design variables  have  sometimes never been
investigated and published  in spite of  the voluminous literature
available.  For  us such a critical variable was  the efficiency with
which shellfish  convert 'different kinds of food  to shellfish meat
(Tenore  and Dunstan,  1973b8, 1973c9).

     Since the individual circumstances surrounding any particular
operation can so easily alter the biology of the system,  the best
approach, even where  apparently good  information exists,  is the actual
testing  of small scale systems under  conditions as close  as possible
to  those to be encountered.  While such testing can be very useful in
reducing the risks and developing nominal relationships and sizing
criteria, there  are  nevertheless  some remaining pitfalls, especially
if  indistinct research objectives are involved.  Even for commercial
operations whose design objectives can be quite unambiguous,, there
are problems of  determining system economics and the optimum scale
for operations as well as risks of major  changes in system  configura-
tion dictated by improvements in  state-of-the-art, market considera-
tions, or institutional factors.

     The engineering  problems to  be encountered include those due to
the corrosive properties  of seawater, toxicity of many materials to
marine organisms, biological fouling  of pipes  and equipment, and the
lack of  any readily available sources of  bioengineering design in-
formation.  On any specific project,  areas involving design risk
should be recognized  and  acknowledged.  Many of these risks, while
not capable of being  completely eliminated, can be reduced by col-
lecting  information  on previous or related endeavors (Clark and
Clark. 196410; Lasker and Vlymen, 196911; Pruder, Epifanio and Malout,
197312;  Strober, 197213), by small scale  testing, and by  careful
design to minimize the consequences of bad judgements.  Due to the
large uncertainties  in aquaculture systems, there is a high premium
on flexible designs and systems with  the  capability to readily under-
go modifications to meet  changing requirements.

DESIGN FEATURES

     The basic configuration adopted  for  the facility designed, at
least in part, as a "pilot  plant" for the previously described con-
cept is  shown on Figure 1.  This  system,  from  two head boxes, is a
gravity  flow system with  the six  ponds, eight  raceways and settling
pond in  order of decreasing elevation.  The settling pond and algae
ponds are prepared sand surfaces  covered  with 20 mil plastic liners,
a relatively inexpensive  form of  construction readily expandable to
much larger sizes.  The raceways  are concrete.  It will be noted that
experiments can be conducted in an indoor wet  lab, on a very flexible
outdoor  test area and in  the larger units, providing a choice of ex-
perimental sizes.  Figure 2 shows the present piping configuration of


                                  379

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the physical plant.  This  figure indicates that the outdoor units can
be operated with a variety of water depths and flow conditions as well
as enabling sequential  on  line operations in physically separated
units.  Services such as raw seawater, filtered seawater, nutrient
supply, temperature  controlled seawater, fresh water, electric power,
drains and oilless compressed air are available in many locations and
could easily be provided at others.  The pipe materials are high
density polyethylene for the main seawater lines to and from  the beach
and polyvinyl  chloride  (PVC) for most of the smaller on-site  piping.
Care was  taken in  the design to assure easy access, for maintenance
and modification,  to as much of this piping as was possible.

     Several  of the  subsystems give the facility considerable added
research  capability. A separate nutrient distribution system, which
includes  three 8,000 gal.  fiberglass tanks, a headbox and piping, can
distribute  any pumpable fluid, such as secondary sewage, an artificial
media,  a  food  slurry or even a "pullutant", to individual ponds or
raceways.  The four  impervious-carbon heat exchangers provide a
significant controllable seawater heating capability.  This capability
can remove  the effects  of  seasonal water temperature variants as an
uncontrolled  variable in experiments, enables year round operations
and enables reasonably  large-scale research on thermal effects to be
performed.

      The  sizes of  the larger outdoor units were chosen to provide
results and experiences applicable to much larger facilities  and still
have an acceptable initial capital cost.  Due to the multi-objective
character of this  facility the relative number and sizes of the com-
ponents are not necessarily those indicated by scaling up the sewage-
algae-shellfish system. If other research objectives were  to be
sacrificed, this  complex could ultimately treat the effluents (if
available)  of up to  500 people and perhaps produce around  four tons
of shellfish meat  annually. However, it must be remembered that the
primary output of  this  facility is technical information and  that  the
economics of a research group and its facilities are vastly different
 from those  necessary for  commercial operations.  A facility built  for
commercial  use would have  been designed much differently.

EXPERIENCES TO DATE

      The major construction activities were completed  in the  fall  of
 1973 and the seawater system was permanently activated on October  30.
The next day 300,000 juvenile  oysters arrived and initial operation
c ommenced.

      The pond design has  so far  proven very satisfactory.   In spite
of a good deal of  activity there have been no serious  problems with
punctures of the polyvinyl chloride  liners. A cinder block  dropped
on its  corner produced  only a  small puncture  in  one  of the  ponds,
which was easily patched.   Some  of  the ponds  have been frozen so
solidly that people  could  walk  on the  ice  and no damage  to  the  liners

                                  382

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has been observed.  In addition, the liners can be readily cleaned,
even after being heavily fouled with filamentous algae.  Still to be
determined is the long term performance of the liners, especially
with respect to sun damage.  The expected useful life is five years
but other, slightly more expensive, liner materials are stated to not
be subject to this damage.

     Overall, winter operations of the total complex have given prob-
lems far less severe than anticipated.  Performance of the heating
and circulatory system provided for two of the six ponds has been
better than planned.  We have been able to maintain water temperatures
in these as high as 75°F, but more normally set at about 60°F, even
with air temperatures in the low teens and with lower than anticipated
heat additions.  Dense continuous flow marine phytoplankton cultures
of up to 2 x 10  diatoms/ml have been easily maintained for many weeks
in these ponds with a daily harvest of 30% of the culture volume.
The densities of these cultures are considerably higher than those
achieved in our smaller scale experiments.  We believe these 36,000
gallon diatom cultures to be the largest in the world.  Unfortunately,
due to the lack of a large tank truck, these large cultures have so
far been operated with concentrated liquid fertilizers rather than
with secondary sewage as originally intended.

     The phytoplankton grown this winter have been fed continuously
on an in-line basis to 300,000 juvenile oysters and more recently
150,000 juvenile hard clams (quahogs) maintained in raceways supplied
with heated (60°F) filtered (20 micron) seawater.  The temperature
drop across a forty foot concrete raceway, even under very cold con-
ditions and low flow through times (about 8 hours), has been less than
2°F.  It is too early to say anything about the winter growth of
these shellfish, except that it has been substantial.

     Several aspects of our operations, however, have proven trouble-
some.  The five specialized carbon lined pumps with carbon impellers
have provided many problems, mostly in the area of not meeting per-
formance specifications and excessive water leakage at seals.  In
addition, a planned traveling service platform has not yet been
procured, making access and the handling of stacked shellfish trays
and other loads in the raceways very difficult.  This unit and a large
tank truck are currently the only important components still missing
from the system.

     More biologically oriented problems have also arisen.  The
valance heaters installed to heat the building were manufactured with
a coating of some hydrocarbon, which has contaminated the building
but is gradually burning off and, fortunately, does not seem to have
affected our current research.  Undesirable growths on the sides and
bottoms of the ponds and raceways have been less of a problem than
anticipated, but are expected to get worse with the coming of spring
and simmer.  As the cultures in the algae ponds get denser the un-
desirable growth on the sides is markedly reduced.  This is a result

                                  383

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which was not  predicted  from our smaller scale  experiments.  However,
removing  fouling and accumulated organic debris is  still  a  large
potential sink for man-hours and the  development of satisfactory
methods and  equipment to minimize the problem may be one  of the keys
to practical forms of marine aquaculture.

OUTLOOK FOR THE FUTURE

     Several potential problems must  be  investigated and  resolved  be-
fore the  promising work  already done  on  the  use of  treated  sewage  in
marine aquaculture can be beneficially applied  on a large scale.   All
potential public health  hazards (Shuval  and  Gruener,  1973^) must  be
thoroughly  investigated  and where real hazards  are  found  to exist,
means must  be found to eliminate or circumvent  the  problem.  The
threat from enteric virus and the possible presence of some carcino-
genic chemicals are two  areas of particular  importance.   If such re-
search is not done there is little likelihood of acceptance of this
type of  system by either the public or the regulatory agencies.  In
addition, continual small scale controlled experimentation  must be
carried  out with the purpose .of increasing efficiency and learning more
about system dynamics.  The gains from such  experimentation can be
substantial. As an example, during the year  and a half between the
preliminary design phase and the construction of the  ESL, the treated
sewage handling capacity of the ponds (size  being constant)  increased
by more  than 3007. and the required seawater  flow to the raceways was
cut 907..  Additional improvements are a  distinct possibility.

     The  current ESL is  a subscale pilot plant  with multiple objec-
tives.  No  attempt was made in the design to enable its potential
"revenue" to exceed its  costs, since  its small  physical size, the  size
and composition of its staff, and its flexible  research objectives
precluded this possibility.  The facility will,  however,  produce
adequate  quantities of waste-grown seafoods  for testing in  related
research projects and possibly even for  the  critical  function of pre-
liminary market testing.   The emphasis has not  been on maximizing  pro-
duction but  rather on conclusively proving technical  feasibility at a
realistic scale of operation and to exploring the practicality of  such
systems for  large scale  applications.  While  technical feasibility
appears relatively assured, the economics and acceptability of the
ultimate system cannot yet be determined.  They  are contingent on  a
great many  factors including methods  and equipment  still  to be de-
veloped.

     A few years  of experience  with the ESL in  improving  system
efficiency,  researching  risk areas, developing better monitoring and
control methods,  and  reducing production costs  is needed  to provide
the information necessary  for realistic evaluation and decision
making regarding  the  value  of the basic concept.  With this and similar
projects providing the needed design  information, full scale demon-
stration projects  may shortly become  realistic possibilities.


                                   38H

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                              REFERENCES

1.    Dunstan, W. M. and D. W. Menzel.  Continuous cultures of natural
     populations of phytoplankton in dilute, treated sewage effluent.
     Limnol. Oceanogr. 16(4):623-632, July 1971.

2.    Ryther, J. H., W. M. Dunstan, K. R. Tenore and J. E. Huguenin.
     Controlled eutrophication - Increasing food production from the
     sea by recycling human wastes.  Bioscience 22(3):144-152, March
     1972.

3.    Tenore, K. R., J. C. Goldman and J. P. Clarner.  The food chain
     dynamics of the oyster, clam, and mussel in an aquaculture food
     chain.  J. exp. Mar. Biol. Ecol. 12:152-165, 1973.

4.    Dunstan, W, M. and K. R. Tenore.  Intensive outdoor culture of
     marine phytoplankton enriched with treated sewage effluent.
     Aquaculture 1:181-192, 1972.

5.    Tenore, K. R. and W. M. Dunstan.  Growth comparison of oysters,
     mussels and scallops cultivated on algae grown with artificial
     medium and treated sewage effluent.  Chesapeake Science 14(1);
     64-66, 1973a.

6.    Goldman, J. C., K. R. Tenore, J. H. Ryther and N. Corwin.
     Inorganic nitrogen removal in a combined tertiary treatment-
     marine aquaculture system - I.  Removal efficiencies.  Water
     Research 8:45-54, 1974.

7.    Goldman, J. C., K. R. Tenore, and H. I. Stanley.  Inorganic
     nitrogen removal in a combined tertiary treatment-marine aqua-
     culture system - II.  Algal Bioassays.  Water Research 8:55-59,
     1974.

8.    Tenore, K. R. and W. M. Dunstan.  Comparison of feeding and bio-
     deposition of three bivalves at different food levels.  Marine
     Biology 21:190-195, 1973b.

9.    Tenore, K. R. and W. M. Dunstan.  Comparison of rates of feeding
     and biodeposition of the American oyster, Crassostrea virginica
     Gmelin, fed different species of phytoplankton.  J. exp. Mar.
     Biol. Ecol. 12:19-26, 1973c.

10.  Clark, J. R. and R. L. Clark.  Sea-water systems for experimental
     aquariums - A collection of papers.  U.S. Bureau of Sport Fisher-
     ies and Wildlife, Research Report #63, 1964, 192 p.

11.  Lasker, R. and L. I. Vlymen.  Experimental sea-water aquariums.
     U.S. Bureau of Commercial Fisheries, Circular 334, 1969, 14 p.
                                  385

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12.  Pruder, G., C. Epifanio and R. Malout.  The design and construc-
     tion of the University of Delaware Mariculture Laboratory.  Sea
     Grant Report DeL-SG-7-73, University of Delaware, 1973, 96 p.

13.  Strober, W. J.  A small bioassay laboratory designed for experi-
     mental thermal effects evaluation.  Circular No. 72-1, Fisheries
     Research Institute, University of Washington, 1972, 12 p.

14.  Shuval, H. I., and N. Gruener.  Health considerations in
     renovating waste water for domestic use.  Environ. Sci. & Technol,
     7(7):600-604, July 1973.
                                   356

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             POLYCULTURAL WASTEWATER RECLAMATION AT
            CALIFORNIA POLYTECHNIC STATE UNIVERSITY -
                 AN ACADEMIC INSTRUCTIONAL SYSTEM

                             by
                      Dr.  Richard  J.  Krejsa  *
 INTRODUCTION
When  I  attended  Michigan State  College  in  the  early  50's,
it was  called  a  "cow college" partly  because of  the
taunting  disdain of  midwestern  football  rivals,  and  partly
out of  respect for the  college's  self-reliance on  campus
grown food  and dairy products.  I am  now on the  faculty of
California  Polytechnic  State University, which also  once
was a self-sufficient "cow  college".  While it is  not
feasible  to return to complete  self-sufficiency, we  are
actively  exploring ways of  regaining  a certain amount of
integrated  independence wherever  possible.  This presen-
tation  constitutes a progress report  of  one such attempt.

Our polycultural wastewater reclamation  system is  still
more  a  concept than  an  accomplished reality.   It is  part
of an educational experiment based on the  realization that
a multi-disciplinary approach to  societal  and  technologi-
cal problems is  necessary.

Goal

Our goal, basically  is  to make  a  resource  out  of waste-
water,  and  to  educate students  and the public  in the
methods and philosophy  necessary  to do so.

Objectives

The objectives of our proposed  polycultural wastewater
reclamation system are  four fold:


 * Associate Professor, Biological Sciences Department
   California  Polytechnic State University, San Luis Obispo,
   Ca., and Supervisor,Fifth District, County  of San Luis
   Obispo.
** Portions of this  investigation  were supported by  a
   grant from  the Creative  Activity Research Effort  (CARE)
   program at  Cal Poly, and by funds  allocated to  student
   projects through  the Student Enterprise Program of the
   Cal Poly Foundation.
                             387

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    1)   To propose and investigate alternative biological
        and agricultural strategies of secondary treated
        wastewater reclamation and reutilization;
    2)   To monitor changes in water and soil quality
        attendant -with the processing of wastewater
        through our polyculture system;
    3)   To introduce students to practical and innovative
        technologies;
    4)   To educate the general public that "waste" water
        need not be wasted.

Accomplishments To Date

Over a two year period, six successive classes of Agri-
cultural Engineering students built the 50 acre-foot
reservoir  (Fig.l) as a practical class project while
learning how to operate tractors, dozers and other heavy
equipment.  This reservoir will serve as the Biological
Treatment Lagoon.  Other Ag Engineering students designed
and constructed experimental closed-system aquaculture
units for rearing catfish and trout.  Still others con-
structed automatic feeders, and bulk harvesting equipment,
and compiled specific bibliographies on the engineering
aspects of aquaculture.

A student  in  Animal Science grew St. Peter's fish, Tilapia
mossambica, brown bullheads, Ictalurus nebulosus and clams,
Corbicula manillensisC?), in an experimental backyard
polyculture greenhouse!.  A Crops Science student tested
the utility of secondary effluent on certain grass crops.
Students in an Ag Business Management class in communica-
tions surveyed local consumer attitudes on the acceptance
of farm grown catfish and successfully promoted the sale of
farm grown fish at a local chain supermarket.

A Biological Science student compared growth patterns of
cage-cultured channel catfish reared in different popu-
lation densities  (Fig.2a,b) .  Another biology student
(Fig.2c) concocted an experimental fish food made from
dehydrated steer  offal, cafeteria kitchen scraps, fish
meal, and  agricultural crop wastes, and then pelletized  in
our campus feed mill.  The growth rates of cage-cultured
catfish fed on the experimental diet were compared to
those from control fish fed on standard Trout Chow.

Another young biologist studied the behavior of hatching
catfish fry and helped design new incubation and hatching
techniques.  Others yet compared the behavior and growth
of fingerling catfish in normal irrigation water and the
secondary effluent from the California Men's Colony treat-
ment plant, studied the ultrastructure of developing cat-

                             358

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Figure 1.  Site Location of  Cal Poly Reclamation Unit.
Diagram legend - a. CMC treatment plant; b. newly con-
structed 50 acre-foot  Biological Treatment Lagoon:  c.
site of proposed aquaculture units;  d.  10 acre-foot
irrigation ponds;  e. irrigated crop land area; f. Chorro
Creek; mb = City of Morro Bay, 5 miles west on Highway 1;
slo = San Luis Obispo, 7 miles east.
Upper photo - view of Chorro Valley looking east, CMC
Treatment Plant on the right of reservoirs.  Lower photo -
view of reservoir area, looking east.	

fish skin using the techniques of electron microscopy, and
examined the skin of catfish for evidence of bacteriologi-
cal pathogens.

Two students from the School of Architecture and Environ-
mental Design did their senior project on the design of a
simple, efficient experimental fish hatchery using space
frame technology3.  A class of 12 design students spent a
portion of the academic year running structural analyses
of the roof design created by the two senior project
students.  Still another design class of 32 students spent
                             389

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                           (a)
                                                V
                                            (c)
Figure 2   Aquacultural  experimentation  at Cal Poly.
a   Floating Cage culture  density/growth experiment area.
Sheppard Lake: b. Weighing channel  catfish grown in cage
culture: c.  Sampling channel  catfish  grown on experi-
mental diet concocted from wastes.
                             390

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the recent winter quarter attempting to incorporate and
synthesize the multi-purpose aspects of the proposed poly-
culture reclamation project into a functional whole.  They
came up with several innovative design concepts, two of
which are presented here  (Figs. 3,4).

All of these preliminary  and exploratory analyses were done
with minimum expense and  maximum enthusiasm.  The construc-
tion of the new 50-acre foot reservoir was completed last
Spring and it was filled  for the first time last Summer.

The program announces  this meeting as "The First Inter-
displinary Conference  of  Its Kind".  As can be seen above,
our Cal Poly project has  been  interdisciplinary from its
inception over two years  ago.  Even  if the production
aspects of the project were to go no further now, we would
deem the educational value of  the project as worthwhile and
valid.

Historical and Political  Perspective

The current form of the Cal Poly project evolved as the
result of the  interplay of several factors, some of which
have been described above, and others which will be
described from a historical and political perspective below.

Consumptive water uses within  the County of San Luis Obispo,
as well as throughout  the Central Coast region  are  taxing
the capability of the  watersheds and groundwater basins.
With certain exceptions,  local public policy up to  the
early 70Ts has not faced  the water problem  squarely.  Pro-
jected growth  in population and  irrigation  development,
anticipated for  the late  60's  did not fully occur  in all
areas; however in areas  of the County where it  occurred
and carried over  into  the 70's,  overdrafting  and mining
of the local groundwater  supply  has  resulted.   To  citizens
of these  areas,  such  activities  provide  a  false sense  of
security  regarding  the permanence  and reliability  of their
groundwater resources.  As long  as water  can  be imported
from elsewhere,  water  conservation  and  quality  objectives
are overlooked.   There is a  need  for consumer  education.

The problem of water  conservation  and quality  is not
limited  to the  Central  Coast  region4.   Indeed  for  years
California water resources have  been managed  for their
utility   expecially  for  consumptive  uses.   In 1968-69,
for example,76 water  treatment facilities,  each with design
capacities greater  than one  million  gallons daily  (m.g.d.)
serviced  90%  of  the  population of  8  coastal Southern
California counties.   Yet only 3.8%  (45,000 acre-feet)  of

                             397

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                    50 ACRE FT
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230

240
247

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           UPPER
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                                   A,

                                      PATHOLOGY
               ,
               AL 
-------
the total wastewater discharged from these plants were
reused for irrigation of crops and/or recreational parks,
or for planned groundwater recharge.  The remainder was
used only once before its discharge directly into the
Pacific Ocean  (84%) or variously disposed onto land or
into fresh water streams (12.2%) .

As recently as June, 1971. the same memorandum report of
the  California Department of Water Resources stated that,
for the immediate future, the areas of San Luis Obispo
County served by four of the five large capacity coastal
wastewater plants had an abundant supply of local, low-cost
water of good quality, and therefore "increased use of
treated wastewater.... is not probable"5.

            . *
                   A
                  *T
                  HI VOMO,
                               MAIN   LEVEL
Figure  4.   Concept  of a polycultural wastewater reclama-
tion  aquaculture unit designed by architecture student
Greg  Gill.   Simple  concrete structure built into slope  of
rock  outcrop at foot of reservoir.   Basic design of
hatchery implies water issuing forth from "womb of
earth"  where embryonic fish are hatched, reared and
tually  grow large enough to exit the hatchery into the
external raceway culture units.
                              393

-------
But times and attitudes have changed.   Increases in popu-
lation and per capita water consumption, coupled with  an
increased environmental consciousness,  have led to increa-
sing pressure for well-planned and controlled growth
statewide.  The Regional Water Quality  Control Boards
across the state have initiated a series of watershed
basin studies which, when  completed, will result in com-
prehensive water quality plans for each of 16 sub-basins.
In the San Luis Obispo County coastal sub-basin, the
interim  plan of 1971 called simply for  the discontinuance
of effluent discharge into surface fresh waters before
1980^.   The recommended plan now specifies land disposal
with reclamation and reuse for all but  two treatment
plants?.  In these  two exceptions, better ocean outfalls
are proposed but reuse and reclamation  projects are still
listed as potential alternatives.

Three years ago, substantial increases  in overall agricul-
tural irrigation development were not expected to occur
within the county.  Considerable increases in local citrus.
avocado  and wine grape acreages however have occurred.  For
example, along the  coast an 8-fold increase, from 650  acres
in 1970. to 5340 acres by  the year 2000, is projectedS.
Similar  increases are already occurring inland in The  Upper
Salinas  Valley watershed with approximately 1600 new acres
of vineyards in the past two years.

It is increasingly  apparent, from the regional as well as
local point of view, that  as development of additional
agricultural and urban land occurs, however well-controlled,
some means of providing supplemental water, other than by
importation, must be explored.  Wastewater reclamation and
reuse should have a high priority among them.

It has been said that wastes are resources out of place.
In San Luis Obispo County, fully one-half of the 25,000
acre-feet of supplemental  water needs projected for the
year 20008 could potentially be met by reclaiming the
relatively high quality wastewater being discharged from
eleven existing secondary  treatment plants (Table 1). The
polycultural wastewater reclamation concept at Cal Poly
evolved  in part out of recognition of this economic and
political reality.

But environmental forces were also at play.  In 1971, one
of the guidelines set by the Interim Water Quality Manage-
ment Plan was that there shall be no effluent discharged
into areas which possess unique or uncommon cultural,
scenic,  aesthetic, historical or scientific value6. Morro
Bay,  one of the least modified major estuaries on the


                             394

-------
California Coast, is recognized as such a unique place.  So
it was that in early 1971, the Central Coastal Regional
Water Quality Control Board gave notice to the California
Department of Corrections, that the California Men's Colony
treatment plant would eventually have to stop discharging
into Chorro Creek, which flows into the Morro Bay estuary.

Fortuitously, at about the same time that the CMC received
notice to consider land disposal of its effluent, officials
of California Polytechnic State University, were becoming
aware of production capacity problems in the two 200 +
g.p.m. irrigation wells located on its Chorro Creek Ranch
property.  The university property consists of some 600
acres of valley floor adjacent to and downstream (west)
from the prison's treatment plant (Fig.l).  An interagency
agreement was entered into on January 1, 1972 between both
State agencies.  Cal Poly was to take all it could use of
the CMC wastewater effluent for land storage and agricul-
tural irrigation.  The remainder would still discharge into
Chorro Creek.

Construction of a 50 acre-foot capacity reservoir was soon
scheduled and the decision was made to build it  the "Cal
Poly way", i.e., with student class help under the direc-
tion of  the  instructional staff and faculty.  The mechanics
of building  a new reservoir soon became  intermingled with
new ideas for aquaculture and catfish farming being in-
vestigated elsewhere on campus  (see above).  The process
opened up the educational experiment described in the
Introduction.   It has already  involved untold hours of
labor from some  150 students and at least  25 faculty and
staff members  in  10 different  instructional departments
on campus.   Four  of the seven  academic schools have been
involved thus far.

SYSTEM DESIGN, FACILITIES AND  PROPOSED  STUDIES

Treatment Plant

The California  Men's Colony wastewater  facility  is  a
secondary  treatment plant consisting  of  barscreen,  primary
settling, biofilters, secondary  settling,  chlorination and
sludge digestion apparatus.   Design capacity  is  1.5 m.g.d.
Current  average volume  is 0.57  m.g.d.  (Table  1).   Average
temperature  of  the  effluent  at  discharge is  7QOF.   The CMC
plant  is located off  State Highway  1,  about  halfway between
the cities  of  San Luis  Obispo  and  Morro Bay  (Fig.l).   It
services the staff  and  prisoners  of the Men's  Colony,  the
staff  and  students  of  Cuesta  College,  and the  staff  of Camp
San Luis Obispo (Army National  Guard).   There  are  no

                             395

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    Table  1.   Current  Disposition  of  Secondary Waste Water from San Luis Obispo County
               Treatment  Plants*
to
DESIGN AVERAGE3
PLANT LOCATION CAPACITY DISCHARGE
(m.g.d.) (m.g.d.)
Coastal
Cambria u.^o
San Simeon 0.15
Cayucos/Morro Bay 1.70
California Men's Colony 1.50
San Luis Obispo 5.00
South County San. District 2.50
Pismo Beach 1.00
Upper Salinas Valley
San Miguel u.ao
Paso Robles 2.20
Atascadero 0.83
Atascadero State Hospital 0.50
TOTALS 15.93
0.06C
0.05°
1.10C
0.57^
3.99e
1.02s.
0.30f
0.30C
1.06e
0.54C
0.14C
8.86
a. Annual totals divided by 365
b. PP - Percolation Ponds; EP - Evaporation Ponds; SI
Freshwater Stream; OO - Ocean Outfall
c. Period January 1973 - December 1973
d. Period August 1972 - July 1973
e. Period July 1972 - June 1973
f. Period July 1971 - June 1972
* Basic data compiled from Central Coastal Regional
files, San Luis Obispo
AVAILABLE
RECLAMATION
(a.f./yr.)
67
56
1232
638
4368
1142
336
34
1187
605
157
9822
TYPE OF
EXISTING
DISPOSAL13
PP, SI
00
00
FS
EP,SI,FS
00
OO
PP
PP,FS
PP,SI,FS
SI

= Spray Irrigation; FS =
Water Quality Control Board

-------
industrial contaminants in the system, the prison laundry
being the only source of sewerage in commercial proportions.

Chlorinated effluent of relatively good quality (Table 2)
is discharged into Chorro Creek and it flows into Morro
Bay estuary, about 4 miles to the west.  Some percolation
into the groundwater basin occurs.  The City of Morro Bay
obtains a portion of its domestic water supply from wells
in the Chorro Creek basin downstream from the CMC discharge.
These wells and others in the basin have been monitored
over a period of years  (Table 3)9.  The maximum degre-
dation of ground water shown in the tables has been the
result of occasional sea water intrusion rather than from
CMC effluent percolation.

Biologica1 Treatment Lagoon  (BTL)

Wastewater discharging from  the CMC plant will be lifted by
a 450 g.p.m., 15 h.p. pump some 60 feet in elevation and
transported via a 6" P.V.C.  pipe over a distance of 1500
feet to the newly-constructed 50 acre-foot capacity reser-
voir  (Fig.l).  There, primarily high nitrate uptake
forage crops will be grown hydroponically in trains of
floating racks.   The young  shoots of various grasses will
be fed to campus livestock.  A greenhouse will also be
constructed for more controlled hydroponic experimentation.

Aquaculture Unit (AU)

Water from the BTL will flow by gravity directly to an
existing 10 acre-foot capacity irrigation reservoir
(Fig.Id) and/or through an experimental aquaculture unit
of a design similar to those illustrated in Figures 3 and
4.  The AU will consist of an experimental hatchery and
dry labs, a parallel raceway system,  and a series of small
0.1 acre ponds.

Experimental Hatchery - This will provide wet lab facili-
ties for artificial spawning, hatching and rearing of warm
or cold water fishes  (Figs.3,4).  .Initial experimentation
will utilize channel catfish, Ictalurus punctatus, with
which we have had the most experience, and also the hitch,
Livinia exilicauda, a native herbivorous fish from the
Central Valley.  We also hope to raise fresh water clams
and local crayfish.

Dry Laboratory - This portion of the  ru will feature a
water and soil quality analysis lab  (Figs.3,4). Computeri-
zed, remote multi-channel sensing equipment  (Montedoro-
Whitney Corp., San Luis Obispo) will  allow continuous or

                             397

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Table 2.   Mineral Analysis and Effluent Characteristics,
California Men's  Colony Secondary Treatment Plant.
Characteristic
PH
ECxlO^SOC (mmhos/cm)
Total Dissolved Solids f
Total Hardness (asCaCO;)
Total Alkalinity
Calcium (Ca)
Magnesium (Mg)
Sodium (Ha)
Potassium (K)
Iron (Fe)
Carbonate (CO;)
Bicarbonate (HCOj)
Sulfate (SOJ»)
Chloride (C1)
Nitrate (NO;)
Fluoride (F)
Orthophosphate (P0t»)
Boron (B)
B.O.D. Annual Mean (ppm)
B.O.D. Annual Range (ppm)
D.O. Annual Mean
Sol ids, Suspended (ppm)
Solids. Settleable (ml/1)
Grease
Turbidity (JTU)
Col iform (mpn)
1970
7.6
-
672
300
255
28
56
108
8.0
0.21
0
255
32
158
17
3.0
21
0.19
-
-
-
12.4
b
1971
7.7
-
955
384
458
22
59
160
9.2
0.10
0
458
53
233
50
3.9
4.0
0.4
-
-
-
5
1972
7.8
1300
-
342
-
29
66
190
5.4
0.14
0
318
66
291
11
1.8
14.6
1.36
6.0
4-7
7.1
40
d
1973
7.6
1500
1040
325
370
18
59
205
9.0
-
0
370
58
258
18
1.3
14
0.25
9.8
8-13
7.2
9
e
1973
8.2
.
90S
416
377
21
88
197
10
-
0
-
44
243
21
2.4
-
-
-
-
-
-
<0.1 <0. 1 <0. 1 <0.t
1.3
-
<45
1.4
-
<45
a. CMC Ann. Report. Regional Water Qual
c'. 	 " " "
6.8
9.5
<45
ity Control
11
M
3-6
25
<45
Bd.
-
-
-
Feb. 16
June 1 1
July 18
       d.   '	      "      "     "      "     "  July  11
       e.  Regional Water Qua 1ity Control Bd.sample obtained June 27
       f.  Constituents listed in mg/1 unless otherwise specified.
                                398

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 Table 3.  Summary of Selected Mineral Constituents in
 Component Portions of Chorro Creek Basin Water3.

No. Analyses
No. Wells Tested
Mg Avg .
(Range)
804 Avg.
(Range)
Cl Avg .
(Range)
NOs Avg.
(Range)
B Avg.
(Range)
TDS Avg.
(Range)
EC x 106 6 25°C
Avg.
(Range)
SURFACE
WATER
12
68
(27-103)
31
(0-59)
43
(19-77)
4.1
(0-9.9)
0.09
(0-0.20)
433
(234-704)
685
(347-1080)
GROUND
WATER
90
23
114
(9-436)
104
(14-788)
254
(45-2559)
14.5
(0-105.0)
0.13
(0-0.71)
1022
(547-5402)
1643
(990-8264)
CMC
EFFLUENT
16
91
(29-589)
272
(114-865)
23
(0-98.0)
1045
(766-1997)
a.  Abstracted from Tables 9, 10,. 14 - Reference No.  9
                             399

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periodic monitoring of several water parameters at critical
locations  in the circulation pathway, including monitor
wells around the crop irrigation area.  Additional facili-
ties will  include large capacity aquarium units with room
for observation of the natural reproductive behavior of
catfish and other warm-or coldwater species.  A pathology
lab will be set up for studying and monitoring fish dis-
eases and  to investigate the transmission of microbial
pathogens  from the effluent water.

Since one  of the functions of our project is to educate the
public, the architectural design takes into consideration
not only the needs of the students and faculty, but also
the visitor.  Guided or self-guided tours of the facilities
will be designed to educate without disrupting routine
operations.

Irrigation Reservoir

Wastewater from the BTL and the AU will exit to the 10
acre-foot  capacity irrigation reservoir (Fig.Id).  Outflow
water from this storage site will be used to irrigate field
crops in the crops area west of the reservoir site (Fig.le).

Crops Area

The areal  geology of the location indicates an upper
Pleistocene alluvium of sand, gravel and clay.  The slopes
and mounds are of Jurassic Franciscan formation consisting
of sedimentary and igneous rocks.  The creek terrace soils
are high in clay content with low permeability and slow
rate of infiltration.

Studies similar to those done at the Penn State University
Waste Water Renovation Project10 will be undertaken.
Faculty and students from the Soils Science Department will
analyze soils and monitor the crops area before, during and
after effluent is utilized for irrigation purposes.  Deep
cores will be taken and test plots utilizing wastewater
will be compared with those using well water.

Faculty and students from the Crops Science Department will
study yields and quality of field crops under irrigation
with wastewater and well water.

Sludge collected from the fish raceway system will be
analyzed and utilized as fertilizer or for methane gas
generation.  Sludge from the CMC treatment plant will also
be evaluated for its potential fertilizer value.  Ter-
restial and hydroponic crop wastes not otherwise utilizable
will be composted along with sludge.

                             WO

-------
Additional Studies

Utilization of Other Wastes - High protein content fish
carcasses,from the Morro Bay commercial fisheries, are
currently being disposed of by land burial.  We propose
to convert this valuable waste into fish meal.  We will
combine this with high protein material gathered from
terrestial or hydroponic crop wastes.  Aquatic crops of
Wolffia or Lemna spp., which are indigenous to our county,
will also be propagated in our ponds.  With an appropriate
vitamin mix added, we intend to produce a viable pelletized
fish food at our campus feed mill.  With formula variations,
we hope to use it for other campus livestock.

Irrigation and Erosion Controls - Faculty and students from
the Agricultural Engineering Department will design the
irrigation systems, erosion control systems and the overall
hydraulics of the system.

Solar Heating - Faculty and students from Architecture and
Environmental Engineering Departments will design a roof
or wall panels for the aquaculture unit which will feature
solar powered heating of either well water or wastewater
for experimental use in the hatchery.

Food Processing - Faculty and students in the Food Indus-
tries Department will study the technology of processing,
packaging and freezing fish products.

We hope to make our polyculture farm a truly multi-
disciplinary educational unit where faculty can conduct
research, where students can learn by doing, and where
the general public can be exposed to graphic examples of
necessary technologies.

CONCLUSIONS

We believe it is possible to make a resource out of waste-
water by routing it through a series of aquatic and ter-
restial biotic filters.  We believe that known methods of
hydroponic culture, aquaculture and agriculture can be
combined into a polycultural process which is not only
economically feasible but also ecologically desireable.
Furthermore, it is our responsibility as educators not
only to teach our students but to educate the public at
large.

SUMMARY

Only a small percentage of secondary wastewater is being


                             407

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reclaimed or reused in Southern California.

Regional Water Quality Control Boards have adopted a policy
of disposal on land for reclamation or reuse with a goal of
substantially reduced ocean outfalls.

It is suggested that wastewater reclamation and reuse
should have high priority among alternate means of supplying
supplemental water in San Luis Obispo County, especially
in areas where overdrafting and mining of ground water has
occurred.  There is a need for considerable consumer edu-
cation.

The Cal Poly project will utilize secondary effluent from
the California  Men's Colony wastewater treatment plant to
grow harvestable crops hydroponically, aquaculturally and
agriculturally.  It is designed for applied research,
student training and public education.

The aquaculture unit wet labs will facilitate the study of
reproductive behavior, hatching and rearing of warm or
cold water fishes.  The dry lab portions will be equipped
to study pathology of fishes and other organisms growing
in the secondary effluent.  Comparative analysis of soil
and water will occur and remote monitoring of both will be
possible at various critical points in the circulation
pathway.

The student-teacher interaction is a vital component of
this interdisciplinary academic instructional system at
Cal Poly.
 1.  Thuresson, Neil M. Backyard Polyculture.  Senior Project,
    Animal  Science Department, California Polytechnic  State
    University, March, 1973, 48p. On file at  CPSU Library.

 2.  Olson,  Robert J.  Density Factors in Cage  Culture of
    Channel Catfish,  Ictalurus punctatus  (Rafinesque).
    Senior  Project, Biological Sciences Department, Cali-
    fornia  Polytechnic State University, July,  1972. On
    file  at CPSU Library.

 3.  Kupadakvinij, Keith  and Dennis  Scott.   A  Catfish
    Hatchery for Cal  Poly.  Senior  Project, School of
    Architecture and  Environmental  Design,  California
    Polytechnic State University, June, 1972.  16p. +
    plans on file in  School of Architecture Library.
                             402

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4.  California, State of, Department of  Water Resources,
    Wastewater Reclamation, State of the Art.  Bulletin
    No. 189, March, 1973, 43 p.

5.  California, State of, Department of  Water Resources.
    Southern District.  Overview of Wastewater Reclamation
    in Southern California, Study Progress during 1969-70.
    Memorandum Report, June, 1971.

6.  California, State of, Central Coast Regional Water
    Quality Control Board,  Interim Water Quality Manage-
    ment Plan, Central Coast Basin, 1971.

7.  Brown and Caldwell, Water Resources Engineers,  Inc.,
    and Yoder-Trotter-Orlob and Associates.  Briefing on
    San Luis Obispo Coastal Sub-Basin Recommended Water
    Quality Control Plan.   Comprehensive Water Quality
    Control Study, Central  Coast Region, August, 1973.

8.  CDM, Inc., Environmental Engineers, Summary Report,
    Master Water and Sewerage Plan, County of San Luis
    Obispo, October, 1972 as adopted and amended by Board
    of Supervisors, November, 1972.

9.  California, State of, Department of Water Resources,
    Southern District, Water Quality  Conditions in
    Coastal Regions of San  Luis Obispo County.  Memorandum
    Report to Central Coastal Regional Water Quality
    Control Board, October, 1969.

10. Parizek, R.R., L.T. Kardos, et al, Waste Water  Reno-
    vation and Conservation.  Penn State University
    Studies,  No.  23, University Park, 1967, 71 p.
                             403

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PART 7    ADDITIONAL PAPERS CONTRIBUTED FOR PUBLICATION

-------
          COLIFORM AND PHYTOPLANKTON STUDIES IN A BRACKISH WATER
           AQUACULTURE POND FERTILIZED WITH DOMESTIC WASTEWATER

                                   by

                  Robert F. Donnelly and Tommy T. Inouye


                             INTRODUCTION

Two aquaculture ponds, of about 0.15 hectares each, were constructed
within the confines of the City of Arcata sewage lagoon, located at the
north end of Humboldt Bay, California.  These two ponds were placed in
operation June 1971.  One pond (designated North Pond) was the experi-
mental site and was filled with a 50-50 mixture of seawater and sewage
effluent from the sewage lagoon.  The other pond (designated South Pond)
was filled with seawater only and used as the control in some experi-
ments.  Funding for construction of the ponds came from the Wildlife
Conservation Board of California and operating monies were provided by
the California State University, Humboldt, Coherent Area Sea Grant Pro-
gram of the National Oceanographlc and Atmospheric Administration, U.S.
Department of Commerce.

No standards were available for sewage-seawater pond discharge; there-
fore, the Public Health Service indicated that a minimum of 2 weeks re-
tention was needed in the North Pond before the water could be dis-
charged into Humboldt Bay without chlorination.  As a result of this
and other considerations, it was decided that one of the authors (Rob-
ert Donnelly) would conduct a study of the coliform levels of the two
ponds, the sewage lagoon itself, and Humboldt Bay, adjacent to the aqua-
culture ponds.  Concurrently, Tommy Inouye undertook a study of phyto-
plankton identification and enumeration of the same water samples used
for coliform determinations.  At the same time, six other variables,
(pH, DO, salinity, temperature, Secchi disc readings, and depth), were
collected.  Due to the large number of samples collected (192), only
the 48 samples from the North Pond were analyzed for their phytoplank-
ton content.

This paper discusses.the possible interrelations among these variables.
Several authors,  »  »  »      » have demonstrated relationships between
phytoplankton and bacteria.  The bacteria break down organic material
and make it available for phytoplankton utilization and, in turn, the


 Donnelly - Fisheries Research Institute, University of Washington,
            Seattle, Washington  98195

 Inouye -   School of Natural Resources, California State University,
            Humboldt, Arcata, California  95521

-------
phytoplankton give off organic materials and contribute dead cells to
the bacterial substrate.  Some of our data appear to support this cycle.

                          MATERIALS AND METHODS

LOCATION AND TIME OF SAMPLING

Concurrently, samples of coliforms and phytoplankton were taken from the
North Pond during two different seasons of 1972  (April 15 through May 27
and September 27 through October 31).   Collections were made adjacent
to the pond outlet (headgate) six times during each season at approxi-
mately 1-week intervals.  Four depths were sampled each time.  The sur-
face sample was actually 20 cm below the surface, while the bottom sam-
ple was taken 10 cm above the bottom.  The two intermediate sample depths
were equally spaced between these surface and bottom depths.  The water
level fluctuated due to rain and evaporation, resulting in different ab-
solute sampling depths each week.

SAMPLING DEVICE

The sampling device consisted of a 500-ml aspirator bottle, a sterilized
and partially evacuated 125-ml bottle, each  attached to a long handle,
(Figs. 1,  2, and 3).

Five ml  of water  and  a few boiling  chips were placed  in each 125-ml  sam-
ple bottle.  The  bottles  were stoppered  with foam stoppers.   Rubber stop-
pers  of  appropriate size  were drilled to accept a Pasteur  pipette.  The
Pasteur  pipettes  were bent and  flame closed, and then inserted  into  the
rubber stoppers (Fig.  1).

The rubber stoppers with the bent Pasteur pipettes and foam-stoppered
bottles  were autoclaved.   After sterilization, the bottles were placed
over  a flame and  the water within was allowed to boil for a short period
 of time.  The bottles were removed from the flame and, using aseptic
 technique, the rubber stoppers with bent Pasteur pipettes were inserted
 into the mouth of each bottle.   These were allowed to cool, creating a
 partial vacuum.  Experimentation showed that these partially evacuated
 125-ml bottles would obtain a sample of about 90 ml when the bent Pas-
 teur pipette was broken beneath the surface of  the water.  The inside
 diameter of the Pasteur pipette (about 5 mm) was small enough so that
 the sample water was held within by surface tension.

 The aspirator bottle was controlled through a small plastic tube running
 from the  top of the bottle to the operator.  The sterilized 125-ml bot-
 tle was activated by an attached pole struck by a hammer (Fig. 3).
                                    405

-------
                  Kt Dili If
                  W$l[«l
                                  12S«I MTILE
                           *» ink
                          IUIICI SWKI-tintf
                    Sterilized. Evacuated SAMPLE BOTTLE
                                  & COMPONENTS

                  Figure 1.   125-ml sterilized  water sample
                             bottle (complete)  bent Pasteur
                             pipette and  bored  rubber stopper,


TREATMENT OF  THE SAMPLES

Water collected  with the aspirator bottle was  used to determine DO, pH,
salinity, and temperature at the sample  site.   The 125-ml samples were
processed at  the laboratory where approximately 35 ml of  each sample was
inoculated  into  the nine-tube MPN test for  coliforms.  The coliform de-
terminations  were made according to the  procedure outlined in Standard
Methods.    One Secchi disc measurement was  taken on each  day sampled.

The remainder of the 125-ml sample was inoculated with iodine (1 drop
                                   406

-------
                             •ALUMINUM ANGLE

                             BRASS ROD - MwutU

                             RUBBER IANO
                             SEINE TWINE


                             BAR I SPRINGS

                             GUIDE RODS

                             3mm HOLE

                             RUBIER (AND
                           -ALUMINUM BACK PLATE
              SAMPLING DEVICE  for  ACTIVATING
              Sterilized, Evacuated Sample Bottle
             Figure 2.  The  device used for  activating the
                        sterilized and partially evacuated
                        125-ml bottles.

per 10 ml  of sample) for  phytoplankton identification and counting.
This was accomplished with  an inverted microscope and hemocytometer
counting chamber.

                         RESULTS AND DISCUSSION

The data were analyzed by BMD02R computer program in two ways:   1) by
6-week time periods, and  2)  some of the 12-weeks of data were pooled and
analyzed.   The first 6-week sampling period included a period after the
sewage-seawater mixture had been in the North Pond for approximately
2.5 months, while the second 6-week period  started 2 days after  a  sew-
age-seawater mixture was  introduced into the pond.  The coliform levels
                                    407

-------
                          r tr r
                     mrted it 10 en
                          . : . .  :: •
                           IUIEEI TUH
                                                  HAMMER
                                                  MTIYATINS KOO
                                                  v...<
                                                  STUUUUI. EUCUTQ
                                                  SAMPLE lOITtE
                                                  ;.  •  :  .
                            SUBSURFACE  SAMPLER
                               with all COMPONENTS


                      Figure  3.   Assembled apparatus for
                                  obtaining subsurface
                                  water samples.

remained  fairly low during the  first period,  while the levels started
high and  decreased until more water was added from Humboldt Bay during
the second  sampling period (Fig.  4).,. Humboldt  Bay generally had higher
coliform  levels than the North  Pond.

This second period is of interest  because we  wanted to know what was oc
curring in  the water column  during the time the pond was undergoing its
greatest  developmental changes.   After the pond had stabilized, large
fluctuations in organism populations no longer  occurred.

Three genera of phytoplankton were found:   Anacystis,  Ankistrodesmus,

-------
:
         2500

                              9        10
                           Sampling week
L;

                                                                    •-.
                                              ...-o-•
                                           • •'' ' Ai.acystis
 Figure 4.   Anacvstis numbers, coliforms levels, Secchi disc readings
            and Ankistrodesinus numbers  (averages of the four depths)
            during the second sampling period from September 27 to
            October 31, 1972.  Note the sharp rise in coliform levels
            from week 10 to 11 is apparently due to an injection of
            Humboldt Bay water on October 23.
and Gymnodinium. with Anacystis  being most abundant and Gymnodinium
least numerous.  A negative  correlation existed between coliform
levels and Secchi disc measurements  and between coliform levels  and
Ankistrodesmus numbers  (Fig.  4).   A  positive correlation existed be-
tween coliform levels and Anacystis  numbers  (Fig.  4).   All other
variables showed no significant  correlation.
                                  409

-------
                               CONCLUSIONS

The data appears to support the hypothesis that there is a direct rela-
tionship between Anacystis and coliforms.  However, a negative correla-
tion existed for Ankistrodesmus and coliforms.  One study demonstrated
that bacteria utilize the organics secreted by phytoplankton.   In an-
other study, phytoplankton was shown to utilize inorganic nutrients gen-
erated from bacterial breakdown of organic materials in lakes.   This
leads us to believe that there may be a nutrient interrelationship be-
tween Anacystis and coliforms.

                            ACKNOWLEDGMENTS

We wish to thank Dr. George H. Allen for continued support and direction
and Dr. Stephen B. Mathews for assistance with the analysis of the data.
Also, we thank Dr. Ernest 0. Salo and Mr. John S.  Isakson for reviewing
the manuscript.
                                  470

-------
                            LITERATURE CITED


1.  Allen, G. H., G. Conversano, and B. Colwell.  A pilot fish pond sys-
           tem for the utilization of sewage effluents, Humboldt Bay,
           Northern California,  Mar. Adv. Ext. Service.  Sea Grant Pro-
           gram, CSUH:  CSUH-SG-3.  1972.

2.  Kuentzel, L. E.  Bacteria, carbon dioxide, and algal blooms.  J. Wa-
           ter Pollution Control Fed. Vol. 41: 1737-47.  1969.

3.  Saunders, G. W.  Carbon flow in the aquatic system.  In;  Carins,
           John Jr. (ed.)  The structure and function of freshwater mi-
           crobial communities.  The American Microscopical Society Sym-
           posium.  Blacksburg, Virginia,  pp. 31-45.  1969.

4.  Saunders, G. W. and T. A. Storch.  Coupled oscillatory control mech-
           anism in a planktonic system.  Nature New Biology.  230(10):
           58-60.  1970.

5.  Schegg, E.  Relation between plankton development and bacteria in
           Lakes Lucerne and Rotsee.  Schweiz. A. Hydrol. 30:289-96  (ci-
           ted in Biol. Abst. 1970).

6.  Donnelly, R. F.  A study of coliform levels in aquaculture ponds us-
           ing reclaimed water.  M. S. Thesis.  California State Univ.,
           Humboldt.  76 pp.  1973.

7.  Standard Methods for the Examination of Waste and Waste Water.
           Taxas, M. J., A. E. Greenberg, R. D. Hook, and M.  C. Rand
           (eds.)  Amer. Public Health Assoc., 13th ed., Washington, D.C.
           874 pp.  1971.
                                   477

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              MINERAL QUALITY OF FISH POND
                EFFLUENT RELATED TO SOIL
             PROPERTIES AND CROP PRODUCTION

                             by

                       L. H.  Hileman*

INTRODUCTION

Commercial fish production in Arkansas is a $21,000,000 industry
and a steadly growing enterprise.  Catfish are efficient converters
of feed into edible meat high in protein, therefore, a valuable
source of human food.   Farm production is carried out in earth
ponds varying in size from 10 to 20 or more acres usually filled
with water pumped from underground  aquifers.  Where adequate
water is available the fish are grown in a system of flumes referred
to as raceways which allows for continous water movement.  Water
from these catfish production areas,  whether ponds or raceways,
is considered as waste water.  A large amount of this waste water
is discharged into streams,  and rivers.  Enforcement of regulations
pertaining to the discharge of water into streams and rivers caused
fish farmers to consider other  methods of disposal.  A logical consi-
deration would be the use of this waste water for crop irrigation.  A
large number of catfish farmers also  grow other food or fiber crops
such as soybeans, rice  or cotton.  Previous water analysis parame-
ters were concerned with quality factors affecting municipal or in-
dustrial uses.  The use of waste water for irrigation may have little
direct effect on the growing crop but can drastically effect soil chem-
istry thus, indirectly affecting  crop production over a long period of
time.  Certain irrigation water quality parameters will be discussed,
and also data obtained from the chemical analysis of catfish waste
water related to potential effects on the soil and plant growth.
 * Agronomy Department, University of Arkansas, Fayetteville, Ark.
** Part of this work was supported by the Cobb Breeding Corp.
   Concord, Mass,  and Siloam Springs, Arkansas
                              472

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RESULTS AND DISCUSSION

Irrigation water quality is determined by the kind and amount of
mineral elements present in the water,  and the relationship of these
elements to the soil's chemical and physical properties as they affect
both immediate and future crop production.  Frequently, in practice
the major concern is with the immediate effect on the growing crop
while the most damaging to the farmer are the long term effects on
the soil.   Emphasis in this paper will be placed on the long term
effects on the soil and their relationship to plant growth.

Data in Table 1 shows some of the important parameters for deter-
mining the quality of water for irrigation.  Water containing relatively
high amount of calcium and magnesium when applied to soils will,  in
time, cause a condition known as a saline soil.  A saline soil is one
that is high in soluble calcium and magnesium.  Such soils are "salty"
due to the accumulation of these salts near the surface.  The princi-
pal effect of high soluble salts is to reduce the availability of water
to the plant.  A high soluble salt level will also interfer with the
plant uptake of other needed ions such as potassium,  nitrogen,  and
iron.  The calcium and magnesium levels found in catfish waste
water were quite low,  (Table 1). It is interesting to note that the
water was initially rather high in calcium and magnesium (5.1  meq/1)
but this level declined rapidly and reached an equilibrium of near 0. 9
meq/1 during the growth period.  Average calcium plus magnesium
data for seven locations shows low values indicating that no serious
soil salinity problems would be expected when any of these waters
were used for irrigation.

Irrigation water containing high amounts of sodium creates  a very
serious soil problem known as sodic or alkali soils.  These soils are
usually strongly alkaline in reaction, (pH 8. 5 to 10.0) dispersed,
almost impermeable to water, and have poor tilth.  This results in
reduced plant growth because of inadequate water penetration,  poor
root zone aeration and soil crusting.  The large amount of sodium in
these soils also interfers with the plants nutrition by preventing the
uptake of potassium, calcium, and magnesium as well as many of the
micro-nutrients.  It is almost impossible to  reclaim sodic  soils that
have restricted internal drainage.  Data in Table 1,  shows  that this
water was very low in sodium through the first six sampling periods
but then showed a decided increase.  Sodium values as shown in
Table 1 are not considered high, however,  the more important fac-
tor is the proportion of sodium to calcium and magnesium.  This re-
lationship is expressed by the S. A. R. and the S. S.P.  values which
 are shown in Table  1. The S. A. R.  or sodium adsorption ratio  is
                                473

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high when it exceeds 10.0 for soils with good internal drainage and
above 5.0 for soils with poor internal drainage.  The average S. A. R.
for location 1 is 3.4 (Table 2), which is less than 5.0.  The S. A. R.
is based on the soil exchange system which may respond differently
under adverse conditions.  The soluble sodium percentage, S. S.P.
is the soluble sodium as a percent of the total soluble cations (Ca,
Mg, Na, K) in the irrigation water.   S. S.P.  values exceeding 60 are
usually considered high for clay soils with impeded internal drainage.
The water from location 1 would be considered useable based on the
S. A. R. while it would be considered  unsatisfactory based on the S. S.P.
The author's  experience in Arkansas indicates that the S. S.P. is the
more valid for Arkansas soil conditions.  Locations 1,  3,  4, and 6
have high S.S.P. values while locations 8, 9, and 10  are much lower,
Table 2.

The data in Table 2 shows the location variation that  can occur with
respect to the irrigation quality of catfish production waste water.
The results of indescriminate use of these waters can result in the
alteration of plant nutrient uptake,  therefore, altered plant quality,
reduce  crop  yields, and soil physical and chemical damage. Farmers
planning to use catfish waste water for irrigation should obtain a
chemical analysis and irrigation quality evaluation before  applying it
to their soils.

All of us should keep in mind the thoughtful statement of Franklin D.
Roosevelt; "The history of every nation is eventually written in the
way in which it cares for its soil".
                                474

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      Table 1,  Calcium,  Magnesium,  Sodium,  Bicarbonates,
           Total Dissolved Salts, S.A.R.  andS.S.P.
        Values for Catfish Production Water at Location 1,
              1971 for the Two Week Sampling Periods.

Sampling*
1- fill
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29

Ca+Mg
5.1
1.9
0.7
0.8
1.4
0.7
0.6
0.9
0.7
0.6
0.7
1.1
0.8
0.8
0.7
0.6
0.8
0.9
0,8
0.8
0.8
0.8
0.8
0.8
0.9
0.8
0.8
0.9
0.9
Na
meq/1
0.5
0.5
0.5
0.5
0.1
0.05
3.5
4. 1
3.9
3.6
3.7
3.6
4.0
3.5
0.05
0.5
4.1
0.4
3.9
3.8
3.7
3.5
2.2
1.4
3.2
2.3
2.6
2.7
2.9

HC03
3.8
4.2
4.2
4.3
7.0
4.1
3.8
3.8
3.9
3.6
3.7
3.9
4.0
4.2
4.0
4.3
4.5
3.8
3.8
3.6
3.0
3.2
2.8
3.1
3.5
3.6
3.6
3.1
3.0
T.D.S,
ppm
269
307
294
320
64
282
283
269
269
256
269
269
307
294
307
320
320
269
269
269
243
243
192
256
269
269
243
230
243
S.A. R.
ratio
>1.0
^1.0
1.0
1.1
M.O
1.0
6.8
6.0
6.1
7.1
6.0
>1.0
6.0
5.6
1.0
>1.0
5.8
1.3
6.0
6.1
5.6
5.3
3.8
2.4
4.8
3.8
4.8
4.0
4.6
S.S.P.
%
8.9
20.8
41.6
39.2
6.6
6.6
85.4
82.0
84.8
85.7
84.0
76.0
83.3
81.4
6.6
45.4
83.6
30.7
82.9
82.6
82.2
81.4
73.3
63.6
78.0
74.2
76.5
75.0
76.3
* Samples taken at two week intervals.
                              475

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        Table 2.  Average Calcium, Magnesium, Sodium,
        Bicarbonate, Total Dissolved Salts, S.A.R.  and
         S. S. P. Values for Catfish Production Waters
                    at Several Locations,
                       Na            T.D.S  S.A.R.   S.S.P.
Location*   Ca+Mg   meq/1    HCOs    ppm  ratio       %
1
3
4
6
8
9
10
0.99
0.58
0.86
0.53
1.60
1.13
1.60
2.4
4.1
2.7
4.3
0.8
0.5
0.9
3.8
3.0
3.3
2.8
1.9
1.6
2.1
265
405
269
383
221
145
226
3.4
7.6
3.8
9.0
1.0
1.0
1.0
70.6
87.2
75.0
89.6
33.0
31.2
36.0
* 1, 3, 4, and 6 were in the Coastal Plain of Mississippi.  Loca-
  tions 8,  9, and 10 were in the Mississippi Delta of Arkansas.
                              476

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                  THE DIALECTICS OP A PROPOSAL

                               ON

            BIOLOGICAL CONTROL 0? iSUTHOPHICATION IN

                         SEWAGE LAGOONS
                           S. T. Lin*
                            ABSTRACT

The existing lagoons for the deposition, of municipal sewage face
the problems of extreme eutrophication and of the effluents being
highly polluted for discharge into public waters.  An elaborate
mechanical engineering plant if established to purify the sewage
water would be very expensive in both initial cost and mainten-
ance.  The present proposal is to take the advantages of biolog-
ical control by utilization of natural performance of pure plank-
ton feeding fishes , filamentous algae eaters , bottom feeders and
scavengers to purify the sewage water.  It is more economical
this way than any mechanical engineering design.  In addition.
biological control has many advantages in fitting in the prin-
ciples of environment improvement and protection and in produc-
tion of animal proteins.

                          INTRODUCTION

BACKGROUND INFORMATION AND GENERAL CONSIDERATIONS

The formulation of this dialectics is the result of many long
discussions with J. M. Malone Jr., Manager of J. M. Mai one and
Son Enterprises of Lonoke, Arkansas, and after some observations
made on the sewage lagoons in Lonoke and England, Arkansas.

The most common phenomenon in these setrage holding lagoons is the
process of eutrophioation as a result of the growth of phytoplank-
ton which will reach in summer warm days to -the highest level
through the support of the organic and mineral nutrients coming
in with the domes tie sewage.  However eutrophication will not


 ^Former Fisheries Biologist of FAO of the United Nations and
  Professor of Fish  Culture, National Taiwan University, Taiwan,
  China.

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occur if the domestic sewage is too thick with sludge or mixed
with industrial waste, some substances from which will kill the
algae.  Otherwise at the highest level of eutrophication no more
space and dissolved oxygen are available for further expansion^
meanwhile toxic substances secreted by the animals and plants
are accumulated to so high a concentration that the algae and
zooplankton can no longer survive resulting in total death of
most of the biota in the lagoon followed by decomposition to pro-
duce the worst situation of pollution which may last for a consi-
derable length of time before the lagoon can return to suitable
conditions for the growth of phytoplankton again.  This is local-
ly known as'a turnover.  It is a biological turnover, to be sure,
but it oust be distinguished from the limnological turnovers
during the spring and fall due to thermal difference and wind
action.  Under the present circumstances the effluents from many
such highly polluted lagoons are being directly discharged into
public waters.

For this reason alone it is highly desirable if such excessive
eutrophication can be prevented by introduction of certain plank-
ton feeders to graze down the exuberant algae so as to avoid a
turnover and moreover a system of new ponds be built to purify
the effluents through similar biological process.  This so far
as dialectics go can be satisfactorily achieved.

While eutrophication is advancing, not only the planktonic algae
are in exuberant bloom but also zooplankton, bottom worms and
algae, snails and insects are thriving to the highest degree of
abundance.  So in addition to the introduction of phytoplankton
and zooplankton feeders, omnivores of bottom feeding habit and
even scavengers like common carp and carpsuckers which consume
waste organic matter in suspension and on the bottom are necessary
to be added to the sewage ponds so that a full balanced extent of
biological control can be accomplished.  Naturally the fishes so
introduced will produce reasonable amount of organic waste in
the form of faeces, but such excreta which have been partly
digested and fermented in the digestive tract of the fish are
easily and rapidly decomposed by bacteria into simple gases,
water, minerals and soluble organic substances.

Actually what happen in the sewage lagoon and the ponds is a
series of transformations of inorganic components activated by
the energy of the sun in the phytoplankton into organic matter
and then back to inorganic components by animals .and bacteria.
The first transformation, of course, involves the process of
photosynthesis in which th* mineral nutrients are taken up by the
phytoplankton to form organic substances with CO- and H_0 as
                                475

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basic materials of life.  Next come the zooplankton, worms, molluscs
insects and fish to feed on the phytoplankton, but as the popu-
lation of the phytoplankton is so large, these animals can only
consume a small portion of it.  In the third phase the large
animals will prey on the small ones.  At last when the plants and
animals die their bodies will be decomposed by bacteria to simple
organic and mineral components, HJ? and C02 which the algae will
be able to utilize again.

In the instance when no efficient plankton feeding fishes are
present, the phytoplankton and zooplankton will increase rapidly
to the highest degree with a  consequence of total death of the
planktons caused by population explosion shock.  But with the
introduction o\ the phytoplankton feeding  fishes to graze down
a substantial part of the phytoplankton and to keep the biological
community in balance, a turnover would be  prevented as illustrated
in figure 1*

Now  let us review some of the information  in  literature as how
far  this principle of biological control has  been applied  in the
field of fis h culture with  the  purpose to improve wildlife
environment in rivers and lakes  and to increase fish production
in ponds•

REVIEW OF LITBRATUEE

There are many kinds  of aquatic  animals which feed  on planktonic
algae.  Oysters,  clams and  mussels, for  example,  are  efficient
algae consumers.  Based on  this  feeding habit Ryther ejt  ad (1972)
envisaged the possibility  of using tertiary sewage  for oyster
culture  under  cotrolled eutrophication: He offered  a very interest-
ing  dialectics  in which he  suggested that  a town of 50,000 people
could, with  its  51  ha aqua culture-sewage  treatment  system, raise
an annual  crop  of over 900  tons  of oyster meat or nearly 90,000
hectoliters  (250,000 bushels) of whole oysters worth, in today's
market as  a luxury table  oyster, upwards  to 55 million.   But
upon further search of information it is  revealed that a certain
fishes because of their large size and free swimming habit may
be even more adequate for the control of phytoplankton.   As the
studies  of  Lin(l969) indicated,  the silver amur or silver carp
 (Hvpophthaltniohthys. molitrix) and the bighead (Aristichthys nobil is)
 which grow to 10 or 15 kgs in a few years  under favorable conditions
 are  pure and efficient plankton feeders.  The silver annir because
 of its spongy-plate-like gill-rakers that can filter minute part-
 iculates in water feeds principally on macrophytoplankton as well
 as microphytoplankton, while the bighead with closely set but
 separate gill-rakers filters out only the macrophytoplankton and
 zooplankton for food.


                                  479

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   600
   5OO
o>
P<
   400
   300
o
00

•£ 200
•H
8
o
  100
                       •Biological turnover or
                       total death of phyto-
                       plankton due to popula-
                       tion explosion shock
                       without fish
Pish
satiety
according
to
seasons
                                  Phytoplankton population
                                  under control with fish
                         Month
    Figure 1 - Hypothetical curves of phytoplankton
        population trends in sewage lagoon before
        ( solid line )  and after (  Broken line ) the
        stocking of fishes (  double lines ).
                            H2Q

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Mukhamedova and Sarsembayev (1967) found by experiments that
silver carp fry of 1.5 g consumed food equal to 11% of its body
weight and the young of 5.8 g 12^ and Omarov (1970) carried out
experiments further on the feeding patterns of silver carp "weigh-
ing 328 to 3^80 g and revealed that the fish ingested food in 24
hours at 23 C with a DO of 4.23 ml/1 of 19^ of i£s body weight on
the average.  At higher temperature (e.g. 26°-30 C) the consump-
tion rate will certainly be increased.  Apart from this information
we are not aware so far that there have been any comprehensive
studies on the quantity of plankton being consumed by the silver
carp per unit body weight per day under conditions of different
seasons.  Information on the quantitative feeding of the bighead
is entirely lacking.

It has been observed, however, that the grass carp (Ctenopharyn-
godon idella) known as pure herbivore feeding on macrophytes can
consume aquatic weeds 5° to 100 or even 200 ( as claimed by some
fish farmers) per cent of its body weight a day dependent on the
size of the fish and water temperature.  This means that a large
fish would eat comparatively more weeds than a smaller one.  Here
we may assume that both the silver carp and bighead would consume
phytoplankton 20 to 50 per cent of their weight per day in water
temperature 26  to 30 C.  It follows that if a lagoon isstocked
with 3000 kg/ha of the two fishes each more than 500g, then
600 to 1500 kg/ha per day or 120 to 300 tons in 200 days chiefly
of phytoplanlton could be consumed by the fishes a year.  It is
estimated that under optimal conditions a freshwater pond or a
sea water lagoon has the potentiality of producing 160 or 200
tons respectively of phytoplankton per ha/year (Tamiya, 1957 and
Ryther et, sd^, 1972).  In the case of sewage lagoon if a quarter
to half of this production could be removed, probably a biological
turnover would be avoided and this may be effectively accomplish-
ed by rational stocking with the above mentioned fishes together
with b3a ck carp (Mylopharyngodon p_iceua) and some other bottom
algae and worm feeders such as grey mullet (Mugil cephalus) and
mud carp (Girrhinus molitorella) also.

It is significant that Cyanophyta constitute the major part of
food bolus ( over 60^,) in the digestive tract of silver carp (Lin,
1969 and Chiang, 1971) and for this reason the fish will be an
effective agent for controlling the blue green algae which are
always abundant in the sewage lagoons.  In China over two million
ha of freshwater ponds are in existence; many of them were built
centuries ago.  Most of them especially those lying in the Bearl
fiiver and Yangtze River deltas are heavily fertilized with dom-
estic sewage, animal manure plus supplementary feeding and are
stocked with silver carp, bighead, grass carp, black carp, mud
carp and common carp (Lin, 1954* Hickling, 1971J and Chinese


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Pish Culture Experience Committee, 1961).  As a result of such
polyculture excessive eutrophication is always placed under con-
trol and consequftntly the so—called biological turnover very
seldom occurs.

The carp pond complex at Munich and Berlin serving as sewage pur-
ification fields is well known (Schaeperclaus, 1961 and Hickling,
1971).  After the sewage effluent is diluted "by 3 or 4 to 1 with
freshwater it is sprayed into ponds where the mixed effluent is
purified through natural process and can be safely discharged
into rivers.

The use of undiluted domestic sewage waste water from a sewage
treatment plant at Kielce, Poland, for the culture of Crucian
carp (Caraaaius carassius) with satisfactory results is even
more impressive (fhorsland, 1971)*  The effluent from the treat-
ment plant (by activated sludge method but without clorination)
was led into five experimental ponds, three of which were stock-
ad with carp and two as control.  Fish yield without supplemen-
tary feeding was up to 1,317 kg/ha, the highest yield ever ob-
tained in Poland.  Allen (1970) advocated the constructive use
of sewage water for fish culturej Huggins (1970) and Konefee
(1970) experimented on the use of tertiary treatment ponds to
raise channel catfish and- fathead minnow with encouraging results.
Tsai (1973) studied fish sensitivity in sewage polluted streams
of Maryland with results hinting strongly on the potentiality
of using sewage waste water for fish culture.

                  OBJECTIVES OP THE PROPOSAL

In trying to solve the problems of eutrophication and that of
the purification of the effluents coming out of the sewage
lagoons it is considered necessary to test the hypothesis or
dialectics in the best practical manner possible and thereupon
it is proposed to initiate a pilot project with the following
objectives in view:

1. The project will be designed to develop different patterns
   of fish communities  each species in which will perform a
   distinct function In controlling a certain element or elements
   in the sewage waste water.  The combined functions of different
   species should eventually result in purification of the sewage
   water.

2. It is further designed to test whether aquatic plants in
   combination with fishes would enhance the purification
   process.
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3. Meanwhile the feeding habits of the silver araur and bighead
   will be studied  and determined quantitatively.  This know-
   ledge  is particularly important in forming a basis for stock-
   ing rate design.

4. The project  would provide sufficient data to show ever more
   strongly that sewage waste water constitutes a great potent-
   ial source for fish production which could be utilized for
   both human and animal food.

                     A PROPOSED PILOT PROJECT

With the  above  objectives in view and on the basis of the in-
formation discussed before it appears very feasible that the
experiences and results obtained in the past can be applied to
the management  of the sewage lagoons in the United States with
a good chance of success.  After a preliminary survey of the
sewage lagoon system in Lonoke and England, Arkansas, we are
convinced that  a great deal can be learned from a pilot project
and therefore we strongly feel that a project of this nature
should be started as soon as possible.

SITE FOR  THE PILOT PROJECT

It is proposed  oo use the sewage lagoon of Lonoke or any other
town or city for the establishment of the pilot project in the
first year.  When it proves a success after a period of test,
one or two or even three other lagoons may be selected for next
period's  test.  In the third period, some three or four years
later, if the results of the first two period's experiments are
satisfactory, work will be extended to as many lagoons as practi-
cal*

The sewage lagoon of Lonoke has a superficie of eight hectares
(20 acres) and  a depth of 1.5 meters.  It receives an inflow of
domestic  sewage at the rate of some 500,000 gallons a day from
a population of 4,000 people.  On June 3, 1973 the lagoon was
observed  to be  at a high level of eutrophioation but not up to
the stage of turnover.  Anaoystis and other blue green algae
were abundant; some small fish (probably Q ambus ia) were seen
along the margins of the lagoon and common carp were reported
also to be present.

SIGNIFICANT ADVAHTAQES OF THE PROJECT

Although  it would be difficult to make any precise evaluation
of the project at this moment its implementation would obvious-
ly have many advantages:  (l)  It is an economical project.. The


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cost of construction and maintenance of any mechanical engineer-
ing sewage treatment plant will be always higher than this pro-
posed "biological control plant.  Houghly speaking the establish-
ment of amechanical engineering plant may cost over a million
dollars while that of a "biological control plant will cost only
half or even less as much.  The maintenance cost will be also much
more in the engineering plant than in a biological control plant;
(2) if the purification ponds are to be well arranged and con-
structed with roads and trees are planted around, no smell will
be emitted from the lagoon and ponds and such area may well be-
come a suitable spot for recreation purpose; (3) the sewage
purification pond system when well stocked with fish can be open
to public for sport fishing; (4) fish is one of the principal
sources of animal, protein for human consumption.  If all the
sewage lagoons in the United States are to be placed under simi-
lar management fish crop would be considerable, which can be
rationally harvested and processed to serve as animal and human
food; (5) the project would turn all the hazards of the domestic
sewage ponds into a natural purification system that would be
in perfect harmony with any balanced environment.

                ORGANIZATION AND MANAGEMENT

Any private fish farm in the United States which has the facili-
ties and expertise to propagate and cultivate the silver amur,
the bighead, the black, the white amur and the common carp can
be trusted by contract by a city or town government to manage
the project.  Such private fish farm under contract will take
the responsibility to design, develop and maintain the pilot pro-
ject.  At the request of the city government the ulnvironment Pro-
tection Agency of Federal Government should subsidize the Pro-
ject by grant funds.

An essential task of the private farm as a managing agent is to
propagate all the fishfls required to stock the biological control
sewage system anfl to replace the fishes after their removal at
harvest able size.

When this pilot project proves a success at the end of a year or
so, it is envisaged that work would be continued and extended
to other city sewage systems under similar working and organi-
zation relationship and financial arrangement between EPA, city
government and the private fish farm as managing agent.

                    METHODS AND PROCEDURE

CONSTRUCTION OF PURIFICATION PONDS
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The first step to be taken for the construction of the  purifica-
tion ponds is to make a topographic survey of the area  concerned
and then basing on the topographic map a pond system will  be
designed and constructed.

As a usual practice one hectare of sewage lagoon is provided  for
a population of 500 people.  For example, for a town of 4,000
people like Lonoke of Arkansas, a lagoon of 8 ha or 20  acres  at
200 persons per acre with about 1.5 meter in depth has  been
built.  Despite the lack of data it is assumed that the concen-
tration of the nutrients in the secondary effluents from the
sewage lagoon ranges quite close to the national average which
shows in micron moles per liter: 242 NO , 1180 SH , 264 PO ,
and 656 SiO  (Ryther «t jal, 1972).  Concentration of such level
is, of course, still too high for discharge into public waters.
In order to reduce the concentration it is propos ed to construct
six ponds each one ha in superficie and two meters in depth,
below the existing sewage lagoon of 8 ha to receive the effluent
for further purification, the proposed arrangement of which is
shown in figure 2.

The superfioie ratio between sewage lagoon and each purification
pond of a system  of six in this case is approximately 8:1.  How-
ever the exact proportion should be determined in the future by
experience and research.

PROGRAM OP STUDIES

Determination of  Hater Quality

The water quality of the sewage lagoon will be studied according
to the Standard Methods for Examination of Water and Waste Water
edited by American Public Health Association, 1971.

After the six ponds are built and filled with sewage water but
before the stocking of fishes, determination of water quality
should be made (of both the lagoon and all the ponds) at least
once a month according to the following criterias
    Conductivity, total chlorine, detergent, ammonian nitrogen,
    nitrite and nitrate nitrogen, PO  , SiO_, Gu, Zn, C02, DO,
     COL, BOD, alkalinity, hardness, acidity, turbidity, tem-
    perature in air and water, eto.

Water samples will be taken at selected spots as indicated in
figure 2.  A 2-liter sampling  cylinder will be made for this
purpose.

Sjbudy of Fishes


                                425

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          U)
 (x)
              Sewage lagoon
                ( 8 ha )
                  U)
                              ire
                         tflow gat a
                                   Inflow gate
        tion of water
              flow
           P4
P6
                                 6 new
                                 purification
                                 ponds
Figure 2 - A sketch showing  the existing sewage lagoon
     (above) and the air new purification ponds (below)
     (X) indicates the  selected spot for water quality
     and plankton sampling.
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Observation will be made on the daily movement of the  fishes
especially in relation to oxygen contents and their growth rates.
As all the fis hes stocked except the common carp are  unable  to
spawn in captivity, popuplation study will be a simple matter.
Even the common carp because of the depth of water and polluted
conditions, may not be able to reproduce as well.  F'ish samples
will be collected with nets from the lagoon and purification
ponds once every three months during the period from April to
October for weight and length measurements.  In each sampling
it is expected to obtain 20 to 50 silver amur, 5 white amur,  5
bighead, 5 black carp and possibly 3 common carp for examination.

Plankton and Benthos.

The same water from samples collected for water quality deter-
mination will be used also for plankton study.  Phytoplankton
and zooplankton concentration, identification and number of each
kind per liter will be determined according to limnological
methods.  Benthic biomass study will follow the method described
in Hallock and Ziebell (1970).

Bacteria

Attempt may be made to determine whether coliforms are present
at the outlet of pond 6.

Use of Aquatic Plants for Purification

Aquatic plants are to be grown in ponds P5 and P6 for the pur-
pose  of testing how effective phosphate and other nutrients left
over  would be absorbed by  them.  The water quality examinations
at tha inflows and outflows of the two ponds  will be compared
to determine  this effect.

SELECTION OS1 WISHES FOR STOCKING

The fishes as described below all possess  the characters  that
would meet the biological  requirement for sewage waHe water
purification, but  the most important common merit to them all
is their, tolerance to low  dissolved oxygen, content; they  all
thrive fairly well in a DO of 2  to 3 ppmQand  would grow well in
4 to  5 ppm at watar temperature  about 28 C; they can even sur-
vive  for  sometime below 1  ppm.

Silver amur - This silver  fish is a vary efficient phytoplankton
feeder, fast growing to attain 5 to 10 kg  In  a few years.  It
lives in  the upper layer of water, fast swimming, agile,  active
and always jumping out  of  water  when disturbed;  large fish can
stand lower DO than small  ones;  a warm-water  fish but tolerates

                                427

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temperature "below 4 C.  Because of the special spongy structure
of its gill-rakers it can filter out phytoplankton less than
20 microns for food.

Boghead - A slow moving fish but fast growing, attaining large
size up to 10 or 15 kg in a feir years; a 10-pounder of three
years old is not uncommon.  The fish has a very large head, hence
its name, and a wide mouth, living in the upper layer of water,
gaping all the time to suck in phytoplankton, zooplankton and de-
tritus for food.  A warm-water fish y«y tolerant to water tem-
perature below 4 Cj it survives under ice.

Black carp - A very large fish found in lakes and rivers of
China up to 50 kg but in captivity it usually grows to some
10 kg in a few years.  It prefers to live close to the bottom
of deep water so that it oan pick up snails, mussels and crus-
taceans for food.

White amur or grass carp - This fish is well known for its
remarkable ability to consume satiably aquatic weeds, filamentous
algae, land grass and leaves of many land plants.  It roams every
where in search of food} a hungry fish will take mixed feedstuff,
meat, pieces of cloth or shoe leather when they are thrown into
the $ond, and yet it prefers fresh, tender vegetable food when-
ever they are available.  It attains some 5 kg in three or four
years on vegetable food alone, tolerant to water temperature
below 40.

Common carp - A fish of longivity, perhaps a unique character
among all fishes known; a color carp was recorded in Japan to
live 215 years in a monastery.  It inhabits most of the time
near the bottom* sucking up worms and detritus mixed with mud.
Immediately following the sucking the mud is ejected and the
worms and digestible part of the detritus are retained and
swallowed.  As a result the bottom soil is thus disturbed and
activated.

FISH STOCKING DESIQHS ?0£ EXPERIMENT

The design is based roughly on the estimates of phytoplankton
production and the eating capacity of the plankton feeders.
There will be designs of different fish communities for any
particular sewage lagoon system.

When the six purification ponds each about one ha in superficie
are built we would have a system of seven ponds including the
sewage lagoon*  According to the figufres given by Tamiya (195?)
and Ryther et. al (1972) the sewage lagoon at Lonoke of 8 ha is
capable , as estimated, to produce 1280 tons of phytoplankton

                               428

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a year, about 128 tons of which might be simultaneously convert-
ed to zooplankton.  However to simplify calculation we may assume
that the lagoon would produce a total of 1300 tons of plankton
in all ayear or 6.5 tons a day if we count only 200 days a year
as the active production period.  Theoretically if all the plank-
ton are to be removed, 13,000 to 32,500 kg of plankton-feeding
fish should exist all the time in the lagoon and they would con-
sume plankton 20 to 5°$ of their body weight.

Evidently it is neither possible nor desirable to deplete the
lagoon of all the phytoplankton by stocking too many fish at
one time, for a considerable quantity of phytoplankton must be
left to do away with the noxious gases, excessive minerals and
organic substances and to provide dissolved oxygen and C0p and
consequently proper pH for the balance of life in the lagoon.
For this reason it is proposed to stock the lagoon with a mini-
mum rate of fish to feed on the plankton or empirically a rate
of 1000 kg/ha or 8000 kg of fish for the whole lagoon of 8 ha,
provided that the DO is sufficient for the fish to thrive.
However, wherever the sewage lagoon is found to be insufficent
in DO, no fish should be introduced and the effluent that flows
into the ^rification ponds should be diluted with freshwater
to improve the conditions for plankton growth and fish life.
Anyhow at this rate of stocking, the silver amur, bighead and
white amur of about 250 g would grow to one kg within a year
which would be sufficient to consume enough plankton so as to
prevent a turnover in the hot summer. As the fish grow and the
lagoon is found overcrowded with fish the big ones should be
removed and small ones introduced to replaced them.

A test fish stocking system of the lagoon and six ponds is
suggested here as shown in table 1 for reference.

This table is given only to indicate a possible system of fish
stocking .  Modifications will have to be made when the waters
of the lagoon and ponds are analised with attention being paid
particularly to DO, SH  and other poisonous substances.  As the
primary sewage waste water is continuously flowing into the
lagoon and out from it carrying plankton at the rate of some
2.5$ of the holding capacity of the lagoon and 10 to 20$ of
that of the purification ponds from one to another, the phyto-
plankton concentration must be accordingly reduced and there-
fore the fish stocking rate should be proportionately lowered.

ADDITIONAL MECHANICAL DEVICES IF N3C2SSARY

It is hoped that by this process of biological purification the
effluent coming out of Pond 6 would be close enough to the EPA
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  standards which stipulate as  30 ppm of BOB,  30  ppm of CO]),  30 ppm
  of suspended solids, 200 coliforms  per ml  and a pH reaction of
  9.  However in view of the fact that after a considerably long
  period the accumulation of solids from the domestic sewage  in
  the lagoon bottom will be too much  for satisfactory functioning
  of the biological control system and the plankton and suspended
  solids may become higher than the standards, it may be  necessary
  to consider, first of all, the installation  of  a sedimentation
  system with a fermentation tank (activated sludge method) to
  remove the solids before the  city sewage is  pumped into the
  lagoon.  This is one part of  the common installations in many
  mechanical engineering sewage treatment plants.

         Table 1.  A proposed stocking rate  for the Lonoke
                   sewage pond  system
                 Quantity of each species  to be introduced
Lagoon
 and   Area ———•	
ponds  (ha) Silver Bighead Black White  Common Aquatic
             amur          carp  amur   carp   plants
                                                           Total
fewage
agoon
PI
P2
P3
P4
?5
P6
Grand
total
8
1
1
1
1
1
1
14
6,000
3,500
2,500
1,500
1,000
1,000
500
16,000
1,000
500
500
500
300
200
100
3,100
400
50
50
50
50
50
50
700
200
50
50
50
50
—
— —
400
200
100 —
50
50 —
50 —
— Selected
— plants
450
7,800
4,200
3,150
2,150
1,450
1,250
650
20,650
                 PROSPECTS 0? FUTURE DEVELOPMENT

 According to the records on municipal sevage system of Arkansas
 kept in the Department of Pollution Control and Ecology, State
 of Arkansas, January, 1972, there were 622,000 people who used
 "oxidation ponds" or lagoon for sewage treatment which cover a
 total of some 3,110 acres or 1,260 ha .  If 945 ha. more ponds
 are constructed to purify the effluents from the sewage logoons
 as described in the above proposed biological control project
 the total area will be increased to 2,205 ha.  Country-wide
statistics on sewage lagoons are not available at the moment, but
 should similar design be extended to the whole country at the
 same rate of one-third of the population or 70 million out of
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210 million people, a total of sewage-aquaculture area  of
247,350 ha would be required.  In this aquaculture system
primarily for the purification of sewage waste waters, if fish
are stocked at the rate as shown in table 1, it would be possible
to harvest a crop of 494,700 tons in total of marketable fish at
the rate of 2000 kg/ha/year.

Objections may be raised against the allocation of land for the
construction of the biological purification system.  Some may
think that land is too valuable for such a project and one  or
two hectares for every 300 people is excessive.  But  contra-
dictions exist between clean air and water and the limited  space
of land.  The urgency for the resolution of such^contradictions
allows us no alternative but to adopt a system Awould be most
economical and fit for maintaining the harmony and balance  of
natural environment for human life.  It is therefore logical to
argue that if clean air and water are desired sufficient land
space must be provided for nature to work.  Moreover consider-
ation must be given to the high cost of establishing and main-
taining a compact  chemical and mechanical engineering plant
against that of an economical biological control  one.  In the
matter of environment protection principle, space is a basic
factor for the maintenance of clean air and water, for unless
sufficient spaoe is allowed for nature to work in oxidation,
reduction, sun energy activating photosynthesis  and bacterial
decomposition, all the wastes produced by men  and animals cannot
be recycled back to their original harmless components.  For
this reason it is  not hard for us to  realize that in  city and
rural  community plannings, adequate land space and open air
must be made available and amply reserved for parks, gardens,
forest, recreation fields, roads, houses, hospitals, schools,
sewage waste water disposal, factories and agriculture fields.
When  the  fact  that such  planning is closely linked  to public
health and environment protection  is  kept in mind,  one  would
 agree  that the provision of  one  or two hectares  of  land for  the
 treatment of  domestic sewage of  every 300 people is  nothing
but a matter of  absolute necessity.

                          REFEREHCES

 Allen, Q.H.  1969.  A preliminary bibliography on the utilization
        of sewage in fish culture.   FAO Fish Circ. 308.
  lien, G.H. 1970. The constructive use of sewage with particular
        reference to fish culture.  Constribution to the FAO
        Technical Conference on Marine Pollution and its Effects
        on Living Resources and fishing.  FIRtMP/70/R-13- Rome,
        9-18 December, 1970.
                                437

-------
 Chekiang Fisheries Bureau. 1957. Pond fish culture.  Finance
       and Economics Press, 226 pages.

 Chiang, ¥an. 1971.  Studies on feeding and protein digestibility
       of silver carp, Hypophthalmiohthys molitrix (C.and V.).
       JOBS Fisheries Series Ho. 11, p. 96-114.

 Chinese Fish Culture Experiences Committee. 1961.  Chinese Fresh-
       Water fish culture.  Science Press, Peking, 612 pages.

 Falck, T. 1934-  The construction of sewage fish ponds as relief
       work.  Gresundh. Ing. 57:228 (Abstract on Sewage Work Jour.
       1934, 6,  4.).

 Federal ¥ater Pollution Control Administration. 1968.  Water
       quality criteria.  U.S. Goveramnet Printing Office, 234
       pages.

 Hallock, R.J. and C.D. Ziebell, 1970.  Feasibility of a sport
       fishery in tertiary treated wastewater.  Jour.  Water Poll
       Control Fed.,  42(9)«1656-1665.

 Hiokling, C.F. 1966.   On the  feeding process in the white amur,
       Ctenopharyngodon idella. Jour. Zool. 148:408-409.

 Hickliag, C.F. 1971.   Fish culture.   Faber and Faber, London,
       317 pages.

 Huggins, T.£. and S.W. Bachmann,  1970.   Production of channel
       catfish (Ictulurus  punotatus)  in tertiary treatment ponds.
      U.S. Govt. Res. Develop. Kept. 70(9): 132.

Hynes, H.B.N., I960.  The biology of polluted waters.  Liverpool
      University Press, England,  190 pages.

Katz, M. and A.R. Ganfin, 1952.   The effect  of sewage pollution
      on the fish population  of a midwestern stream.  Trans. Amer.
      Fish. Soo., 82:156-165.

Konefes, J.L. and R.¥. Bachmann,  1970.   Growth of fathead
      minnow (Pimephales  promelas) in tertiary treatment  ponds.
      Proo. Iowa Acad. Sci.,  77:104.

Kraus, Harjorie, 1971.  Molecular process  in lagoon management.
      Remarks made at Chester County Commissioner's Symposium
      on Lagoons. Holiday Inn, Lionville, Pa., 2 pages.
                               432

-------
Lin, S.Y., 1940 •  Pish, culture in ponds  in the Hew Territories
       of Hong Kong.  Jour. Hongkong Fish.  Res. Sta., 1(2): 161-
       163.

Lin, S.Y., 1954*  Chinese systems of pond stocking.  Indo-Pacif ic
       fisheries Council Proceedings, p.  65-71.

Lin, S.Y., 1969.  The feeding habits of  silver carp, bighead
       and mud carp.  JCHB Fisheries Series No. 8, p. 49-66.

Mckee, J.E. andH.¥. Wolf, 1963.  Water  quality criteria.  Calif.
       State Water Quality Control Board Publ. 3-A, 548 pages.

Mukhamedova, A.F. and Zh.G. Sarsembayev ,  1967.  On the daily
       feeding pattern and food consumption of underyearling
       silver carp.  Tr. Volgogradskogo  otd. Cos. N.-i. in-ta
       Ozern. 1 reohn. rybn. kh-va, _3_.

Omarov, M.O., 1970.  The daily food consumption of silver carp,
       Hypophthalmiohthys molitrix (Val.).  Jour, of Ichthyology,
       10( 3) » 425-426.

Prowse, O.A., 1969.  The role of cultured pond fish in the con-
       trol of eutrophxcaTion in lakes and dams.  Verh. Internat.
       Verein., 17:714-718.

Ryther, J.H., 1959*  Potential productivity of the sea.  Science,
       130:602-608.

Ryther, J.H., W.M. Duns tan, K.R. Tenore and J.S. Huguenin, 1972.
       Controlled eutrophication - Increasing food production
       from sea by recycling human wastes.   Bioscience, 22(3):
       144-151.
Ryther, J.H. and W.M. l)unstan, 1971-  Nitogen, phosphorus and
       eutrophication in coastal marine  environment, Science,
       171:1008-1013.

Schaeperclaus, W., 1961.  Lehrbuch der teichwirtschaf t.  Verlag
       Paul Parey, Berlin, 582 pages.

Tamiya, H., 1957.  Mass culture of algae.  Ann. Rev. Plant
       Physiol., 8:309-334.

Thorsland, Anders B., 1971.  Potential uses of waste waters and
       heated effluents.  EIPAC Occ. pap., (5): 23, p. 1-7.
                               433

-------
Tsai, Chu-fa, 1973-  Vater quality and fish life below sewage
        outfalls.  Trans.  Amer. Pish. Soc., 102(2):281-292.

Willis, Duddley, 1970.  A  talk on effective, inexpensive, proven
        method of recycling human waste and preventing water
        pollution.  The Archive, Downingtown. Pa., ¥ed. Nov. 18.
        1970.

tfolny, P., 1967.  Fertilization of warm water ponds in Europe.
        W.P.P.C., (3)*64-8l.

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         THE  HARVESTING  OF ALGAE AS  A  POOD  SOURCE

         PROM  WASTEWATER USING NATURAL AND  INDUCED

                  FLOCCULATION TECHNIQUES


                            by


Joseph L. Pavoni1, Steven  W.  Keiber2,  and Gary  T.  Boblitt^
                     Conference on the
          Use of  Wastewater In the Production  of
                      Pood and Fiber

              Trade  Winds Motor Inn - Central
                  Oklahoma City, Oklahoma
                      March 6-b, 1974
1.  Associate Professor of Civil and Environmental Engi-
    neering, University of Louisville, Louisville,
    Kentucky  40205

2.  Environmental Engineer, Hazelet and Erdal Consulting
    Engineers, Louisville, Kentucky

3.  Environmental Engineer, Kentucky Department of Trans-
    portation, Frankfort, Kentucky  40601
                             435

-------
         THE HARVESTING OF ALGAE AS A FOOD SOURCE

         FROM WASTE WATER USING NATURAL AND INDUCED

                  FLOCCULATION TECHNIQUES

                            by

Joseph L. Pavoni1, Steven W.  Keiber2, and Gary T. Boblitt3


INTRODUCTION

The autotrophic nature of algae has recently led to in-
creased speculation as to the usefulness of these micro-
organisms in various nutrient removal schemes applicable
to effluents from existing wastewater treatment plants.
Since algae characteristically utilize inorganic compounds
in the production of protoplasmic material, several ter-
tiary treatment processes have been developed which focus
upon' the "stripping" of basic nutrients from solutions via
normal algal metabolism.  Whereas algae represent a con-
centrated mass of protein, fats, and carbohydrates, recent
interest has also been expanded to include algal cultiva-
tion for possible commercial food usages.  Therefore, a
nutrient removal process which utilizes algae metabolism
for "stripping" purposes represents a means of processing
unwanted community wastes while economically, continuously,
and quickly providing a relatively pure potential food pro-
duct.

One of the serious drawbacks in any nutrient removal sys-
tem involving algae is the separation of these microorgan-
isms from the corresponding liquid phase.  Regardless if
one is considering nuisance conditions in surface waters,
nutrient "stripping" processes, or the commercial produc-
tion of al^ae for food supplies, feasible methods of har-
vesting algae in all cases becomes of utmost importance.


1.  Associate Professor of Civil and Environmental Engi-
    neering, University of Louisville, Louisville,
    Kentucky  no20«

2.  Environmental Engineer, hazelet and Erdal Consulting
    Engineers, Louisville, Kentucky

3.  Environmental Engineer, Kentucky Department of Trans-
    portation, Frankfort, Kentucky  40bOi


                            436

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Among the various mass-liquid  separation processes that
have been Investigated with regard to their potential for
algae 'Harvesting include:  centrifugation, filtration,
flotation, ion-exchange, sedimentation, straining, chemi-
cal treatment,  and  biof locculation.   V.lhile the majority of
these methods are quite costly, properly managed floccula-
tion techniques nay offer a feasible, as well as economi-
cal, solution to algal-liquid  separation.  The purpose of
tills investigation  was therefore focused on describing
the feasibility of  harvesting  algae as a food source from
wastewater treatment  systems using natural and induced
flocculation techniques.

LITERATURE REVIEW

Unlike bacterial aggregation,  very little published re-
search information  Is available pertaining to algal agglu-
tination.  Initiating a discussion of algal flocculation
mechanisms necessitates an understanding of algal surface
charge.

Most algal cells (with the exception of certain filamentous
forms) fall in the  size range  of 5 to ^0 urn.  Although
algae are larger than true colloids (1 to 100 nm), they
nevertheless possess many surface properties similar to
true colloids.  Discrete algal cells are known to form sta-
ble microbial suspensions, possess a chemically reactive
cellular surface, and possess  a net negative surface charge
(1) (2) due to the  ionization  of functional ionogenic
groups.  Algae may  be considered as hydrophilic bio-col-
loids since algal suspension stability depends not only on
the forces interacting between the particles themselves,
but also on the forces interacting between the particles
and. the water.

Algal_Bioflocculation Literature

Due to the limited  amount of research conducted on algal
biofIccculation, documentation in the published, literature
of actual floe formation Is scarce.  The agglutination of
5ii..El§Q2.l2.2:l1iriL stagnatile into settleable floes resembling
aerated activated sludge was observed by Eogan, Albertson,
et_ al (3) during a  I960 investigation of phosphorus removal
by algae.  It was postulated that the enclosure of the cell
by a broad gelatinous sheath (4), usually characteristic of
these filamentous algae,  may have promoted entanglement of
filamentous algal appendages with consequent enhancement of
bioflocculation.
                            437

-------
A review of algal harvesting processes by Golueke and Os-
wald (5), in 1965, resulted in the attainment of biofloc-
culation under specific environmental conditions.  The
necessary requirements were an actively photosynthesizing
shallow culture, a relatively warm day, and sunlight.  How-
ever, as in the previous investigation, no hypothesis for
the algal bioflocculation mechanism was proposed.

In 1967, Schuessler (6) proposed an algal bioflocculation
model in which naturally produced polysaccharide polymers
would bridge between discrete algal cells.  In his studies,
bioflocculation potential was correlated with the algal
growth phase - maximum algal removals being observed during
declining growth.  Schuessler pointed out that declining
growth was a period of increased polysaccharide production
and excretion, thereby affording optimal bridging between
linearly extended polysaccharide polymers and algal cells
with resultant bioflocculation.  Experimental results also
noted that high algal removals due to bioflocculation were
observed at both low and high ph levels.  For the low pli
band from 1.0 to M.O,  in which algae are in close proximity
to their isoelectric point, effective bioflocculation was
reported with best results at ph 3.0.  For the high pH band
from ph 11,0 to 13.0,  the algal removal efficiency was
greater than that observed in the low pK region and was
attributed to the formation of hydroxyl and carbonate pre-
cipitates that served to enmesh the algae with resulting
removal.

In 1969, Tenney, Echelberger, et_al_  (7) postulated a bio-
flocculation model In which high molecular weight exocellu-
lar metabolites, which are of sufficient length, bridge
between discrete algal cells, thereby initiating the bio-
flocculation process.   They contended that the microbially
produced polymers initially might enhance floe formation,
while in later growth phases the accumulation of this ex-
tracellular material could be acting as a protective col-
loid.

While Investigating further the effects of algal exocellu-
lar polymer production on the bioflocculation process,
Pavoni  (b) reported, in 1971, that algal cell aggregation
appeared to be governed by the physiological state of the
microorganisms.  Observations indicated that floe formation
was restricted to the declining growth phase.  In this
study, direct correlations were developed relating exocel-
lular polymer production and algal agglutination.  Pavoni,
therefore, postulated surface coverage phenomenon as the
mechanism of algal bioflocculation,  in which case surface

                             438

-------
potential reduction was not necessarily a prerequisite for
agglutination.

The excretion or exposure of natural polymers on the algal
surface during photosynthesis is widely documented in the
literature with the implication by several investigators
(9) (10) that the mechanism of algal bioflocculation is the
bridging function of naturally produced polymeric species.
It, therefore, seems appropriate to discuss the specific
types and quantities of these algal exocellular materials
referred to in the literature.

In 1849, Kutzing's original description of Navlcula pelli-
culosa  (Synedra minutissima and pelliculosa)» according to
German  (11), made reference to an algal gelatinous cap-
sule -  "Individua in gelatina membranacea nidulantia" -
which has given the organism its present specific name.

In 1851, reports by Magin (12) indicated that through the
use of dyes Bailey found an external envelope of complex
carbohydrates to exist around the cell walls of diatoms.
Liebisch (13) showed these exocellular secretions to have
the same chemical nature as pectic compounds in 1929.

Various fresh and salt water algal species were observed
to secrete organic materials by Krough (14) and Aleev (15)
during the 1930's.  In 1950, Locker (16) noted the presence
of slime on the surface of ponds containing an abundance of
Navicula pelliculosa.  She noted the capsule of this spe-
cies,  when cultured in the laboratory, sustained a posi-
tive carbohydrate stain.

Microscopic examinations of Anabaena, an algal species from
which Bishop, Adams, et al (1?) isolated an exocellular
polysaccharide, showed many of the filaments were enclosed
in a mucilaginous substance which was readily sloughed off
into the medium.  This report of nitrogenous material se-
cretion by blue-green algae was supported in 1952 by Fogg
(18) who found polypeptide and amide secretion by young
cultures of Anabaena, a species of blue-green algae.  That
same year Hough, Jones, et al (19) observed amylopectin-
like polymers in Qscillatoria while the species Nostoc
yielded a mucilaginous, complex, acidic polysaccharide
composed of a minimum of six distinguishable monosaccha-
rides.

In 1954, Bailey and Neish (20) found that polysaccharide
reserves, composed of amylose and amylopectin, constituted
up to  20 percent of the dry weight in some algal cultures.

                            439

-------
Secretions of peptides, amides, and amino-nitrosen were
observed by Fogg and Vfestlake (21) the next year, and their
report included emphatic remarks as to the importance of
such secretions to the ecology of fresh waters.

Lewis and Rakestraw (22) found carbohydrates in seawater in
quantities of from 0.1 to 0.4 mg/1 and attributed it to the
secretion products of planktonic algae.  While Lev/in (23)
found no evident capsule around actively dividing Mavicula
pelliculosa cells, he did observe the formation of a gela-
tinous capsule, composed of glucuronic acid residues, fol-
lowing the cessation of cell reproduction.

Polysaccharide and organic acid secretion by green algae
was reported by Tolbert and Zill (24) and Allen  (25) in
1956.  Extending this specific research with unicellular
and colonial green algae, Lewin (26) observed that all
Chlamydomonas species studied liberated some soluble poly-
saccharides into the growth medium.  This material was
found to constitute 25 percent of the total organic matter
produced by the cells of Chlamydomonas mexicana.  In all
but one species, galactose and arabinose were the main
components of the polysaccharides.  Lewin also postulated
other possible algal metabolites including free organic
acids and polypeptides.

Collier (27) demonstrated carbohydrate production in bac-
teria-free cultures of Prorocentrum, in 1958» while dis-
cussing the importance of naturally produced organic com-
pounds in surface waters.  Krauss (2b) also reported find-
ing an exocellular polysaccharide material excreted by the
genus Oscillatoria.  The same year Fogg and Boalch  (29)
noted extracellular polysaccharide and nitrogenous con-
pounds in Ectocarpus confervoides and concluded that both
types of exocellular products are liberated from healthy
cells.

Carbohydrate accumulation, occurring initially during de-
clining growth and continuing into the endogenous growth
phase, was observed by Guillard and Wangersky  (30).  They
stated that cell demise liberated comparatively large
amounts of carbohydrate which could account for the pre-
sence of this material during the active growth phase.
Guillard and Wangersky, noting that algal photosynthetic
reserves amass during stationary growth under circumstances
unfavorable for the utilization of external carbohydrates
as an energy source, also postulated that these reserve
materials consist largely of other materials such as poly-
peptides and organic acids, which may also be present in

                             440

-------
the growth medium.

Mer?,, Zehnpfenning, et al  (31) investigated species of
9l}^^y.Q™™&*L» Chlgrell.gV Chloracoccurn, and Scengdesmus, in
196*2, and noted some production of soluble exocellular
organic natter by the majority of autotrophic algal species
studies.

More recently, Moore and Tischer  (32) reviewed the three
classes of organic compounds known to be liberated by
freshwater algae - organic acids, nitrogenous material such
as pclypeptides and free amino acids, and carbohydrate
polymers.  Their investigation was specifically concentrat-
ed on the exocellular polysaccharide content of eight spe-
cies of green and blue-green algae.  Results indicated
total polysaccharide production in amounts equal to 10 to
40 percent of the algal dry weight.

In summary, a large variety of organic substances have been
detected in the exocellular products of algae.  These
materials can be classified into three general categories:
proteins, polysaccharides, and nucleic acids.  With the
structure of most of these substances being polymeric and
with these materials possessing a net negative charge in
neutral Dh ranges, the exocellular products may be con-
sidered to respond as naturally produced anionic polyelec-
trolytes.

There is, however, a scarcity of information regarding the
role of these exocellular products in the algal agglutina-
tion mechanism.  Consequently, any refinement of the algal
agglutination process would entail investigations into the
composition of the exocellular polymers, the mode of poly-
mer release from the cell, and the role of cell surface
potential.

Chemical Flocculation of Algae

The feasibility of removing algae from water and wastewater
by chemical flocculation techniques has -been studied by
several investigators.  The available literature, although
limited, indicates the apparent feav^ibility of utilizing
this technique for algal removal.  In 1958, Cohen, Rourke,
et_ ajl (33) conducted studies involving the agglomeration of
Chlorella cells with synthetic organic polyelectrolytes.
Results indicated that good flocculation was achieved while
using a cationic polyelectrolyte, whereas no flocculation
was observed with the anionic polymer.
                            447

-------
Golueke, Osvjald, et_ al_ (3*0 Indicated excellent aggregation
   Chlorella and Scenedgsmus with cationic polyelectrolvtes
in 196*1.  In these studies the requisite polyelectrolyte
dose was insensitive to pH in the range of 5.0 to 10.4;
however, increasing concentrations of dispersed algal cells
increased the requisite polyelectrolyte dose proportionate-
ly.  Algal flocculation with anionic or nonionic oolyelec-
trolytes was not investigated.

More recently field scale pilot plant studies by the Dow
Chemical Company have served to demonstrate further the
feasibility of utilizing cationic polyelectrolytes as algal
flocculants.

In 1969, Tenney, Echelberger, et_ al_  (7) investigated the
feasibility of removing algae from water and wastewater
with synthetic organic polyelectrolytes.  Representative
cationic, anionic, and nonionic synthetic organic poly-
electrolytes were used as flocculants.  Under their experi-
mental conditions, chemically induced algal flocculation
occurred with the addition of cationic polyelectrolytes,
but not with anionic or nonionic polymers, although attach-
ment of all polyelectrolyte species to the algal surface
was shown.  It should be noted, however, that anionic and
nonionic polymeric flocculation was only studied at pK 7.0.

Obviously, very little information Is available which be-
gins to delineate the parameters involved in the chemical
flocculation of algae using anionic or nonionic polyelec-
trolytes.  It would appear that a basic need exists, not
only for the development of quantitative data regarding
the effectiveness of selected anionic and nonionic poly-
electrolytes as algal flocculants, but also for the evalua-
tion of the basic mechanism of algal-f locculation inter-
action and the associated parameters affecting this inter-
action.  Also, since synthetic organic anionic and nonionic
polyelectrolytes most probably closely resemble natural
exocellular algal polymers, a correlation may exist between
synthetic polymer-algal interactions and natural polymer-
algal interactions.

EXPERIMENTAL PROCEDURE

The general experimental approach employed throughout this
investigation was to select scientifically established
techniques that would not only allow proper evaluation of
basic theoretical parameters, but also would be flexible
enough  to thoroughly support  reasonable variations in
experimental protocol.  Analytical procedures chosen were

-------
sufficiently sensitive and specific for the scope of this
investigation but, nevertheless, were not overly sophisti-
cated to the point of being nonapplicable to the wastewater
treatment field.

Laboratory scale batch algal culturing reactors were used
throughout this investigation.  The initial algal inoculum
was obtained from the inside wall of a secondary clarifier
at the Hite Creek Wastewater Treatment Plant in Louisville,
Kentucky.  A typical algal culturing system consisted of a
40-liter glass Jar, a stone air diffuser which supplied
both aeration and complete mixing, a large cooling fan, and
a bank of flourescent lamps developed specifically to stim-
ulate photosynthesis.  This light source imparted a culture
illumination of 400 foot-candles to the algal surface with
an energy dominance in the blue and red regions of the
visible light spectrum.

An initial algal concentration of approximately 10 to 20
mg/1 was inoculated into 25 liters of a standard liquid
algal medium (35), and daily pll adjustments were performed
when necessary to insure a pH between 7.0 and 7.5.  All
evaporation losses were made up with distilled water .each
day previous to sample withdrawal.  Culture temperature was
maintained at 22°C±2°C.

Once an algal batch reactor was begun, the experimental
procedure consisted of recording several parameters includ-
ing:

     1.  algal mass  (dry weight basis) in accordance
         with the procedures of Engelbrecht and Mc-
         Kinney (36).

     2.  culture flocculation as a function of either
         cake filtration time or percent transmission
         at 520
     3.  algal cell surface charge using a Riddick
         Zeta Meter (37).

     4.  extraction of exocellular polymer for quan-
         titative monitoring  (dry weight basis) and
         fractional composition analysis.

Exocellular polymer extraction techniques utilizing ethanol
v/ere employed similar to the work of Ueda, et al  (38).
Three samples secured from batch reactors were centrifuged
at 32,500 g for 15 minutes to affect shearing of polymeric

                             443

-------
material  from  the algal surface.  The supernatent from
this  centrifugation was added to 95 percent ethyl alcohol
so  that the  final ratio of volume was two parts ethanol to
one part  supernatent.  The supernatent-ethanol mixture was
refrigerated for 24 hours at 4°C after which time a white
fibrous precipitate was observed.  Quantitative monitoring
of  microbial polymer production was accomplished at this
point by  performing a membrane filter suspended solids test
on  two of the  original three samples.

The white precipitate formed in the third sample was then
separated from the supernatent-ethanol liquid phase by
centrifuging at 32,500 g for 15 minutes and dissolved in
distilled water.  This polymer solution underwent qualita-
tive  colorimetric analysis for polysaccharide  (39), pro-
tein  (40), ribonucleic acid (RNA) (41), and deoxyribonu-
cleic acid (DNA) (42).

Plocculation of algal dispersions with synthetic organic
polyelectrolytes was performed utilizing standard jar
testing procedures (7).  Algal suspensions were secured by
harvesting algae from batch cultures by means of centri-
fugation at 32,500 g for 15 minutes and resuspending the
algal pellet  in a 0.5 percent aqueous sodium chloride
solution.   This centrifugation procedure was repeated to
insure proper contaminant  removal.   Organic flocculants
used were produced by the  Dow Chemical Company and included
cationic polyelectrolytes  (C-31 and C-32), anionic poly-
electrolytes  (A-22 and A-23), and nonionic polyelectrolytes
(N-ll, N-12,  and N-17).

RESULTS AND DISCUSSION

Algal Bioflocculation Studies

Initially, laboratory batch fed algal cultures were nur-
tured to study the relationship between algal growth,  cul-
ture turbidity, exocellular polymer production, exocellular
polymer to algal mass ratio,  exocellular polymer composi-
tion, and cell surface charge.   If  current polymer bridging
models are to be expanded  to  explain algal biofiocculation,
then it should be possible to correlate algal culture
turbidity decrease with an increase in exocellular polymer
production.  Additionally, algal cell surface charge would
be expected to remain constant  throughout the entire growth
cycle.

The first study was comprised of a  batch fed heterogenous
algal culture and was focused on duplicating and refining

-------
several correlations that had previously been shown to
exist for algal biof locculation phenomenon.  A microscopic
analysis of this culture was performed to ascertain the
predominant al.ir.al species present in the medium.  Chpjrella
and a filamentous strain were observed in abundant numbers;
however, a determination of which algal type predominated
and the exact identification of the filamentous strain were
not made.

 he data frcn culture 1, as presented in Figures 1 and 2,
clearly demonstrate several significant aspects of biologi-
cal f locculation.  Algal agglutination (culture turbidity
decrease) was observed to occur only after the algae had
entered endogenous growth stages (see Figures 1A and IB).
The dependence of the algal biof locculation process on the
physiological state of the algae agrees with previous
investigations by Favoni (8) and parallels results obtained
for similar bacterial systems  (43).  Filtration time
(sec Figure 1C) was also observed to be a qualitative mea-
sure of algal biof locculation in agreement with the work of
La-'or and liealy  (i\>\}.  The filtration time in this culture
was observed to reach a maximum prior to endogenous growth
and decrease steadily from that point as biof locculation
phenomenon increased.

If current polymer bridging models are to be expanded to
explain algal biof locculation, it should be possible to
correlate algal culture turbidity decrease with an increase
in exocellular polymer production.  As indicated in Figure
2A, the algal exocellular polymer concentration in culture
1 increased significantly during endogenous growth (a peri-
od of enhanced agglutination).  The inference, as in bac-
terial systems, is that this exocellular polymer is the
primary cause of biof locculation during endogenous growth.
Since the ratio of exocellular polymer to algal mass sharp-
ly increases during culture aggregation (see Figure 2B),
the polymer bridging model of biof locculation is further
supported.  Correspondingly, the increase in this ratio
indicated that surface coverage phenomenon may have been
responsible for increased agglutination.

Another important aspect of the biof locculation process is
the mode of polymer release from the algal cell.  Algal
aggregation was noted as being limited to endogenous growth
with a concurrent increase in exocellular polymer concen-
tration during the growth phase.  Since restricted growth
is a period of massive cell lysis, algal autolysis may have
been the mechanism of polymer release.

Whereas culture 1 was investigated solely for the purpose

                            445

-------
I

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•H
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    150
     100
      50
     40
     50
    100


     25



     20




     15



     10
         -
                            A.   Algal Mass
                            B.   Culture Turbidity
                            C.   Filtration Time
• •»• —
1
1 1
1 1
                           10
                                   15
                                             20
25
                            Days of Growth
          Figure 1
                     Culture 1 Data Depicting  (A) Algal
                     Growth, (B) Culture Turbidity,  and
                     Filtration Time.
                                                         (C)
                             446

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c
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  0






1.0




0.9





0.8





0.7




0.6





0.5





0.4





0.3





0.2
A.  Exocellular Polymer Concen-

    tration
                              B.  Exocellular  Polyme^  to Algal

                                  Mass Ratio
            0
            Figure 2
                      10
       15
20
25
                              Days  of  Growth
                 Culture 1 Data Depicting (A) Exocellular

                 Polymer Production and (B)  the Ratio of

                 Exocellular Polymer to Algal Mass.
                                447

-------
of refining the  characteristics of previously observed
phenomena, culture 2 was developed and monitored to deter-
mine the algal cell surface charge through the entire
growth cycle and the fractional composition of extracted
exocellular polymer.  Knowledge of cell surface charge
and exocellular  polymer compositional makeup could signifi-
cantly shed light on the understanding of the overall 'bio-
flocculation process.

A-microscopic analysis of culture 2 revealed the presence
of a dense population of Chlorella with only occasional
sightings of filamentous strains.  Therefore, Chlorella
were determined as being the predominant species in this
culture.

The results of culture 2 relevant to the refinement of pre-
viously observed bioflocculation phenomenon paralleled
those of culture 1,  i.e.,  a correlation was observed be-
tween the endogenous growth phase and enhanced aggregation
(see Figures 3A and  3B).  Filtration time was again ob-
served to serve as a qualitative indicator of biofloccula-
tion phenomenon  (see Figure 4A).

If polymer bridging  theories are to be expanded to explain
algal bioflocculation,  cell aggregation should be indepen-
dent of surface potential  reduction.  The algal cell zeta
potential was  monitored  in order to evaluate the algal sur-
face charge during the  complete growth cycle of culture 2.
Figure 4B shows that  no  significant changes in zeta poten-
tial were observed during  any algal growth phase.  The sta-
bility of the  cell surface charge indicated that surface
potential reduction  was  not a prerequisite in the biofloc-
culation mechanism.   Since a bioflocculation model based on
some measure of charge neutralization would involve the
formation of tightly  compacted cell aggregates,  the inde-
pendence of surface  charge and algal bioflocculation ob-
served in this investigation added support to the polymer
bridging theory in which discrete cells are loosely bound
in a three-dimensional matrix.

Algal exocellular polymer  produced in culture 2  was also
extracted and  weighed  to develop polymer/algal mass ratios.
Unlike results observed  in culture 1, the polymer concen-
tration in culture 2  remained virtually constant throughout
all growth phases with only a minor increase detectable
during endogenous growth (see Figure 5A).   Although the
exocellular polymer  to algal mass ratio increased during
endogenous growth,  this  increase appeared incremental when
compared to that obtained  in culture 1 (see Figure 5B).

-------
                            A.  Algal Mass
 I

 CO
 co
tn
C
O
•H
W
CO
•H
6
W
C
to
4J
C
(U
O
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0)
500   _




400



300


200   -



100  h



  0

  4
      8

     10
 20
 40
                  10
                            B.  Culture
                                Turbidit-*
                       20       30      40

                        Days of Growth
50
         Figure  3
                Culture 2 Data Depicting  (A) Algal Growth
                and  (B)  Culture Turbidity.
                               449

-------
                              A.   Filtration Time
o
0)
CO

-------
    250
o
•H
-P
                           A.   Exocellular Polymer
                               Concentration
VH tP


0) I
o
r* ^-"H.
o w
CJ -H
  CO
    200
    150
    100
Oi
      50
 rH
 O
 cu
 OJ
  to
  to
  I
   (C
   Cn
 o
 o
                           B.  Exocellular  Polymer to Algal
                               Mass  Ratio
                  10
         Figure  5
                          20
                                   30
                                            40
                            Days of Growth
                                                     50
                     Culture 2 Data Depicting  (A)  Exocellular
                     Polymer Production and  (B) the Ratio  of
                     Exocellular Polymer to Algal  Mass  (from

                     Weight Analysis).
                               457

-------
 It  is  believed,  however, that the extracted polymer  in
 culture  2  was  contaminated with inorganic material.
 Friedman,  et al  ('45), while isolating exocellular polymer
 from Zoogloea  ramigera, observed the presence of inter-
 fering materials in  the polymeric precipitate.

 Consequently,  another polymer production curve was devel-
 oped for culture 2 by adding the concentrations of the
 individual components (polysaccharide, protein, RNA, DNA)
 found  in the fractional composition tests.  This curve,
 presented  in Figure  6A, shows a more significant increase
 in  exocellular polymer concentration during endogenous
 growth,  and the  ratio of exocellular polymer to algal mass
 also shows a marked  increase during late growth (see Figure
 6B).

 In  comparing the polymer production curves obtained from
 weighing the polymer and from the fractional composition
 analysis (refer  to Figures 5A and 6A), a substantial dif-
 ference  in polymer values can be observed.  This difference
 was postulated as arising from the precipitation of inor-
 ganic  constituents which increased the polymer totals in
 the weighing procedure,  but had no effect on the specific
 compositional analysis techniques.  To determine whether or
 not inorganic Interference was occurring, several observa-
 tions were made  of precipitated polymer.  Following filtra-
 tion of  a polymer sample,  examination of the membrane fil-
 ter indicated the presence of not only long strain polymer-
 ic fibers,  but also fine,  granular particles.  Consequent-
 ly, a membrane filter containing the filtered precipitate
 was combusted in a muffle  furnace at 600°C.  Since the fil-
 ter itself  left no ash,  the residue remaining represented
 inorganic contaminants present in the sample.  It is impor-
 tant to note that a significant amount of residue was ob-
 served after combustion.   Therefore, it can be assumed that
 inorganic precipitates were formed during the ethanol ex-
 traction in agreement with Friedman, et_ al_ (45).

 Of major importance in a description of the algal biofloc-
 culatlon process, as  developed from the polymer bridging
model,  is the composition  of the exocellular polymer.  Sam-
 ples of the polymer-ethanol mixture were centrifuged to
 separate the polymer  from  the liquid phase.  The  exocellu-
 lar polymer was then  added to 20 ml of distilled  water and
 fractional  composition analysis techniques were performed.
The analysis was designed  to categorize the polymeric com-
ponents into the broad classifications of protein,  RNA,
DNA, and polysaccharide.   Figures 7A, 8A, 9A, and 10A
represent the concentrations of the major components of


                            452

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 u
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       50




       40




       30




       20




       10





        0


      0.4
        0.3
                               A.   Exocellular Polymer

                                    Concentration
        0.2
        0.1
                                B.   Exocellular Polymer to Algal

                                    Mass Ratio
                      10
                             20
30
                                                40
50
                                Days of Growth


              Figure 6   Culture 2 Data Depicting  (A) Exocellular

                         Polymer Production and  (B)  the Ratio  of

                         Exocellular Polymer to Algal Mass  (from

                         Fractional Composition Analysis).
                                  453

-------
ff
c
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-p
-p
c
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u
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         25
                             A.   Protein  Content in
                                  Exocellular  Polymer
•H

-------

o
•H
4J
c

-------
c
o
•H
-P
cti
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-P
C
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O
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u
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          5
          4
       A.   DNA  Content in Exocellular
            Polymer
                                B.  DNA to Algal Mass  Ratio
  to
  to
  m
             0
             Figure  9
        Days of Growth


Culture 2 Data Depicting  (A)  the  DNA
Fraction of Exocellular Polymer and (B)
the Ratio of DNA to Algal Mass.
                                     456

-------
e

i

c
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•P

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0)
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8




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      0.04
      0.03
      O.t)2
      0.01
                                A.  Polysaccharide  Content  in

                                    Exocellular  Polymer
                                B.  Polysaccharide  to  Algal  Mass Ratio
* 1
) 10
v —^r-— — -
1 1
20 30
1
40
•
1
50
             Figure 10
                       Days of Growth


              Culture  2  Data Depicting (A) the Poly-

              saccharide Fraction of Exocellular Polymer

              and  (B)  the Ratio of Polysaccharide to

              Algal  Mass.
                                   457

-------
exocellular polymer observed throughout the algal growth
curve.  These data show the total concentration of each
component to increase during endogenous growth and the
ratio of each component to algal mass to also increase
during this growth phase (see Figures 7B, BE, 9B, and 10E).
Consequently, it appeared that all major components of the
exocellular polymer were involved in the increase in poly-
mer production associated with algal bioflocculation; how-
ever, it should be noted that protein and RNA appeared most
prevalent in the compositional makeup of the exocellular
polymer from this culture.

To substantiate bioflocculation phenomena observed in cul-
ture  2, a third culture consisting of Chlorella as the
predominant algal type was investigated.  Data from culture
3  (see Figures 11 through 16) closely paralleled that
developed for culture 2.  Inorganic contamination of ex-
tracted exocellular polymer was again observed.

The conclusions of the algal bioflocculation studies may be
summarized as follows:

Algal agglutination appeared to be governed by the physio-
logical state of the microorganisms.  Algal flocculation
was not observed to occur until the microbes had entered
into a state of restricted growth  (endogenous growth
phase).

A direct correlation was found to exist between exocellular
polymer production and microbial aggregation.  Since exo-
cellular polymer to algal mass ratios dramatically in-
creased during algal flocculation, surface coverage pheno-
menon may be interpreted as the mechanism by which bio-
logical flocculation occurs.

Cell  surface charge investigations indicated that surface
potential reduction is not a necessary precursor to algal
bioflocculation.

The major compositional makeup of exocellular algal consti-
tuents associated with bioflocculation may be classified
into  four general categories of organic polymers:  poly-
saccharides, proteins, RNA, and DNA, thereby implying nega-
tive  polymer charge characteristics.

The mechanism of algal bioflocculation is interpreted as
resulting from the interaction of high molecular weight
exocellular polymers, which have sufficiently accumulated
at the microbial surface during endogenous growth.  These


                            458

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 35




 30




 25




 20




 15






0.7




0.6




0.5




0.4




0.3




0.2




0.1
                                Exocellular Polymer

                                Concentrat ion

                                       *
                                                      I
                              B.  Exocellular  Polymer to Algal

                                  Mass Ratio
                              10
                               15
                                             20
25
                               Days of Growth
             Figure 12   Culture 3 Data Depicting (A) Exocellular

                        Polymer Production and (B)  the Ratio of

                        Exocellular Polymer to Algal Mass  (from

                        Fractional Composition Analysis).
                                 460

-------
tr>

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 20


 18


 16


 14


 12


 10


  8



0.4


0.3


0.2


0.1
                                A.  Protein Content in Exocel-
                                    lular Polymer
                               I
I
                                B.   Protein to Algal Mass Ratio
                               I
                              10        15       20

                                Days of Growth
                                                  25
             Figure  13   Culture 3 Data Depicting (A)  the Protein
                         Fraction of Exocellular Polymer and (B)
                         the  Ratio of Protein to Algal Mass.
                                 461

-------
I
  flj

  H
  <
      15
10
       5
     0.2
     0.1
                      A.
RNA Content in Exocellular
Polymer
                           1
                                       J_
                     1
                       B.   RNA to Algal Mass Ratio
                           10       15
                             Days of Growth
                                       20
                     25
          Figure  14  Culture 3 Data Depicting  (A)  the  RNA
                     Fraction of Exocellular Polymer and
                      (B) the Ratio of RNA to Algal Mass.
                               462

-------
z
Q
10
W
(0
CP
         4   I-


         3


         2
      0.05


      0.04


      0.03


      0.02


      0.01
                              A.   DNA Content in Exocellular
                                  Polymer
                                                         I
                              B.   DNA to Algal Mass Ratio
                                Days of Growth

              Figure 15  Culture 3 Data Depicting  (A) the DNA
                         Fraction of Exocellular Polymer and
                         (B) the Ratio of DNA to Algal Mass.
                                  463

-------
 tr>
 e
 i

 0)
•H
 0
 0
i-H
O
o«
o
JG
O
O
fOl
CO
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  to
  (0
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tn
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rH
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    3



    2
 0.1



0.08



0.06



0.04




0.02



   0
            0
                              A.   Polysaccharide Content in
                                   Exocellular Polymer
                              B.  Polysaccharide to Algal
                                  Mass  Ratio
                       10        15
                              Days  of  Growth
                                             20
25
            Figure 16  Culture 3 Data  Depicting (A)  the
                       Polysaccharide  Fraction of Exocellular
                       Polymer and  (B)  the Ratio of Poly-
                       saccharide to Algal Mass.
                                 464

-------
polymers electrostatically or physically bond, ana nubsc-
auently bridge, the cells of the dispersion into a three-
dimensional matrix of sufficient magnitude to subside under
quiescent conditions.  Surface potential reduction is not
necessarily a prerequisite for agglutination in this model.
Algal flocculation can most conveniently be viewed in terms
of surface coverage relationships.

Chemical_Flqcculation Studies

Batch algal reactors were utilized in this study to supply
algal suspensions for chemical flocculation testing that
would be similar to algal samples found in typical waste-
water treatment systems.  The function of the batch reactor
was to simulate the growth of an incremental volume of
mixed algae as it passed through an oxidation pond.  The
particular algal growth phase was not a ma.lor consideration
in this study but rather the algal concentration at any
specific time.  Most chemical flocculation experiments uti-
lized algal suspensions directly from the batch reactors
with no pre-treatment to simulate as closely as possible
flocculation phenomena that might be observed at an opera-
tional oxidation pond.

The composition of a "typical" algal culture utilized dur-
ing the chemical flocculation investigation Is given in
Table 1.

             Table 1.  Typical Composition of
               Mixed Algal Cultures Utilized
           During Chemical Flocculation Studies
   Broad Group

Green Unicellular
Algae or Small
Group  (2-4 cells)

Blue-green
Filamentous

Green Filamentous

Diatoms


Green Colonial
  Genera

Chlorella
Euglena
Scenedesmus
Oscillatoria

Ulothrix

Fragilarla
Synedra

Volvox
  Number
of Species

    2
    1
    2
    3

    2

    1
    1
Percent
ELomass
  40

  29


  29

   1
                             465

-------
Effect of Hydrogen Ion Concentration on Algal Autof loccula-
tion - Before attempting to determine the feasibility of
flocculating algal suspensions with synthetic organic poly-
electrolytes, it was necessary to determine the effect of
hydrogen ion concentration on algal autof locculation.  This
data would then serve as a reference while conducting opti-
mum pH and dosage studies for each polymer.  Consequently,
an algal suspension at a concentration of 166 mg/1 was
flocculated at varying pH levels (from 1.0 to 11. U) with no
polymer addition.  The resulting data, as shown in Figure
17, depicts maximum algal autoflocculation phenomena occur-
ring at a pH of 3.0.  This study indicates that natural
flocculation is maximized when the surface charge of the
algal biocolloid is minimized, i.e., near the isoelectric
point or point of zero charge (8).  At the algae's isoelec-
tric point  (pH 3.0) the electrostatic repulsive forces will
be reduced to a minimum, thereby allowing the biocolloids
to assume interparticle distances sufficient for van der
Waal's forces to promote optimum flocculation.
Alga^Fl occ ulation with Synthetic Organic Poly electro-
lytes - The polymer flocculation of algal study was intend-
ed to investigate the effectiveness of various cationic,
anionJc, and nonionic synthetic organic polyelectrolytes
on the aggregation of various algal suspensions.  The ex-
tended segment theory of polymer flocculation would predict
that to achieve optimal flocculation for a given algal sus-
pension, an optimal concentration of poly electrolyte must
be added at an optimal pK level.  Addition of polyelectro-
lyte at pH levels other than optimum or at concentrations
greater or less than the optimum will not achieve the maxi-
mum degree of flocculation.  The pE will affect the surface
charge density of the biocolloid and various polymer physi-
cal characteristics including linear extension, degree of
ionization, and charge density.  Polymer dose will control
biocclloid surface coverage.

Consequently, the initial study focused on the effect of pH
on the cationic polymer flocculation of algae.  Standard
Jar tests were performed in the study at a constant cation-
ic polymer (C-32) dosage of 2 mg/1, a constant algal con-
centration of 161 mg/1, and pK values varying from 1.0 to
11.0.  Results indicated that optimum flocculation appeared
at pH 3.0 (see Figure 18).  This appears reasonable since
the algal biocolloid exhibits a net charge of approximately
zero at this pH level and, therefore, interparticle separa-
tion distances are at a minimum.  Additionally, the cation-
ic polymer possesses a high positive charge at this pH
level and is, therefore, well extended.  Similar phenomenon

                             466

-------
      50
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      40
30
20
      10
                             Algal  Content =166 mg/1
              1
'
                             I _ I _ I - 1 - 1 - 1 - L
               123456789  10  11  12



                                   PH


          Figure  17  Relationship Between Algal Bioflocculation

                      and Varying pH Values.
                                 467

-------
ITS

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 90




 80




 70




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                           Algal Content = 161 mg/1

                           Dosage of C-32 =2.0 mg/1
                                    I   I    I
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                                PH


         Figure 18  Algal Plocculation with Cationic Polymer

                    C-32 at Varying pH Values.
                             468

-------
were observed with another cationic polymer, C-31.

To determine the optimum cationic polymer dosage for algal
flocculation, a study was conducted at the optimum pH
(3.0), a constant algal concentration (166 mg/1), and vary-
ing dosages of C-32  (from 0 to 20 mg/1).  The results, as'
shown in Figure 19,  illustrate that optimal flocculation
occurs with a C-32 dose of 1.0 to 5.0 mg/1.  These data
correspond to the current polymer bridging theoretical
framework that states that flocculation is enhanced by
polymer addition up  to a certain dosage (approximately 50
percent surface coverage of the algal cell), and that addi-
tion of polyelectrolyte beyond that dosage results in re-
stabilization of the algal biocolloid.

Anionic polymers were the next group of polymers studied.
Again, standard jar  testing procedures were utilized to
determine the optimum pH level for anionic flocculation of
algae utilizing a constant A-23 dosage of 5 mg/1, a con-
stant algal concentration of 166 mg/1, and varying pH lev-
els from 1.0 to 11.0.  Results indicated an optimum pK of
3.0, similar to that obtained with cationic polymer sys-
tems  (see Figure 20).  However, whereas the mechanism of
cationic polymer attachment was apparently electrostatic
(due to opposite algal-polymer charges), the anionic poly-
mer is thought to attach by chemical means, i.e., hydrogen
bonding or anion interchange.  It again appears reasonable
that optimal flocculation phenomena occur at pll 3.0 (the
algal isoelectric point) since this is the point at which
the biocolloids possess a minimum separation distance.
Similar pH phenomenon was observed with another anionic
polyelectrolyte, A-22.

To determine the optimum anionic polymer dosage for algal
flocculation, a study was conducted at the optimum pH of
3.0, a constant algal concentration of 166 mg/1, and vary-
ing A-23 dosages from 0 to 20 mg/1.  The results, illus-
trated in Figure 21, indicate an optimum dosage of 1 to 2
mg/1 A-23.  Algal flocculation at the optimum A-23 dosage
was only slightly improved over a system with no polymer
(about 10 to 15 percent).  This poor increase in floccula-
tion efficiency appears to be due to the fact that the neg-
ative polymer is of  insufficient length to bridge between
the algal cells (i.e., to overcome the electrostatic
repulsive separation distance and attach itself to the
colloidal surface).  Optimum algal flocculation most pro-
bably occurs at pH 3.0 because at this point the net nega-
tive surface charges on both the algal cell and polymer are *
sufficiently reduced to allow some bridging to occur.  It


                            469

-------
     90
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04
     80  _
     70  -
     60
     50
      40
                           Algal Content =  166  mg/1
                           pH = 3.0
                        6     8    10    12   14


                        Dosage  of C-32 (mg/1)
                                                16   18
20
          Figure 19  Algal  Flocculation at Optimum pH (3.0)
                     with Varying Dosages of Cationic Polymer
                     C-32.
                              470

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                      Algal Content =  166  mg/1

                      pH = 3.0
60  _
    50
     40
                       6    8   10   12   14


                       Dosage of A-23  (mg/1)
                                           16
18
20
         Figure 21  Algal Flocculation at Optimum pH (3.0)
                    with Varying Dosages of Anionic  Polymer

                    A-23.
                             472

-------
should be noted that similar flocculation phenomena were
observed with another anionic polymer, A-22.

Optimum pH and polymer dosage studies were also performed
with nonionic polymers, i.e., polymers having a net charge
of zero.  To determine the optimum pH for algal floccula-
tion with a nonionic polymer, N-12, a standard jar test was
performed at a constant algal concentration of 153 nig/1, a
constant N-12 dosage of 2.0 rag/1, and varying pH values
from 1.0 to 11.0.  The results of this study again indicat-
ed an optimum pH of 3.0 (see Figure 22).  The poor floccu-
lation achieved with the nonionic polymers can again be
attributed to insufficient polymer chain length.  Optimum
flocculation again occurs at the point where the electro-
static repulsion forces are minimum and, therefore, al-
low some polymer bridging to occur.  At the elevated pH
ranges the repulsive forces produce biocolloid separation
distances large enough to prevent polymer adsorption onto
the surface of the algal cell.

To determine the optimum dose of N-12 for algal floccula-
tion, a study was conducted at the optimum pH  (3.0), a
constant algal concentration of 171 mg/1, and a polymer
dosage varying from 0 to 20 mg/1.  The results, shown in
Figure 23, indicate an optimum dosage of 5.0 mg/1.  This
nonionic polymer at its optimum dosage achieved an in-
crease in algal removal over that achieved by the anionic
polymers.  However, it was again postulated that the non-
ionic polymers were of insufficient length to bridge the
electrostatic separation distance effectively.  Although
the biocolloid-nonionic polymer repulsion forces are as-
sumed to be minimal due to the net nonionic polymer charge
of zero, the surface charges of both the polymer and algal
cell still appear to exert a significant effect upon algal
flocculation with nonionic polymers.  It should be noted
that similar flocculation data was developed with two
other nonionic polymers, N-ll and N-17.

In summary, Figure 24 illustrates the general effect of pH
upon algal flocculation with various polymers.  With each
polymer tested the flocculation phenomenon followed the
same general pattern.  The major factor controlling these
results appears to be the isoelectric point  (i.e., point
of zero charge) of the algae which occurs at a low pH lev-
el and dominates polymer flocculation mechanisms.  The
effect of optimum polymer dosage followed a pattern in
that dosages beyond the optimum resulted in decreased al-
gal removal.  This effect appears to arise from biocolloid
restabilization due to excessive surface coverage with
                            47J

-------
flj
>
0)
   100


    90


    80


    70


    60
&   50
    40
<
-P
0)
o
n
£   30


    20


    10
                          Algal Content = 153  mg/1
                          Dosage  of  N-12 =2.0 mg/1
           I	I    I    I    I
           12   34   5   67   89  10  11  12

                               PH

       Figure 22   Algal Flocculation with Nonionic Polymer
                   N-12  at Varying pH Values.
                            474

-------
(0
>
o
e
QJ
OS
tr>
H
<

4J
C

O

Q)
0<
    70  _
60
    50
                      Algal Content  =  171  mg/1

                      pH  = 3.0
     40
1 j
2 4
1
6
1
8
1
10
1
12
1
14
1
16
1
18 2
                       Dosage of N-12  (mg/1)


         Figure 23  Algal Flocculation  at Optimum pH (3.0)
                    with Varying Dosages  of  Nonionic Polymer

                    N-12.
                              475

-------
It)
i
s
(0

rH
<
 C
 Q)
 O
 H
 0)
100


 90



 80



 70



 60



 50



 40



 30



 20



 10



   0
                        • Algal Bioflocculation

                        A Dosage of C-31 =  15 mg/1

                        • Dosage of A-23 =  5 mg/1

                        V Dosage of N-17 =  2 mg/1
                                               10  11  12
                                pH
        Figure  24   Effect  of Varying  pH Values on Algal
                    Flocculation  Synthetic Polymers.
                             476

-------
polymer.

Algal Flocculation at Naturally Occurring pH Levels - Since
the majority of naturally occurring biological systems
operate in the neutral pH ranges, it was decided to con-
duct some polymer flocculation tests at pH 7«0«  The ini-
tial study conducted at pH 7.0 was to determine the effi-
ciency of algal flocculation with cationic polymers C-31
and C-32.  Results indicated optimum C-31 and C-32 polymer
dosages of 15 rog/1 and 20 mg/1, respectively (see Figure
25).  The significant difference between these tests and
the ones previously conducted at the optimum pH of 3.0 is
that the dosages required for optimal algal flocculation
are considerably increased.  This is probably due to in-
creased biocolloidal electrostatic repulsive forces since
the algal biocolloid becomes more negative as the hydro-
gen ion concentration decreases.  Also, the cationic poly-
mer will be less extended at higher pH values.

Algal flocculation studies were also performed with certain
anionic and nonionic polymers at pH 7.0 to determine the
effect of neutral pH levels on algal removal with these
polymers.  The results of these studies, as shown in Figure
26, indicate that very little polymer bridging is occurring
probably due to insufficient polymer length.  Also, the
electrostatic repulsive forces are significantly increased
on the algal surface at pH 7.0 compared to pH 3.0.  These
factors plus the inability of anionic and nonionic polymers
to perform a charge neutralization function result in poor
algal removal.

Algal_ Flocculation wl_th__Pqlvmer_s and Trivalent Metal Ion
Additives - The remainder of this investigation concen-
trated on algal removal with anionic polyelectrolytes, in
particular Dow Purifloc A-23.  Since in the previous stud-
ies flocculation was not achieved with polymer addition
alone, it was decided to add a trivalent metal ion to act
as a bridge between the negative algal particle and the
negative polymer.  It was felt that the trivalent metal ion
could achieve one or both of the following functions.
First, the metal ion could reduce the algal cell surface
potential, thereby allowing the negative polymer to over-
come the electrostatic separation distance and attach it-
self to the algal surface, thereby allowing bridging be-
tween the various cells.  Or secondly, it was felt that the
metal ion could serve as a bridge between the algal cell
and anionic polymer forming a biocolloidal-metal ion-poly-
mer-metal ion-biocolloid complex.  If either or both mecha-
nisms were achieved, good settling was expected to result.


                            477

-------
        Dosage of Cationic Polymer (mg/1)

Figure 25  Algal Flocculation with Cationic Polymers
           C-31 and C-32 at pH 7.0.
                    478

-------
to
s
g
<1>
PS
rH
(C
C
0)
U
H
0)
CM
    30
20
10
      0
                 10
                      Algal Content =  400 mg/1
                      pH = 7.0
                           lAnionic Polymer A-22

                           iAnionic Polymer A-23

                           Nonionic Polymer N-12

                             i     I     '	I	L
                        20        30

                       Dosage  (mg/1)
                                                40
50
         Figure 26  Algal  Flocculation with Anionic and
                    Nonionic  Polymers at pH 7.0.
                              479

-------
Alum  (aluminum sulfate) was chosen as a representative
netal ion because of its wide use in the field of water and
wastewater treatment.  The initial study utilizing alum
involved the addition of varying alum concentrations  (from
0 to  100 mg/1 as Al^"1") to algal samples at a constant pH of
7.0 and a constant algal concentration of 170 mg/1.  The
results indicated an optimum dose in the range of 15 to 20
mg/1  as depicted in Figure 27.  Algal flocculation was
achieved primarily by charge neutralization with the posi-
tive  aluminum metal ion.  If the positive metal ion is add-
ed in excess of the optimum dosages, it can cause restabi-
lization of the colloidal suspension.  This occurs by ad-
sorption of the positive metal ion into the diffuse layer
around the algal cell in such quantities that the net
charge on the particle becomes positive, thus causing
electrostatic repulsion.  The optimum dosage of the metal
ion occurs when adsorption of the positive ion has re-
duced the surface potential to its lowest point, thereby
allowing van der Waal's forces to dominate and cause  floc-
culation.

To determine algal removal phenomena with A-23 and alum, a
study was conducted at the optimum alum dosage  (20 mg/1), a
constant pH (7.0), a constant algal concentration of  176
mg/1, and a varying A-23 dosage  (from 0 to 20 mg/1).  The
results, depicted in Figure 28, illustrate an optimum A-23
dose  of 1.0 mg/1.  The removal of algae was considerably
improved over a similar system without the alum addition
 (see  Figure 26).  This would indicate that the metal  ion is
capable of serving as a bridge between the negative algal
colloid and the negative polymer.  Also, the alum-polymer
system improved flocculation over that of alum alone  (see
Figure 26).

The second trivalent metal ion utilized was Fe3+ in the
form  of ferric chloride.  An initial study was performed
to determine the optimum iron dosage.  This test was  con-
ducted on algal samples at a constant concentration of
166 mg/1, a constant pH of 7.0, and an iron dosage varying
from  0 to 100 mg/1 as Fe^ .  The results, as shown in
Figure 29, indicate an optimum iron dosage of 50 mg/1.
 Iron  dosages above 50 mg/1 caused restabilization of  the
biocolloids and thus hindered the flocculation process.
This  phenomenon is similar to that observed with the  alumi-
num.

To determine the optimal A-23 dose for algal removal  with
an iron additive, a study was conducted as a constant pH
of 7.0, a constant algal concentration of 170 mg/1, a con-


                             480

-------
     50
i
0)
ITS

r-<
<
-P

CD
o
i-l
     40
     30
    20
    10
     0
                           Algal Content =  170 rag/1
                           pH = 7.0
                                                          «.
                             I
_L
I
I
                  20        40         60         80

                   Dosage of Alum as A13+  (mg/1)
                                                          100
        Figure 27   Algal Flocculation with Varying  Dosages
                    of Alum at pH 7.0.
                             481

-------
     80
«t
4J
c
Q)
O
H

-------
(C

I

-------
stant Fe3+ dosage of 50 mg/1, and varying dosages of A-23
ranging from 0 to 20 mg/1.  The results, depicted in
Figure 30, show that over 90 percent algal removal is
achieved with an A-23 dose of 1.0 mg/1.  Although the algae
was effectively removed with the addition of the Fe^4" and
A-23, the mechanism of removal did not appear to be solely
that of polymer bridging.  Due to an excess of phosphate
ion in the culture medium, it appeared that the Fe^* was
precipitating the phosphate causing an enmeshment of al-
gae and polymer in the iron-phosphate precipitate.  Re-
stabilization of the biocolloidal system did not seem ap-
parent from the results but would probably occur at exces-
sively high polymer dosages.  Even though these results de-
pict effective algal removal, such removals would not be
achieveable in actual systems since naturally occurring al-
gal systems usually contain about no more than 4 mg/1 to
5 mg/1 of phosphorus, whereas the culture media contained
several hundred mg/1 of phosphorus.  Therefore, the next
series of studies were performed to determine whether or
not these high removals could be achieved in a harvested
algal culture.

The first investigation conducted on a harvested culture
 (i.e., a culture with a phosphate content less than 1.0
mg/1) was to determine the optimum iron dosage.  A study
was conducted at a constant pH (7.0), a constant algal
concentration (166 mg/1), and varying dosages of Fe^*
 (ranging from 0 to 500 mg/1).  The results, shown in Figure
31, indicate an optimum iron dosage of 200 mg/1.  The algal
removal curve again shows a peak with decreased removals
occurring when iron is in excess of the optimum.  However,
the significant factor is that the optimum iron dosage is
considerably higher ('J times as great) in the harvested
suspension as opposed to the "dirty" suspension.  This
would seem to indicate that algal enmeshment in the iron-
phosphorus precipitate did occur in the previous study.
Also, the percent algal removal achieved by the iron at
the optimum dose was reduced by about 50 percent as corn-
pared to the removals achieved in the "dirty" suspension
(nee Figure 29).
T
 he next investigation was intended to determine the per-
cent algal removal with A-23 and iron in the harvested
culture.  A study was performed at a constant algal con-
centration (166 mg/1), a constant pK (7.0), the optimum
iron dosage (200 mg/1 as Fe^ ), and varying dosages of
A-23 varying; from 0 to 20 mg/1.  The results depict an op-
timum A-23 dosage occurring at 0.5 mg/1 with subsequent
reduction in percent removals due to excessive A-23 doses

-------
   100
    90
nj

O
e
CD
PS
                          Algal  Content  =  170  mg/1
                          Iron as  Fe3"1" = 50  mg/1
                          pH  = 7.0
    80  .
c

-------
It)

i
0)
OS
Cn
-P
c
0)
o
M
0)
(X
                          Algal Content  =  166 mg/1

                          pH = 7.0
                 100       200        300        400


                   Dosage of Iron  as  Fe3+  (mg/1)
500
         Figure 31  Algal Flocculation  in a Harvested Culture
                    with Varying  Dosages of Iron at pH 7.0.
                              486

-------
causing restabilization of the algal biocolloids  (see
Figure 32).  These removals are markedly reduced from
those illustrated in Figure 30.  These data again support
the theory that algal enmeshment in the iron-phosphorus
complex was occurring.  Although enmeshinent of the algal
cell in the phosphate precipitate appeared to be the major
removal mechanism, some bridging probably occurred between
algal cells as well as between the iron-phosphate precipi-
tate and the algal cells.

However, flocculation did occur in the harvested culture
indicating that the anionic polymer-ion bridge was capable
of removing algae in suspension.  The mechanism of algal
flocculation appeared to be a combination of charge-neutra-
lization and bridging or a combination coagulation-floccu-
lation phenomenon.

Two final studies were performed to determine the effect
of iron addition on algal flocculation with cationic and
nonionlc polymers.  The initial jar test was conducted at
a constant algal concentration of 170 mg/1, a constant ph
of 7.0, a constant iron dosage of 50 mg/1 as Fe^*, and
varying dosages of C-31 ranging from 0 to 15 mg/1.  The
results of this study, as shown in Figure 33, indicate an
optimum C-31 dosage of 1.0 mg/1 as compared to approxi-
mately 15 rng/1 previously reported in Figure 25 for a
similar system without iron additive.  Apparently, the
iron-phosphate complex is enmeshing the algae to such an
extent that the polymer dosage required for destabilization
of the algal suspensions is greatly reduced.  However, the
addition of the cationic polymer significantly enhances al-
gal removal beyond that achieved with iron additives alone
due to its ability to electrostatically attach to the algal
biocolloid, thereby forming polymer bridges.

The last study conducted was intended to determine the
effect of the optimum iron dosage on algal removal with a
nonionic polymer.  This Jar test was conducted at a con-
stant algal concentration of 168 mg/1, a constant pH of
7.0, a constant Fe-^  dosage of 50 mg/1, and varying dosages
of N-ll from 0 to 20 mg/1.  The results, shown in Figure
3^, illustrate an optimum polymer dosage occurring at 0.5
mg/1.  Algal removals observed were vastly improved over
those obtained in algal suspensions flocculated with N-12
alone (see Figure 26).  It is postulated that this im-
proved algal removal is due to algal enmeshment in an
iron-phosphate precipitate.  However, the M-12 was ob-
served to Improve algal removal beyond that obtained
                             H87

-------
(0

I
Q)
OS
tn
-u
c
0)
o
    70
                          Algal Content = 166 mg/1

                          Iron as Fe   = 200 mg/1
    60
50
40
    30
     20
                   I
I
I
I
I
                       6    8     10   12   14


                       Dosage  of A-23 (mg/1)
                                            16    18
                              20
         Figure 32
               Algal Flocculation with  the Optimum Iron

               Dosage  (200 mg/1) in  a Harvested Culture

               and Varying Dosages of Anionic Polymer

               A-23 at  pH 7.0.
                             488

-------
   100
                          Algal  Content = 170 mg/1

                          Iron as Fe3+ = 50 mg/1

                          pH = 7.0
    90
o
e
0)
en
rH
<

-P

0)
o

(1)
    80
    70
     60
     50
        Figure 33
                        Dosage of C-31 (mg/1)


                   Algal  Flocculation with the Optimum Iron
                   Dosage (50 mg/1)  and Varying Dosages of

                   Cationic Polymer C-31 at pH 7.0.
                             489

-------
(0
g
E
0)
(C
0>
.H
4J
C
0)
o
H

-------
with iron addition only.

In summary, the mechanism of algal removal using nonionic
arid cationic polymers appears to be enneshment. in the
phosphate precipitate being formed as a result of iron
addition.  Algal removals observed in cationic polymer-
iron systems were considerably greater than those in non-
ionic polyrner-irori systems since the cationic polymer
electrostatically bridged, between the algal cells, whereas
the nonionic polymer had to rely on adsorption phenomenon
for attachment to the biocolioid surface.

The conclusions of the  chemical fiocculation studies may be
summarized as follows:

The optimum pH for algal agglutination with synthetic
organic polyelectrolytes appeared to be in the range of pH
2.0 to 4.0, thereby implying that polymer fiocculation
mechanisms are governed by the algal cell surface charge.

Optimum dosages of all  synthetic polymers at optimum pFi
levels appeared to be  in the range of 0.5 to 5.0 mg/1 with
subsequent restabilization of the algal suspension at
higher polymer dosages.

At neutral pH levels  the cationic polyelectrolytes utilized
appeared  to effectively flocculate the algal suspension
through a combination  of polymer bridging and charge neu-
tralization, whereas  anionjc and nonionic polyelectrolytes
failed to efficiently  flocculate the algae at pK 7.0 due to
their inability to bridge  the repulsive separation distance
and chemically bond to  the cell  surface.

The addition of trivalent  metal  ions to algal suspensions
flocculated with  polyelectrolytes was observed to signifi-
cantly enhance algal  fiocculation phenomena compared to
algal' removals obtained with either  trivalent metal ion or
polymer  addition  only.   It is postulated  that the trivalent
metal ion enhances  fiocculation  phenomena by reducing the
algal cell  surface  charge  (reducing  biocolioid separation
distances)  and possibly serving  as  a bridge between the
algal cell  surface  and  the polyelectrolyte.

CONCLUSIONS

It appears  that algae utilized  in nutrient removal schemes
for wastewater treatment may provide a  potential food pro-
duct  if  they can  be  efficiently  harvested.  Natural and
induced  fiocculation  techniques  apparently may promote
                             497

-------
efficient algae-liquid separation at reasonable costs.
The results of this investigation imply that algae har-
vesting by means of bioflocculation or chemically in-
duced flocculation techniques can be accomplished in a
very efficient manner under certain conditions.  It, there-
fore, seems imperative that further research be focused  on
algal bioflocculation and chemically induced flocculation
mechanisms on a pilot scale to develop pragmatic opera-
tional data which could be utilized to accomplish effective
algal harvesting as a food source.

AC KNO WLEDGEMENT

This investigation was supported by a National  Science
Foundation Engineering Research Initiation Grant GK-32732
awarded to Joseph L. Pavoni.

The kindness of the Dow Chemical Company for supplying
samples of synthetic organic polyelectrolytes is appreci-
ated.

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     Flocculation," Journal Water Pollution Control  Federa-
     tion, 44_, 414 (March 1973).

44.  LaMer, V. K., and Healy, T. W., "Adsorption-Floccula-
     tion  Reactions of Macromolecules at the Solid-Liquid
     Interface," Reviews of Pure and Applied Chemistry,
     13., 112 U9637:

45.  Friedman, B. A., Dugan, P. R., Pfister, R. M.,  and
     Remsen, C. C., "Fine Structure and Composition  of the
     Zoogloeal Matrix Surrounding Zopgloea ramigera,"
     Journal of Bacteriology. 96, 2144 (1968).

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                   CRITICAL VARIABLES IN FOOD-ITEM

       POPULATION DYNAMICS IN A WASTE-WATER AQUACULTURE SYSTEM

                                  by

                           Joseph E. Powers*

INTRODUCTION

Increases in human populations and the associated increases in domestic
wastes have made it essential that beneficial methods of disposal of
these wastes be developed.  One suggestion was the addition of treated
sewage to aquaculture ponds  (Allen*).  Nutrient-rich sewage waters
added to an ecosystem would generally increase the primary production
and lead to higher productivity in the system as a whole.  But, in
order for such a strategy to be effective in an aquaculture system,  it
is mandatory that the majority of the energy that results directly from
the sewage addition be channelled into the species being cultured and
not lost to other populations within the system.  This implies that
simple straight-chain food-webs would be most effective in aquaculture.
Therefore, in managing aquaculture systems, it is imperative that there
be an understanding of the key variables associated with the population
dynamics of the major food organisms.

An aquaculture system fertilized by domestic sewage was constructed
adjacent to Humboldt Bay, California (Allen, Conversano and Colwell2)
for use in rearing salmon (Oncorhynchus kisutch) to the smolt age,
i.e., the age at which they migrate to the ocean.  Ultimately such a
system will provide a return run of,adult salmon to Humboldt Bay.**

The primary food organism which developed within this pond was the
gammarid amphipod Anisogammarus confervicolus Stimson.  Powers^ re-
ported on the structure and general results of a simulation model des-
cribing the dynamics of this amphipod population.  That simulation
model is now used to give evidence as to the nature of the critical
variables affecting the dynamics of the amphipod population.  This
evidence is formulated into a possible management strategy and some
generalizations concerning management of these types of aquaculture
systems are made.


 *Department of Fisheries and Wildlife Sciences, Virginia Polytechnic
  Institute and State University, Blacksburg, Virginia.
**The ponds were constructed by the Conservation Board of the
  California Department of Fish and Game and are operated under
  National Sea Grant Funds, California State University, Humboldt
  Coherent Area Project, Northern California Coast.
                                  497

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THE AQUACULTURE SYSTEM

The Humboldt Bay aquaculture system consists of a 1.4 hectare pond lo-
cated within the perimeter of a 55-acre oxidation pond.  Tertiary-
treated domestic sewage could be introduced from the oxidation pond to
be mixed with sea water from Humboldt Bay.

Major animal populations existing in the pond (other than the salmon
and amphipods) were two species of fish:  sticklebacks (Gasterosteus
aculeatus) and topsmelt (Atherinops affinis).  These fish entered as
larval or egg forms through a metal screening device which strained
the inlet water from Humboldt Bay.  Holes in the device were six milli-
meters in diameter.  These apertures were the means by which the amphi-
pods gained access to the pond, as well.

THE SIMULATION MODEL

The model consists of a system of difference equations that are updated
each of 22 time periods.  Each period corresponds to three days of
actual time.   The amphipod population was divided into age classes and
at each time period the vital statistics of ration size, growth, preda-
tion by fish populations and natural mortality, i.e., mortality other
than predation (assumed to be the result of the direct effect of temp-
erature and salinity)  were computed for each age class.  Inputs into
the model consisted of initial population estimates for the fishes and
amphipods, temperature and salinity measurements, predation rates of
fish on the amphipods, amphipod reproduction rates and particulate
organic matter (POM)  measurements, all of which were taken directly
from studies  of the aquaculture pond (POM measurement method was that
of Lovegrove^).

The initial simulation used these inputs exactly as they were measured
in the pond.   Subsequent runs were made with perturbations of most of
the inputs in order to discern the key relationships.

RESULTS

Most of the amphipods that entered the pond through the Humboldt Bay
inlet were adult forms  (Figure 1) and, therefore of too large a size
to be utilized by the small fingerling salmon.  In order to maintain a
stable food-item population in the system, the population must be
allowed the time to develop.  The importance of this lag time and its
approximate length can be observed in Figure 2.  This figure actually
displays two lag times:  1) the lag between conversion of the sewage
nutrients to POM, and 2) the subsequent conversion of POM to amphipod
biomass.  The pond was  filled with sewage water and sea water seven
days before the initial time used in the simulation.  Approximately
30 days lag time was needed for POM build-up and 45 days for amphipod
build-up.

                                  498

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 GO
 c
 CO
 CO
 CO
 0)
 C
 (U
 o
 (-1
 CD
     O
     00
                       18
              36
                    54
                              Time in Days


        Figure 1.   Changes in Amphipod  Size Distribution with
                    Time.
                            POM
CN


 60
 g
     o „
     CO
             .	,	.	,	.   Amphipods
                12
~T"

 24
—T

 36
                                                               • Cv|
                                                              CM I
                                                                O
                                        00
                                        a
                                     o  -H
                                        CO
                                        C
                                        V
                                        Q

                                        n
                                        CO
48
60
                           Time in Days
        Figure 2.   Particulate Organic Matter and Simulated
                    Amphipod Biotnass Density versus Time.
                              499

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  Table 1.   Effects  of Salinity  on Amphipod  Growth Rate
Salinity (ppt)





Average Growth
Rate (mg dry/day)

Age
(days)
3
6
9
12

20
Ave Weight
(mg dry)
0.376
0.468
0.581
0.718
0.038
24
Ave Weight
(mg dry)
0.377
0.471
0.587
0.728
0.039
16
Ave Weight
(mg dry)
0.373
0.462
0.569
0.697
0.036
   Table  2.   Effects  of  Density on Amphipod Growth Rate
Amphipods/m^







Age
(days)
3
6
9
12
15
188.27
Ave Weight
(mg dry)
0.376
0.469
0.583
0.722
0.889
782.23
Ave Weight
(mg dry)
0.311
0.384
0.476
0.588
0.721
Average Growth
Rate (mg dry/day)               0.043            0.034
                            500

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X
CO
60
W
•O
O
ex
E
o
 •
o
                   18
—i—
 54
                 27      36      45
                   Time in Days
Figure 3.  Simulated  Egg Production versus Time
63
                                 507

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A decrease in the lag time would be a desirable goal in an aquaculture
system.  This could be attained by starting with an amphipod population
composed primarily of young animals and/or by having an increased
growth rate of the amphipods.  Manipulations of several of the system
variables could produce such changes in growth rate.

Salinity changes caused flux in the growth.  Results from computer runs
at  three different salinities on day 24 of the simulation are shown in
Table  1.  The average growth rate of 15-day-old smphipods appeared to
be  greatest at a salinity of 24 parts per thousand  (ppt).  Similarly,
the effects of population density on day 45 of the simulation are
shown  in Table 2.  Quadrupling the amphipod density resulted in a 26.4
percent decrease in the growth rate over the first 12 days of life.

Another cause for this lag in biomass production is the reproductive
process  (Figure 3).  Total reproduction did not rise appreciably until
day 36.  This rapid increase was caused by growth into reproductive age
of  the amphipods which, themselves, were produced in the pond.  It was
at  this stage that the size distribution of the pond began to stabilize.

DISCUSSION

A major concern in managing an aquaculture system is that of establish-
ing an adequate food source for the species being cultured.  In the
Humboldt Bay salmon culture project, it appears that this can be
achieved most effectively by maintining a brood stock of amphipods and
by  controlling the salinity.

Maintenance of a brood stock would mean assuring the survival of a
sufficient number of amphipods from one culture session to the next,
i.e.,  the pond should not be completely drained allowing some habitat
in  which the amphipods could reside.  Also, the pond should be refilled
as  soon as possible after the partial draining to further minimize
mortality.

Salinity manipulations could be used to maximize amphipod growth rate.
 If  the initial salinity was kept at 24 ppt, the amphipods would grow
 fast and  quickly establish  the population.  This salinity  (24 ppt) was
 shown to be  optimum  from  the laboratory studies using  in  fitting data
 to equations  in  the  simulation model5.  Then  salinity  could be  lowered
 (the proportion  of  freshwater could be  increased) to meet the physiolo-
 gical needs  of  the  salmon.   Temperature would  affect growth rate as
 well, but it is  unlikely  that temperature  control would be economi-
 cally feasible.

 A large number  of  sticklebacks  and topsmelt entered the  aquaculture
 system and grew to a size that  was capable of exploiting  the  amphipods.
 This introduced  the element of  interspecific  competition to  the salmon.
                                   502

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This aspect should be eliminated  from  the  system.  Since the incoming
water cannot be screened  any more finely  (or  else the amphipods could
not enter) the time of  the  year the  ponds  are filled should not coin-
cide with the times in  which large numbers  of eggs and larval fish
appear in Humboldt Bay.

CONCLUSION

Sewage water can bring  added productivity  to  an aquaculture system, and
by judicious management that productivity  can be shunted into the
desired species.  The food-item must be allowed to develop a sufficient
population.  The time needed for  this  development can be decreased by
controlling system variables at the  optimal levels for the food-item.
Once the food-item population is  somewhat  stable and able to with-
stand environmental pressure,  the system variables should be converted
to levels optimal for the species being cultured.  Species which do not
contribute directly to  the  cultured  species should be eliminated from
the system entirely.

REFERENCES

1  Allen, G. H. The Constructive  Use of Sewage, with Particular Ref-
   erence to Fish Culture,   FAO Technical  Conference on Marine Pollu-
   tion and its Effects on  Living Resources and Fishing.  Paper No.
   Fir: Mp/70R-13, 1970.

2  Allen, G. H. , G. Conversano and B.  Colwell.  A Pilot Fish-pond
   System for Utilization of Sewage  Effluents, Humboldt Bay, California
   Marine Advisory Extension Service,  Sea  Grant Program, California
   State University, Humboldt, CSUH-SG-3,  1-25, 1972.

3  Powers, J. E. The Dynamics  in  a Salmon  Culture Pond.  Simulation.
   21(4): 69-72, October  1973.

4  Lovegrove, T. The Effects of Various Factors on Dry Weight Valves.
   J. H.  Faser, ed.  Contributions to  Symposium on Zooplankton Produc-
   tivity, Rapports et  Proces-Verbaux  Des  Reunions, 153: 86-91, 1961.

5  Powers, J. E. Ibid.
                                  503

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       THE FEASIBILITY OF PENAEID SHRIMP CULTURE  IN BRACKISH PONDS
                   RECEIVING TREATED SEWAGE  EFFLUENT*

                          William L.  Rickards**

                              INTRODUCTION

 The study described herein was  one  part of  an extensive investigation
 of the ecological structure and functioning of brackish water pond
 systems receiving treated sewage effluent.   The  studies were con-
 ducted at the University of North Carolina  Institute of Marine Sciences
 in Morehead City, N. C.  from 1969 through 1972.   Details concerning
 the results of the four-year program were presented by Odum and
 Chestnut,1Kuenzler and Chestnut,3*3  and Kuenzler, Chestnut and Weiss*
 in project annual reports.

 Experimental facilities  for the program consisted of six 0.1 acre
 ponds, three of which received  brackish tidal creek water plus
 treated sewage effluent  while the remaining three ponds received
 only brackish water and  served  as control units.

 The long-range goals of  the overall  sewage  study program included
 the development of methods  of aquaculture which  would utilize the
 effluent of sewage treatment plants  as  well as providing a screening
 device for organisms with the ability to withstand the environmental
 fluctuations which occur in ponds receiving the  effluent.

 During the two previous  years of the pond project, penaeid shrimp
 had been stocked in both the control ponds  and the sewage ponds.
 The results of these stocking attempts  were described by Beeston5>6.
 In both cases, the shrimp survived  and  grew in the control ("C")
 ponds but failed to survive  in the sewage  ("P") ponds.  At the
 time, it was the opinion of project personnel that failure of the
 shrimp to survive in the "P" ponds  was  due  to the very low levels
 of dissolved oxygen in the  water at night (Smith7) as well as rather
 wide diurnal pH fluctuations (Laughinghouse and  Kuenzler6).

 Despite the failure of penaeid  shrimp to survive, aquacultural interest
 in them because of their economic value prompted the study described
 below.  The objectives of this  study were to modify the oxygen and pH
 regimes in one of the "P" ponds and to  subsequently stock the pond
 with penaeid shrimp to determine whether or not  factors in addition
 to oxygen and/or pH had  been responsible for the previous shrimp
 mortalities.
 *This study was sponsored by Office of Sea Grant, NOAA, U.S. Dept.
  of Commerce under Grant No. 2-35178, and the State of North Carolina,
  Department of Administration.
**University of North Carolina Sea Grant Program, 1235 Burlington
  Laboratories, North Carolina State University, Raleigh, N. C.  27607
                                  504

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                      I.   HABITAT IMPROVEMENT  STUDY

In an attempt  to  alter  the "P"  pond  habitat so  that penaeid shrimp
would be able  to  survive,  P-2 was selected for  a  study in which the
pond was aerated.   It was  hoped that a  relatively small amount of
aeration would alleviate  the  extremely  low dissolved oxygen encountered
in the early morning  hours by other  investigators (Smith7).  Since
penaeid shrimp generally  require dissolved oxygen levels of at least
2.0 ppm, 3.0 ppm  was  considered a desirable level to attempt to
maintain in P-2.

METHODS

Prior to aeration,  pond maintenance  procedures  involved routine daily
measurement of dissolved  oxygen, temperature, and pH in the evening
and early morning.  In  addition, a diurnal dissolved oxygen curve
was determined on 20-21 June, 1971.   On 14 July 1971, aeration of
the pond was begun by means of  a 3/4 horsepower,  oil-less compressor
and 200 feet of perforated plastic air  hose.  The hose was arranged
in a four-leaf clover pattern so that all areas of the pond received
aeration.  During the period  14-22 July, dissolved oxygen, pH, and
temperature fluctuations were monitored.  Temperature and pH were
measured by recording instrumentation at the  pond.  Dissolved oxygen
was determined by Winkler  titration  of  samples  fixed at the pond
and returned to the laboratory.

RESULTS AND DISCUSSION

Prior to aeration,  the  dissolved oxygen varied  diurnally from 0.0
mg/1 to about  13.5  mg/1 as shown in  Figure 1.   Values plotted in
Figure 1 are averages of  triplicate  measurements  taken at different
depths and locations  in the pond.

Following aeration, fluctuations in  the diurnal curve had moderated
(Figure 1).  Neither  the  daytime peak nor the early morning low
point reached  those experienced without the aeration.  Of greatest
interest was the  maintenance  of an average minimum dissolved oxygen
value of 3.4 mg/1  over  the seven day period of  measurement.

In addition, the  pH which  had at times  varied from 7.5 to nearly
10.0 on a diurnal  basis was now being maintained  within a much
narrower range, 7.6 to  8.3 Water temperatures  did not differ
noticeably between  the  two periods being compared.

Use of the compressor and  perforated air line did not stir up the
bottom sediments.   This could have been a problem since increased
turbidity' in the system would have resulted in  lower rates of photo-
synthesis by the  phytoplankton  possibly pushing the  system past the
point of compensation where production  balances respiration.  This
would not be desirable  since  phytoplankton production is the basis

                                  505

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    16
    14
     10
  »  8
  E
 5   6
 0
 >•
 X
 o
 2   2
                   , NO AltATtON - JO-JI JUNI


                   . WITH AltATION • 15-31 JUIT
      o
      o
                          TIME
Figure 1.  The  average dissolved oxygen in milligrams  per liter

           on a diurnal basis for Pond P-2 before and  during
           aeration.
                                  506

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for the food chains being investigated as sources of aquacultural
products in systems receiving treated sewage effluent.

CONCLUSIONS

Aeration of pond P-2 by  the means employed maintained dissolved
oxygen well above 3.0 mg/1 which had been accepted as the minimum
desirable level.

Aeration had no detectable effect on temperature, but diurnal
fluctuations in pH were  moderated as had been desired at the beginning
of the study period.

As a result of the modification of  the pond environment into one
which was more favorable for penaeid shrimp, it was decided to
undertake studies to determine whether or not shrimp could now
survive and grow in the  "P" ponds.

                          II.  SHRIMP SURVIVAL

The objective of this aspect of the study was to determine whether
or not penaeid shrimp could survive in the modified environment of
pond P-2.  If survivors  were found, growth would then be measured.

METHODS

On 20 August 1971,  juvenile white  shrimp  (Penaeus  setiferus) were
seined from Hoop Hole Creek, a  tidal slough  on  the  sound side of
Bogue Banks near Atlantic Beach, N. C.   The  shrimp were transported
to the laboratory and transferred  to a holding  pen  through which
water from Bogue Sound  circulated.  The  shrimp  were held overnight
and were released into  ponds C-2 and P-2  the next morning.  Thus,
on 21 August, C-2 received 221  juveniles  and P-2 received  211
juveniles.

Additional juvenile P.  setiferus were  added  to  the  same ponds on
13 September bringing  the total  numbers  of  shrimp  to  321 in C-2
and 287  in P-2.  No attempt was made  to  stock  the  ponds to carrying
capacity.  A sample of  35 of  the  juveniles were preserved  in  10%
formalin for weight and length determinations.

On 10 November  seine hauls were made in each pond  stocked.  Sampling
recovered  60  shrimp from C-2 and  33 shrimp  from P-2.  Weight  and
length were  determined  for each shrimp.

Because  shrimp  were released  into  the  ponds  on two occasions,  21
August  and  13  September, those shrimp  recovered on 10 November were
in the  ponds  for either 82 or 59 days.   Data discussed  in  the
following  sections  are  based on an average stocking data  of  1
September giving an average duration in the ponds  of 70 days.

                                  507

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RESULTS AND DISCUSSION

Figure 2 shows the average initial and final weights of the shrimp
recovered from P-2 and  C-2.  Whereas shrimp in both ponds were
stocked at the same average weight (1.4 grams) those in P-2 grew  to
a slightly greater average weight  (14.8 grams) than did those in
C-2  (13.45 grams).  Average shrimp lengths were also somewhat larger
for  shrimp from  P-2 than for those from C-2 (Table 1), 121.5 mm
as compared  to 113.3 mm.

The  increase in  weight  of shrimp in P-2 was 13.4 grams in approximately
70 days or 0.97  mm in length per day.  Such growth compares favorably
to that of shrimp in natural populations in North Carolina  (McCoys
McCoy and Brownio Williams**).  Thus, it would appear that  the "P"
pond environment may be made suitable for growing penaeid shrimp
simply by aeration of the water.

Differences  in the numbers of shrimp recovered from P-2 and C-2
may  have been due to several reasons.  However, the most likely
reason would be  the difference in bottom type between the two ponds.
C-2  has a fairly firm bottom in which seining is much easier than
in P-2 which has a very soft, muddy bottom into vhich the shrimp
can  burrow and which makes seining very difficult.

In addition, mortality  of the stocked shrimp may have been  greater
in P-2 than  in C-2.  This could not be verified since it was impossible
to recover all of the shrimp in either of the ponds.  The presence  of
numerous blue crabs in  P-2 could have resulted in greater losses  of
shrimp in that pond than in C-2 in which fewer blue crabs were seen.

Also shown in Figure 2  are the changes in heads-on count  (number  of
shrimp per pound) which occurred during the study.  When stocked,
the  juveniles were approximately 300 count heads-on, and they were
approximately 30 count  in P-2 and 50 count in C-2 on 10 November.
Shrimp of 30 count weight are well within a marketable size range.
Thus, shrimp of  coranercial size were produced in the pond receiving
treated sewage effluent.

Supplementary feeding was not employed during the study, and it  is
assumed  that the shrimp were feeding on the numerous small  organisms
and  accumulated  detritus in the ponds.

CONCLUSIONS

Penaeid shrimp were able to survive and grow in the aerated "P"  pond.
It is now logical to proceed with studies of penaeid shrimp food
chains utilizing treated domestic effluent and production dynamics
in aquacultural  applications.
                                  505

-------
         16


         15


         14


         13


         12


         II
        10
     O)
    X
    O
    flL
irt

ui
O
<   4
at
UI

<   3


    2
         1 -
         15   25

           AUG.
                          C2

                          P2
                    14   24

                    SEPT.
 14   24

OCT.
                                                    I,
                      30
                                                       80
                                                           130
                                                           Q
                                                           Z
                                                           3
                                                           O
                                                           D.
                                                           180
                                                           230 O
                                                               Z
                                                               O
                                                               I
                                                               in
                                                           280 Q
                                                           325
3   13

 NOV.
Figure  2.   Changes in the average weight and heads-on  count
            (number of shrimp  per pound) during the study  period
            for shrimp stocked in ponds P-2 and C-2.
                                 509

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Table I.  Data obtained from shrimp stocked in ponds P-2  and  C-2.
          Shrimp lengths were measured from the tip  of  the telson
          to the base of the rostrum.  Data ranges are  in parentheses,
                    SHRIMP IN POND P-2       SHRIMP IN POND  C-2

Number stocked             287                     321

Average No. days            70                      70
   in pond               (59-82)                  (59-82)

Average initial weight     1.40                    1.40
       (grams)             (0.4-3.1)               (0.4-3.1)

Average initial length       57                       57
       (mm)                (36-75)                  (36-75)

Number sampled               33                       60
  10 Nov.

Average final weight       14.77                    13.45
     (grams)             (5.0-21.0)                (4.0-21.0)

Average final length         122                      113
      (mm)                 (92-139)                 (84-130)
                                   510

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Shrimp growth in the "P" ponds approximated that found in natural
populations of penaeid shrimp in North Carolina.  Thus, there
appeared to be no detrimental effects on growth resulting from the
extremely eutrophic environment employed.

It appears that such eutrophic environments may be benefically
employed as grow-out ponds  in shrimp culture  operations since shrimp
of marketable size were produced.   In addition, it may be possible
to obtain three crops from  such grow-out facilities in North Carolina
depending on the duration of  favorable water  temperatures in a
particular year.
                            Literature  Cited

1.  Odum, H.T. and A.F.  Chestnut.  1970.  Studies  of marine estuarine
        ecosystems developing with treated sewage wastes. Univ. of
        North  Carolina.  Sea Grant  Project Ann. Kept.  1969-1970.364  pp.

2.  Kuenzler,  E.J. and  A.F. Chestnut.  1971a.  Structure  and  functioning
        of estuarine  ecosystems exposed  to treated sewage wastes.
        Univ.  of  North  Carolina.  Sea Grant Project Ann.  Rept.
        1970-1971. 345  pp.

3.  Kuenzler,  E.J. and  A.F. Chestnut.  1971b.  Structure  and  functioning
        of estuarine  ecosystems exposed  to  treated sewage wastes.II.
        Univ.  of  North  Carolina.  Sea Grant Project Ann.  Rept.
        Supplement.  1970-1971. 50 pp.

4.  Kuenzler,  E.J.,  A.F. Chestnut, and C.M.  Weiss,  1973. Structure
         and  functioning of estuarine ecosystems exposed to treated
         sewage wastes,   III,  1971-1972. Univ. of North Carolina,
         Sea  Grant Pub.   UNC-SG-73-10. 222 pp.

 5.   Bees ton, M.D. 1970. Crustaceans and  fishes in the Sea Grant
         ponds. In Odum, H.T.  and  A. F. Chestnut, 1970. pp. 272-279.

 6.   Bees ton, M.D. 1971. Decapod crustaceans and fish populations in
         experimental ponds receiving treated sewage wastes, in
         Kuenzler E.J. and  A.F.Chestnut,  1971a. pp. 182-204.

 7   Smith  M  1971.  Productivity of marine ponds receiving treated
         sewage,  in Kuenzler, E.J.  and A. F. Chestnut, 1971a. pp. 24-41.

 8   Laughinghouse, W.R. and  E.J.  Kuenzler. 1971. Insolation, pH, and
         turbidity, in Kuenzler, E.J. and A.F. Chestnut, 1971b.
         pp.  3-8.
                                   577

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 9.   McCoy,  E.G.  1968. Migration,  growth,  and mortality of North
          Carolina pink and brown  penaeid  shrimps . N.C. Dept. of
          Conserv.and Devel.,  Div.  of Comm.  and Sports Fisheries.
          Spec. Sci. Rept.  (15):1-26.

10.   McCoy.  E.G.  and J.T. Brown. 1967.  Migration and growth of
          commercial penaeid shrimps in North Carolina. N.C. Dept.
          of Conserv. and Devel.,  Div.  of  Comm. and Sports Fisheries,
          Spec. Sci. Rept.  (ll):l-29.

11.   Williams, A.B. 1955 A contribution to the life histories of
          commercial shrimps (Penaeidae) in North Carolina.  Bull
          Mar. Sci. Gulf and Caribbean, 5_:116-146.
                                   572

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   STANDING CROPS OF BENTHIC FAUNA IN MARINE AQUACULTURE
                 PONDS USING RECLAIMED WATER

                            by

                      Thomas R. Sharp*

INTRODUCTION

In a time of worldwide shortages of food and energy, it is
essential that new methods of human food production be
developed.  One possible method is to produce fish for
human consumption from domestic wastewater  (Allen1).  An
important link between wastewater nutrients and fish pro-
duction is the benthic fauna, a basic food for most fish
(Darnell2).  The placing into operation of two experimen-
tal aquaculture ponds in July 1971 located within the
confines of a city wastewater treatment plant next to the
tidal flats of North Humboldt Bay  (Allen and Dennis3 ,
Fig. 1) provided the opportunity for studying the effects
of treated domestic wastewater on estuarine benthic fauna.

The primary purpose of this study was to determine the
differences in benthic standing crops between the
fertilized pond and the unfertilized pondy  North Pond
was managed as a static estuarine-sewage effluent mixture.
The South Pond was an estuarine pond only, and was tidally
flushed every 2-3 weeks  (Allen and Dennis3, Figures 1-3).

To determine a desirable substrate for fish food produc-
tion in future aquaculture ponds, the benthic standing
crops on four available substrates were studied; substrates
included mud, sand(Hookton soil), river-run gravel, and
oyster shell.  Pens to exclude fish were used to determine
the effect fish predation had on benthic standing crops.
Since this study began with the initial filling of the
ponds, the colonization of the ponds was studied for
comparisons with conditioned pond bottoms  in future
experiments.

METHODS AND MATERIALS

Temporal Changes in Benthos

In order to study fluctuations in benthic  animal nutrients
over time, a transect technique was employed.  A transect
*Department of Fisheries, Humboldt  State  University,
Arcata, California.
**TT-i-io  -investiaation was  supported  by funds from the
Humboldtnstitegu5lv¥rsity Coherent  Area  Sea Grant  Program.

                            573

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line was marked  by  a  rope  stretched  between the  headgate
and the northeast corner of  each pond.  This line  was
located over  areas  of mud  substrate  only  (Sharp1*) .  Monthly,
beginning  September 1971,  five  core  samples were taken at
6 meter intervals along the  transect line of each  pond.
Ten samples were taken on  each  sampling date for a total
of 50  samples over  the course of the study.  The time  of
the study  corresponds to Pond Experiment IB  (Allen and
Dennis3, Table 2).  Thus the early stages of succession
were not covered.

Benthos was  sampled using  a  Carrin core sampler  (Carrin5)
having a diameter of  4 inches  (surface area of about 80 cm2)
Each sample was  washed through  a 0.457 mm Tyler  screen and
the part remaining  preserved in 10%  formalin solution.  The
organisms  were sorted to species and counted.  The speci-
mens of a  species in  a sample were dried at 100°C  for  24
hours  (Hanks6)  and  weighed on an analytic balance.

Standing Crop

In order to  study the standing  crop  of benthos at  the  end
of the rearing experiment, an "in-place" sampling  technique
was used.  An in-place basket sampler similar to that
o f    Hanks6,   with  modifications to accommodate  the  dif-
ferent substrates and changes in water level was employed
 (Figure 1).   The sampler consisted of a 2-quart  polyethelyne
basket with a surface area of 200 cm2 and a volume of  1600
cc.  Triangular  cut-outs covered by  0.457 mm Nitex nylon
were made  into the  sides of  each sampler to allow  for  water
circulation which might occur in the substrates, especially
in shell.  A  brick  and float were attached by two  lengths of
nylon  twine.   The brick prevented the float twine  from
interfering with the  substrate  surface.

"Exclusion pens"  enclosing an area of 4 m2 were  built  on
each of four  substrates within  each  pond (Sharp1*) .  On
July 13, 1971 the in-place basket samplers were  installed
as follows.   At  each  station (substrate within a pond)  six
samplers were filled  with  the appropriate substrate and
placed so  that the  surface of the sampler was flush with
the surface of the  pond bottom.  Three of the samplers
were put inside  the exclusion pen and three outside the
pen (Sharp1*).  Thus,  with  four  substrates in each  pond,
and two ponds, a  total of  48 samples were installed
(Figure 2).

The ponds were initially filled July 21, 1971 and  finally
drained on January  15 and  22, 1972.  The samplers  were
withdrawn from North  Pond December 13, 1971 and  from South


                             574

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                                               STAINLESS
                                                STEEL WIRE
        FIBERGLASS
            RESIN
Figure 1.   In-place Basket Sampler used for .Substrate
           study (top surface  area, 200 cm2; volume
           1600 cc).
                       575

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Oi
                                              SUBSTRATE  STUDY
                                Figure 2.   Flow  diagram of the substrate study.

-------
Pond December 20, 1971, before either pond was drained.

Benthos and sediments in the basket samplers were processed
in the same manner as that used for the transect samples
with the following exceptions:
    1.  Mud, sand, and gravel samples underwent a sugar
floatation technique  (Anderson7) to eliminate the large
volumes of substrate remaining after seiving.
    2.  Due to the high numbers of organisms in North Pond,
all North Pond samples were divided in half using a Folsom
Plankton Splitter and the results doubled.
    3.  For weighing, the three replicate samples on each
test area were pooled and the results averaged.

METHODS OF ANALYSIS

A three-way analysis of variance  (Sokal and Rohlf8) was
employed to analyze the number of benthic animals only
taken in basket  samplers.  The analysis was to test which
treatment  (pond, substrate, or exclusion pen)was signifi-
cant for each of the major species found in the study.  A
standard logarithmic  transformation  (100 log  [X+l] where
X is the number  of animals in each sample) was used to
make the data more homogenous.  Calculated F values for
each treatment were compared with tabulated F values for
a significant level of p=.05.   If the F value was larger
than the tabulated value, the treatment was considered
significant.

A Student-Newman-Kreuls Test  for multiple comparisons among
means based on equal  sample  size  (Sokal and  Rohlf  ) was
used to determine  significant factors within treatments.
If  the treatment being considered was substrate, the
factors considered would  be mud,  sand, gravel  and  shell.

Numbers/M2 and biomass(dry weight/M2) were calculated from
both transect  samples and in-place basket samples.  Wet
weights/M2 were  calculated  from dry  weight data using
conversion formulae  (Thorsen9).   Total numbers/M   and wet
weights/M2 were  calculated  for  ponds, substrates and
exclusion  pens,  using in-place  basket sample data  and for
each  sampling  period  using  transect  sample data.   The
basket  sample  totals  were compared with other studies.

RESULTS

Organisms  Present

The organisms  found  in the ponds  included two species of
polychaete, 
-------
Polydora ciliata Fabricus  (Spionidae) ; and two species of
gammarid amphipod, Anisogammerus conferviaolus Stimpson, and
Corophium spinicorne Stimpson  (Table 1) .  Daphnia spp shells
were found in large numbers but were considered artifacts
from the oxidation pond.  Bryozoans were not quantified.
Great numbers of dipteran insect pupae casts were found
but not considered part of the benthos.  Other species
occurred but in low numbers.

Changes in Standing Crops with Time

Both polychaete populations increased to November or
December and then declined for both ponds (Table 2) .  North
Pond  (with sewage effluent) maintained generally higher pop-
ulations with one notable exception:  C. aapitata standing
crops peaked at the same level in both ponds  (62.8 g/m2 wet
weight in North Pond compared with 62.4 g/m2 wet weight in
South Pond).

The two amphipod populations varied inversely between ponds
 (Table 2).  A. conferviaolus populations showed a continu-
ous buildup in North Pond to the end of the experimental
period with very few organisms occurring in South Pond.
C. epinicorne, on the other hand, started with large
populations in South Pond which continually decreased to
total disappearance by December.  Very few C. spinicorne
were found in North Pond transect samples.

An unexpected relationship between A. eonfervicolus and
P. ciliata was noted.  Both numbers and weights showed
high positive correlations between these two species
(0.42 and 0.52 respectively).  Both species also showed
increases in number with decreases in surface temperature
and salinity (Allen and Dennis3).  The correlation could
be by chance and should be investigated further.

The total disappearance of the benthos on South Pond mud by
December remains unexplained.  I sampled water properties
in January and found low dissolved oxygen directly over
the South Pond mud substrate only (Table 3).  This strongly
suggested a non-uniform mixing resulting in low DO 's as a
possible cause for the disappearnace.  Populations on other
substrates in South Pond seemed uneffected.

Effects of Fish on Standing Crop

No significant difference existed between samples taken
inside and samples taken outside of the fish exclusion
pens in this study.  The lack of survival of salmonids
in the initial experiments  (Exp. IA, Allen and Dennis3)

                           518

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Table 1.  Frequency of occurrence of benthic animals taken by
          two methods of sampling of fauna developed between
          July 1971 and January 1972 in two marine aquaculture
          ponds, one fertilized with treated wastewater.
          Kind of Animal                   Frequency.of Occurrence
                      _    .       Transect Sampling  Basket Sampling
   Group              Species         (N=5Q)   H   y      (N.48)
Polychaete worms

Gammarid
amphipod
Capitella aapitata
Polydora ail-iota
Anieo gammer us
aonfevv-ioolue
97.9
47.9
43.7
83.3
83.3
79.2
                  Corophium  epinioorne    33.3               52.1

Bryozoans                              undetermined, but common

Isopod  (Plabellifera)                      2.1                4.2

Barnacle          Balanue  opp.                               4.2

Hollusk           Lyoneia  aal-iforniaa                        4.2


Fish              GaeteroeteuB
                       aauleatus                              4-2
                     (stickleback)
                                  579

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Table 2.  Monthly average number and wet weight in grains per
          square meter of four major species of benthic animals
          taken in transect sampling of North and South Ponds,
          September 1971 to January 1972.
Species
Capitella
capitata
Polydora
ciliata
Anisogammerue
eonfarvico lus
Corophium
epinieorne
TOTAL
Capitella
capitata
Polydora
ciliata
Anieogammerue
eonferviaolue
Corophium
epiniaorne
TOTAL
Pond
? Sep 71
North 76
0.5
0
0
102
1.1
76
1.1
254
2.7
South 0
0
0
0
51
0.3
2548
8.0
2599
8.3
Date
5 Oct 71
3456
23.4
382
2.6
127
3.9
25
0.8
3990
30.8
1376
4.6
1147
2.8
25
0.5
2140
2.0
4688
9.8
of Sampling
4 Nov 71 6
9019
62.8
1401
9.9
688
11.1
0
0
11108
83.9
14726
62.4
408
0.7
229
5.7
1019
1.8
16382
70.7

Dec 71
6624
43.3
5299
32.7
1274
8.3
0
0
13197
84.4
0
0
0
0
0
0
0
0
0
0

7 Jan 72
1962
13.4
3566
12.7
3745
8.7
0
0
9273
34.8
0
0
0
0
0
0
0
0
0
0
                             520

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Table 3.  Comparison of dissolved oxygen in water  near  pond
          bottoms with surface water near headgates on
          7 January 1972.
Sampling Location
                                  North
      Pond
              South
Center of Exclusion Pens:
        Mud
        Sand
        Gravel
        Shell
13.8
15.0
15.6
14.4
22.0
15.0
12.8
11.6
Distance in meters from
headgate along transect
        10
        20
        30
        40
  Headgate-surface
10.4
12.2
13.2
15.0
12.4
 6.2
 1.2
 2.0
15.0
16.2
                              527

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and the short exposure time of the samples to surviving
salmonids in the later experiments (Exp. IB) may account
for this.  Larval marine fish introduced with bay waters
could pass freely through the mesh used for exclusion pens
and thereby negated studies designed to measure the effect
of salmon predation on benthos.

Organisms on Different Substrates

Only two species were found to vary significantly with sub-
strate  (polychaete, C. eapitata; and gammarid amphipod,
A. confervicolus-, Table 4) .

C. eapitata had the largest standing crops on sand with an
average of 348 organisms/sample.  Since sand tended to be in
shallower water than other substrates, depth of the water
column could have been a factor in the success of C.
eapitata on a sand substrate.  Gravel supported only 54
organisms/sample.  Mud and shell showed no difference in
standing crop with populations between 12 to 13 organisms/
sample.  This result has been somewhat biased by the total
mortality on South Pond Mud.

A. eonfervicolus standing crops were largest on oyster shell
or gravel, (31-38 organisms/sample); no significant differ-
ence was found between these two substrates.  Mud and sand
were shown to support equal populations averaging 6 to 7
organisms/sample.  Particle size appeared to be a factor.
High crops on shell could have resulted from sampling bias
due to water filtration through sampler screens preventing
escapement since shell did not completely fill the entire
sampler as did other substrates.  These results should be
retested as trap sampling on this species showed different
results  (Pollard1°).

Total biomass was highest on the shell substrate  (115g wet
wt/M2) due to heavy incrustation by P. ciliata and the
presence of A. confervicolus.  This was followed by sand
 (86 g wet wt/M2), then gravel  (71 g wet wt/M2) and finally
mud  (45 g wet wt/M2).

Differences in Benthos between Ponds

The two  species, P. ciliata and ^4. confervicolus, had
significantly higher populations in North Pond  (with
sewage  effluent) than in  South Pond  (a tidally flushed
estuarine pond)  (Table 4).  P. ciliata standing crop in
North Pond was 128 organisms/sample compared with 1
organism/sample  in South  Pond; A eonfervicolus populations


                            522

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Table 4.
          Mean numbers of benthic organisms per basket sampler
          for factors within significant treatments (pond,  sub-
          strate or exclusion pen).   Significance of  treatment
          determined by a Three-way Analysis of Variance.
          Significance of factors within a treatment  determined
          by a Student-Newman-Kreuls Test (Sokal and  Rohlf8).
  Treatment
Species
                                          Mean numbers/Sampler*
    North Pond vs.
      South Pond
  Polydora
    ciliata

  Anisogammerus
    confervicolus
 South Pond

     1


     2
            North Pond

               128


                77
    Substrate:
    Samples from
    both ponds
    combined
  Capitella
    oapitata

  Anisogammerus
    aonfervicolus
Mud

 12
Shell

  14
                                     Sand

                                        6
        Mud
          7
Gravel

  54

Gravel
  31
Sand

 348

Shell

  38
   *Values underlined are not significantly different from each
    other at the 5% probability level.
                             525

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were 77 organisms/sample and 2 organisms/sample respectively.
C. aapitata and C. spinioorne standing crops did not differ
between ponds, with C. aapitata having large populations
and C. spinieorne having extremely low populations.

In terms of total biomass North Pond supported a much larger
standing crop  (159 g wet wt/M2) than South Pond (35 g wet
wt/M2).  For all in-place basket samples, the standing crops
I found after about 145 days of rearing ranged from zero in
South  Pond sand to 195 gms wet wt/M2 in North Pond shell
(Table 5).

COMPARISONS WITH OTHER BENTHIC STUDIES

The results obtained for average total biomass in each pond
were compared with several benthic studies (Table 6).  North
Pond had standing crops greater than or equal to standing
crops  of freshwater ponds fertilized with commercial fertil-
izers  (Liang11)/ and standing crops larger than those found
in the adjoining bay  (Carrin5), Buzzards Bay  (Sanders12),
or the English Channel (Holme13).  On the other hand, North
Pond's benthic biomass was smaller than that found in a con-
tinuous flow pond (Hanks6), for an arctic macoma community
(Ellis11*), and for certain intertidal areas  (Sanders, et
al15).

The South Pond had a standing crop equal to that found in
unfertilized freshwater ponds  (Liangri)/ but lower than
standing crops of all other studies considered.

Powers, in his study of the amphipod A. confervieolus in
the aquaculture ponds used mathematical models and estab-
lished a carrying capacity of 300,000 amphipods per pond or
200 amphipods/M2 (Powers1t).  This study shows standing
crops  of much higher magnitude existing for long periods
of time in the ponds.  On each of the substrates in each of
the ponds, A. confervioolus standing crops between 2000-
12,000 organisms/M2 commonly occurred.  Powers, however,
studied the ponds under a different set of conditions
 (Exp.  Ill, Allen and Dennis3).

DISCUSSION

At present, when sewage-effluent mixtures are used in the
fish  ponds, they are  run as a  static system.  If ponds
were  to be fertilized periodically with both bay water and
effluent, say  every 3 to 4 weeks, benthic standing crops
might improve  in biomass and species density.  I would be
interested in  seeing  the effect of such a program.
                             524

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Table 5.  Comparison in total numbers and weight per  square
          meter of benthic biomass as taken in basket samples
          on four substrates between North and South  Ponds
          (6 samples from four substrates in each pond -
          48 total).
   Substrate
    pond
    rmd
    sand
    gravel
    shell
Total Number/M2
                     South  Pond
  6500

 21400
  2900
  1800
Weight in graras/Ms
                                           Dry
 12
  7
  6
                                                      Wet1

Pond
mud
sand
gravel
shell
North Pond
28000
23500
25500
22700
40300

22
16
19
18
34

159
90
106
102
195
35

66
41
35
Calculated from dry weight  by wet weight conversion factors
 for polychaetes and amphipods as listed in Thorsen9.
                              525

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Table 6.  Comparison of North and South  Pond  biomass with
          some values reported in the  literature.
Author
Holme11
Sanders1 *
Sanders,
et alls
Carrin*
Ellis1*
Banks'
Liang11





Present Study


Location and Habitat
English Channel
Buzzards Bay, Mass.
Barnsdale Harbor, Mass.
(Intertidal)
North Humboldt Bay, Calif.
Arctic tnacoma community
Estuarine constant flow pond
Unfertilized freshwater pond
summer
winter
Fertilized freshwater pond
summer
winter
Marine Aquaculture Ponds,
Arcata, Calif.
North
South
Weight (g/M* }
Dry
11.2
12.3
17.6-60.0


136

9.2
7.0

22.4
8.6

21.9
6.2
Wet
55


105
200
1360







159
35
                             526

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With the proper stabilization and aerating system, the
benthic standing crops of  the ponds could conceivably be
manipulated toward the important fish food items such as
A. confervieolus and  away  from  seemingly "weed species"
such as C. eapitata.  Toward this end,  I would suggest that
a slight covering of  the mud substrate  (and perhaps even
the sand) with oyster shell or  gravel might reduce C.
oapitata habitat and  improve A. oonfervioolus habitat.
Shell appears to be doing  the  intended  function of
providing shelter to  amphipods.  Further studies  should
be done on A. confervieolus population  dynamics and its
relationship to salmonid  feeding habits.

It is of  interest that  the ponds are  classified as
"polluted" by standards  applied to  non-aguacultural marine
environments  (Reisch17).

LITERATURE CITED

  1.  Allen,  G.  H.   1970.   The  constructive use of sewage,
          with particular reference to fish culture.  FAO
          Technical  Conf .  on Marine Pollution and  Its
          Effects  on Living Resources and Fishing. FIR:
          MR/70/R-13.

  2.  Darnell,  R.  M.   1961.  Trophic spectrum of an estuarine
          community,  based on studies of Lake Pontchar train,
          Louisiana.   Ecology 42:553-568.

  3   Allen,  G.  H. and L. Dennis.  1974.  Report on pilot
          aquaculture system using domestic waste waters
          for rearing Pacific salmon smolts (This conference) .

  4    Sharp,  T.  R.  1974.  Benthic Fauna standing crops in
          estuarine aquaculture ponds using reclaimed water.
          M.  S.  Thesis in  Fisheries, Humboldt State Univer-
          sity,  June.

  5   Carrin, L. F.   1973.  Availability of invertebrates as
          shorebird food on a Humboldt  Bay mudflat.  M. S.
          Thesis in Wildlife, Calif.  State University,
          Humboldt.

      Hanks, Robert W.  1968.   Benthic  community  formation
      Hanks  woe           environment.   Cheasapeake
          Science.  Vol.  9,  No.  3:163-172.

      ar.rioT-.5on   R  0.   1959.   A modified flotation technique
      Ande^°n;oR;i°g  bo;tom fauna samples.   Limn, and Ocean.

          4:223-235.
                              527

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 8.   Sokal,  R.  R.  and F.  J.  Rohlf.   1969.   Biometry;  the
         principles and practice of  statistics in biolog-
         ical research.  W.  H.  Freeman and  Co.,  San
         Francisco. 776 pp.

 9.   Thorsen, G.   1957.  Bottom Communities.   In:Treatise
         on Marine Biology and  Daleoecology,  Vol. I.  ed.
         J.  W. Hedgpeth.   Geological Society of American,
         Memoir 67. pp. 461-534.

10.   Pollard, Robert, 1972.   A  catch per unit effort study
         on a population of Amisogammei'us confervicolus in
         a saltwater pond fertilized with sewage.  Under-
         graduate field study in Fisheries, Calif. State
         University, Humboldt,  (unpublished).

11.   Liang,  J. K.   1966.   Benthic  fauna of fertilized and
         unfertilized ponds in  winter.  In: Proceedings of
         World Symposium on warmwater pond fish culture.
         FAO Fish Dept. 441(3):82-94.

12.   Sanders, H.  L.  1960.  Benthic  studies in Buzzards
         Bay. III.  The Structure  of the soft-bottom
         community.  Limn, and  Ocean. 5(2)  :138-153.

13.   Holme,  N. A.   1953.   The biomass of the bottom fauna
         in the English Channel off  Plymouth.  J. Mar. Biol.
         Ass. U.K. 32:1-50.

14.   Ellis,  D. V.   1960.   Marine infaunal benthos in Arctic
         North America.  Arctic Institute of North America,
         Tech. Paper No.  5:1-53.

15.   Sanders, H. L., E. M. Goudsmit, E. L. Mills, and G. E.
         Hampson.   1962.  A study of the intertidal fauna
         of Barnstable Harbor,  Mass.  Limn, and  Ocean.
         7(1) -.63-79.

 16.  Powers, J. E.   1973.  The dynamics of a population  of
         Anisogammerus confervicolus  (Amphipoda) :  A
         computer  simulation.  M. S. Thesis  in Fisheries,
         Calif. State  University, Humboldt.  Ill  pp.

 17.  Reisch,  D. J.   1973.  The use  of marine  invertebrates
         as indicators of varying degrees  of marine pollu-
         tion and sea  life,  ed:  Mario Ruivo. FAO. Fishery
         News (Books)  Ltd. Surrey,  England.   pp.  203-207.
                             528

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             CONTROLLED EUTROPHICATION:  SEWAGE TREATMENT
                         AND FOOD PRODUCTION

                                  by

          J. Glenn Songer*, N.M. Trieff*, Rodney F. Smith**,
                          and Dov Grajcer***

INTRODUCTION

Natural ecosystems can produce no more than two metric tons per hectare
per year of animals useful to man on a sustained and reliable basis.
Commercial yields, greater than natural yields by 100 times, are
achieved in artificial systems in which animals are provided with food
and flowing water under carefully controlled conditions.  A combination
of natural and artificial systems, utilizing the best aspects of each,
yields the approach toward food production and waste treatment known  as
"controlled eutrophication."!

The essential feature of controlled eutrophication is the physical  sep-
aration and compartmentalization of the producer and consumer levels  of
the community.!  An example of this is the growth of phytoplankton (a
freshwater species of Chlorella) and, subsequently, bivalves.2

A pond one meter deep provides enough carbon dioxide for algal growth
year-round.  Seawater contains, naturally, a quantity of carbon dioxide
in the form of dissolved carbonates and bi carbonates J  Free exchange
with the atmosphere is an additional source.

Several sources of mineral nutrients are available.  Commercial fertili-
zer is capable of providing the necessary nitrogen, phosphorus, and
other nutrients, but is quite expensive.'  Artificial upwelling of the
ocean is also quite costly, but is, nevertheless, very effective; a
pilot project utilizing artificial upwelling has been in operation at
Saint Croix, Virgin Islands, for some time.3

A third and probably more obvious source of nutrients is raw or treated
sewage.  The algae growing ability of sewage is well known from labora-
tory, pilot plant, and full-scale waste stabilization ponds.  Reported
yields have been tremendous.  »5
  *  Department of  Preventive  Medicine  and  Community Health, University
     of Texas Medical  Branch,  Galveston,  Texas
 **  Microbiology Laboratory,  Alta  Bates  Hospital, Berkeley, California
***  Head, Aquaculture Research,  Syntex Corporation, Palo Alto, Calif.
                                   529

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Dunstan and Menzel6 have worked with sewage as the nutrient supply for
continuous culture of algae.  The high nutrient level in sewage allows
for a considerable dilution which is necessary to accomodate the salt
tolerance of marine algae.  The algae is fed to herbivores; the herbi-
vores then become food for man, or alternately, are fed to a primary
carnivore and the nutrients harvested at a higher trophic level.

A system has been developed on the laboratory scale involving the use
of the marine alga Tetraselmis for controlled eutrophication of raw
sewage.  The subsequent algal proliferation was fed to brine shrimp,
Artemia salina.  Net products of the system were reported as (1) brine
shrimp for use as fish food or shrimp food and (2) a purified effluent.7

The brine shrimp were adaptable to life in raw sewage in which an algal
bloom had taken place.  Recommendations for optimal treatment of raw
sewage on this scale are (1) algal growth with artificial light, (2)
addition of Artemia, (3) decantation and additional algal growth, and
(4) final decantation.

Reductions by the process of such parameters as total suspended solids,
five-day BOD, turbidity, odor, orthophosphate, nitrate, nitrite, and
ammonia were to levels equivalent to those achieved by conventional
secondary and perhaps  tertiary treatment.

The objective of the present research is to further study and develop
this system.   The design of a continuous-flow system is undertaken.   In
addition, various water quality parameters are studied and the passage
of certain bacterial groups through the system is  examined in detail.
Water quality parameters chosen for study are:  BOD, total  solids (res-
idue on evaporation),  total  inorganic phosphorus,  and total  Kjeldahl
nitrogen; bacteria chosen were coliforms, enterococci, Salmonella,
Shigella, Vibrio alginolyticus. and Vibrio parahaemolyticus.

It is hypothesized that improvements in all  water  quality parameters
will  approach or surpass required reductions (i.e., a non-polluting
effluent will result).   Coliform bacteria will be  reduced to  less than
one per cent of raw sewage concentrations, as will  enterococci.   Sal-
monella and Shigella will  not be detected.  No halophilic Vibrios"wTn
be isolated at any stage.

MATERIALS AND METHODS

Continuous-flow System

The initial work was done using a static set-up.   A continuous-flow
system was designed and built for use in the present research.

Lighting - Cool-white fluorescent lighting was used (Westinghouse).
Three 1*2 cm, two-tube fixtures with 40 watt tubes  were wired in para-
llel , side by side.

                                  550

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Raw Sewage - Raw sewage was obtained weekly from the main sewage  treat-
ment plant in Galveston and was stored in a 20-liter Pyrex carboy.
Synthetic Sea Salts - Instant Ocean (Aquarium Systems, Inc., Eastlake,
Ohio, 44094) was used.  It was mixed to yield a salinity of 40 o/oo  and
stored during use in a 125 liter Rubbermaid trashcan.
Algae and carboy for culture - The marine alga Tetraselmis chui  was  ob-
tained from the stock cultures of the National Marine Fisheries" Service,
Gulf Coastal Fisheries Center, Galveston, Texas, through the courtesy
of Loretta Ross.  The culture was maintained in a 20-liter Pyrex carboy.
A rubber stopper with a 40 cm length of 15 mm OD glass tubing inserted
into it was placed in a 2.5 cm hole in the bottom of the carboy, the
tubing rising to such a point inside the carboy as would allow for re-
tention of a maximum volume of 18 liters.  This tube thereby provided
an automatic overflow.  Continuous aeration was used.

Brine Shrimp and Hatching - Brine shrimp (Artemia salina) eggs were  ob-
tained from California Brine Shrimp, Inc., Menlo Park, California,
94025.  The eggs were hatched in a circular dish (25 cm diameter, 7.5 cm
depth) according to the method of Needham,^ collected with a large-
tipped pipet, and transferred to the culture container.  An aquarium
(20 cm height, 32 cm width, 76.5 length) was used as a permanent con-
tainer for the brine shrimp.

Pump - Raw sewage, Instant Ocean, and the effluent were pumped with  a
Buchler Polystaltic Pump  (Buchler Instrument Company, Fort Lee, N.J.,
07024).  The tubing was Tygon, 1.60 mm ID.  Continuously variable flow
rates from 2.5 ml/hour to 1000 ml/hour were available.

These components were assembled into the system shown in Figure 1.   The
lights were suspended 10  cm above the algal culture.  Operation of  the
lights was on a full-time basis.  A turnover rate in the algal culture
of 50% per day was desired, so the pumping rate was adjusted to give a
combined flow of raw sewage and Instant Ocean amounting to 6.25 ml  per
minute or 9 liters per day into the algal culture.

Water Quality Parameters

Sampling - Samples were taken by pipet at the various stages (raw sew-
age, algal culture, and effluent) and subjected to the tests.

Biochemical Oxygen Demand - Determinations were done according to Stan-
dard Methods for the Examination of Mater and Wastewater.9  Various
dilutions were used, depending upon the expected order of magnitude of
the BOD value.  Dissolved oxygen measurements were done by the membrane
electrode method using an Edmont Oxygen Analyzer  (Model 60-625, Edmont-
Wilson, Coshocton, Ohio,  43812).
                                   537

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Total Solids (Residue on Evaporation) - Liquid samples  (100 ml) were
evaporated to dryness in vacuo, in preweighed flasks.   Weight of resi-
due in mg/1 was determined by reweighing, subtracting  the initial  value,
and multiplying by 10.

Total Inorganic Phosphorus - Determinations were done  according to Stan-
dard Methods for the Examination of Water and Wastewater,^ using the
aminonaphtholsulfonic acid method.

Total Kjeldahl Nitrogen - Determinations were done according to Standard
Methods for the Examination of Water and Wastewater.9   The procedure was
that for organic nitrogen, omitting the ammonia removal  step.  The iso-
lated nitrogen was determined by titration.

Bacterial Groups

Except there otherwise stated, incubation was at 37°C  for 48 ± 3 hours
and all media was from Baltimore Biological Laboratories, Cockeysville,
Maryland, 21030.  Samples were taken at the various stages (raw sewage,
algal culture, and effluent) and subjected to the various tests.

Coliforms - An MPN/100 ml was determined by the multiple tube fermenta-
tion technique using lauryl  sulfate broth.  Transfers  were made from
positive tubes to EC broth;  incubation was at 44.5°C in a water bath.
Growth and gas in 24+2 hours indicated the presence  of fecal coliforms
and confirmed the MPN.

Enterpcocci - Azide dextrose broth served as a primary medium yielding
an MPN/100 ml.   Positives were transferred to ethyl violet azide broth.
Turbidity and the appearance of a purple button constituted a positive
test; streaks were made to Columbia CNA blood agar (composed of Columbia
CNA agar base containing colistin and nalidixic acid with 5% sheep blood
added;.  Typical colonies were picked to Enterococcosel  broth where
blackening of the medium in 24 ± 2 hours gave a completed test and con-
firmed the MPN.

Salmonella and Shi gel!a - Growth in Selenite-F broth was considered a
positive presumptive test; streaks were made to MacConkey agar.  Typi-
cal colonies were picked to Kligler's iron agar slants and incubated
for 18 hours.  Cultures giving reactions typical of Salmonella or Shig-
ella were subjected to biochemical testing in the API-20 Profile Recog-
TnTTon System (Analytab Products, Inc., New York, N.Y.,  11514).  A cor-
rect profile confirmed the presence of Salmonella or Snivella.

Vibrio parahaemolyticus and Vibrio alginplyticus - Enrichment cultures
fin alkaline peptone broth from Difco Laboratories, Detroit, Michigan,
48232) were streaked to TCBS agar.  Appearance of typical colonies in
24 hours indicated the presence of Vibrios.  Suspect colonies were sub-
jected to biochemical tests, again using the API-20 Profile Recognition


                                  532

-------
System.  Three per cent NaCl water was used as diluent.  A correct pro-
file confirmed the presence of V. parahaemlyticus (#4346106) or V. al-
ginolyticus (#4146124).        "	               -  —

RESULTS AND DISCUSSION

Water Quality Parameters

BOD analysis of raw sewage yields the comparatively high and variable
values normally associated with it (Figure 2).  Samples from the algal
culture compare closely with raw sewage in BOD values.  This is reason-
able, since sewage organics are simply converted, for the most part,
into algal cells.  Effluent BOD's (average 64.7) are somewhat higher
than expected, but represent a considerable improvement over raw sewage
(average 76.5% removal of BOD).

Interpretation of the measurements of total solids (Figure 3) is con-
fused by the fact that the carrying water (Instant Ocean) contains an
average of 45,014 mg/1 of residue on evaporation.  A correction factor
of 22,507 mg/1 to account for the contribution from the Instant Ocean
was applied to each of the measurements of total solids in the algal
culture and effluent (i.e., 45,014 divided by 2, since Instant Ocean  and
raw sewage were mixed 1:1).  This yielded the corrected lines (Figure 4)
which show a consistent increase in this parameter from raw sewage to
effluent.  The increase between raw sewage and the algal culture is pro-
bably attributable in part to the presence of algal cells and also to
some slight evaporation.  The subsequent increase in the effluent is
believed to be almost wholly attributable to evaporation.  Measurements
indicate nearly a 5 o/oo increase in salinity alone from the 1:1 mixture
of raw sewage and Instant Ocean to the effluent.  Since it is virtually
impossible to control evaporation, measurements of this parameter inevi-
tably face the need of correction and must be interpreted on that basis.

The data for total inorganic phosphorus shows a pattern which is often
observed in stabilization ponds (Figure 5).  Phosphates (ortho- and
poly-) in raw sewage are removed by algae, by incorporation into cell
material and/or by precipitation (due to high pH).  In the effluent,
the phosphorus concentration increases, probably due to a pH drop in  the
brine shrimp tank.  Also, brine shrimp fecal pellets settle out and
likely contribute to the concentration of dissolved phosphorus by verti-
cal exchange as they are broken down by phosphatizing bacteria.  Per
cent removals in the algal culture averaged 65.5%, but the high was
81.5%.  The decrease, from raw sewage to effluent, averaged 39%, and
effluent values (average 5.07 mg/1) are somewhat lower than those repor-
ted by Neel^° in a report on the operation of a stabilization pond
sy s tern.

Total Kjeldahl nitrogen values (Figure 6) are quite different from those
expected.  Extremely low values in raw sewage measurements indicates


                                   533

-------
extensive denitrification and either loss as nitrogen gas or oxidation
to nitrites and nitrates.  The drop in total nitrogen values for the
algal culture is likely due to settling out of many sewage organics and
further oxidation of nitrogen compounds (an insufficient concentration
of phosphorus leads to nitrogen excess with subsequent conversion to
nitrites and nitrates).  Effluent values are somewhat elevated from
those of the algal culture; nitrogenous wastes of the brine shrimp are
probably the greatest contributors to this increase.  Remnants of algal
cells suspended in the water, as well as numerous small brine shrimp,
could not be removed before making determinations and no doubt contribu-
ted  substantially to the values.

Bacterial Groups

Figure  7 shows the MPN/100 ml values for coliforms at various stages of
treatment.  As expected, a disappearance of fecal coliforms is evidenced
on  passage  of raw sewage through the system.  Figure 8 shows similar
patterns for the enterococd.  No serotypes of Salmonellae of Shigellae
were detected at any stage (Table 1).

Vibrio  alqinolytlcus was detected in the effluent, but at no other point
In  the  system.  Infiltration of seawater (at exceptionally high tides)
into Galveston's sewage treatment plant is the most probable explanation
for its presence.  The environment 1n the brine shrimp tank is favorable
for its multiplication.

CONCLUSIONS

This research has yielded valuable baseline data for further study of
this sewage treatment system.  The incorporation of a  continuous-flow
system  into previously static laboratory studies has helped to emphasize
some of the problems which may be incurred  in a scale-up to pilot  plant
size.

Further study of raw sewage is needed to determine  the presence of phy-
 totoxic compounds.   It is  apparent that a build-up  of  such substances
occurs, since great  reductions in the algal population were observed
from time to  time.   A unicellular alga  (such as Tetraselmis chul used
 here)  is best for  consumption by brine shrimp, but  It  may be  that  a
mixed  culture of marine  dinoflagellates will respond better to the
 stresses incumbent in using raw sewage as  a nutrient source.  Experi-
ments  to test this are  in  preparation.

The applicability  of this  system  to  sewage treatment and food production
 is, for the most  part,  certain.   Further work  is  necessary, however,  to
optimize conditions  for  treatment  of sewage and  multiplication of  brine
shrimp.
                                   534

-------
Previous research on this system included the use of a second algal
culture to remove wastes of the brine shrimp.  The elimination of this
step simplifies the procedure considerably, but additional treatment is
apparently required to further improve the quality of the effluent.
System modifications are being studied at present to overcome this and
other problems, including, perhaps, a "mopping up" stage, as reported
by Rhyter.'

Effluent BOD values, though much improved over raw sewage, are somewhat
above the standard required of secondary treatment plants (i.e., 20
mg/1).  This required value is certainly approachable, if not surpas-
sable, by this system, perhaps if an additional passage through an algal
culture were performed.

A better method must be devised for the study of inorganic salts (total
solids) for reasons previously discussed.  The potential for solids
removal cannot be assesed without a correction for the salinity of the
carrying water, as well as an allowance for evaporation.

Total inorganic phosphorus values are affected by a number of factors
which should be controlled.  A careful monitoring of pH in the algal
culture and brine shrimp tank should yield information on the precipi-
tation-dissolution of phosphates.  Design changes in the system should
include a method for removing solid wastes of brine shrimp from the
brine shrimp tank.  This should eliminate the loss of phosphorus from
bottom sediments to the effluent.  Possible uses of the sediment as a
fertilizer are being investigated.

Inability to maintain a continuously fresh supply of raw sewage is in
large part responsible for the low total Kjeldahl nitrogen values.  At
least, initial studies should be done on the relationship between
nitrite-nitrate and ammonia-organic nitrogen.  An increase in concen-
tration of nitrite-nitrate or even their presence in notable quantities
would be an unexpected finding if raw sewage were kept fresh.  This is
true since denitrification is normally limited to the ammonia stage
in the presence of active algal growth.

The bactericidal effect of this treatment is obvious.  Elucidation of
the mechanism which brings about bacterial demise would be of help in
controlling system operation.
                                   535

-------
LITERATURE CITED

1.  Rhyter, John H., W.M. Dunstan, K.R. Tenore, and J.E. Huguenin.
         Controlled Eutrophication-Increasing Food Production From the
         Sea by Recycling Human Wastes.  Bioscience.  22:144-152,
         March, 1972.

2.  Tamiya, H.  Mass Culture of Algae.  Ann. Rev. Plant Physiol.  8;
         309-334, 1957.

3.  Roels, O.A., and R.D. Gerard.  Artificial Upwelling.  Marine Tech.
         Soc.  Proc. Conf. Food-Drugs From the Sea.  102-122, 1970.

4.  Gotaas, H.B., W.J. Oswald, and H.F. Ludwig.  Photosynthetic Recla-
         mation of Organic Hastes.  The Scientific Monthly.  79:368-
         378,  1954.

5.  Rhyter, John H.  Potential Productivity of the Sea.  Science.
         130:602-608, September 11, 1959.

6.  Dunstan, W.M. and D.W. Menzel.  Continuous Cultures of Natural Popu-
         lations of Phytoplankton in Dilute, Treated Sewage Effluent.
         Limnology and Oceanography.  1_6:623-632, July, 1964.

7.  McShan, M., N.M. Trieff, and Dov Grajcer.  Biological Treatment of
         Sewage Using Algae and Artemia.  JWPCF.  In press, 1974.

8.  Needham, J.G., P.S. Galtsoff, F.E. Lutz, and P.S. Welch.  Culture
         Methods for Invertebrate Animals.  New York, Dover Publica-
         tions, 1937, 590 pp.

9.  American Public Health Association, American Water Works Associa-
         tion, and Water Pollution Control Federation.  Standard
         Methods for the Examination of Water and Wastewater.  Washing-
         ton,  American Public Health Association, 1971, 874 pp.

10. Neel,  Joe  K., J.H. McDermott, and C.A. Monday, Jr.  Experimental
         Labooning of Raw Sewage at Fayette, Missouri.  JWPCF.   33:
         603-641, 1961.
 ACKNOWLEDGEMENT

 This research was supported by the James W.  Mclaughlin Fund,  University
 of Texas Medical Branch, Galveston, Texas
                                  536

-------
                                                  Effluent
t
      Peristaltic
        Pump
t
 Raw
Sewage
                t
              Instant
              Ocean
\
                                        ft
                                 Algal
                                 culture
           Brine shrimp tank
      o
      o
      o
      o
      o
     o
     o
           SCHEMATIC OF CONTINUOUS - FLOW SYSTEM
                      Figure 1
                         537

-------
u>
CO
                    BIOCHEMICAL OXYGEN DEMAND (mg/l)
                 3r
             "
             X
 o - Raw Sewage
 • -Algal Culture
 A -Effluent
S.E.-Standard Error of the Mean
                                   2        3
                              Experiment  Number
                                   )?± one S.E.
                                     Figure 2

-------
<0
                              TOTAL SOLIDS (RESIDUE ON EVAPORATION) (mg/l)
                                  5r
e
o>
o
II
x
                                         o - Raw Sewage
                                         • - Algal Culture
                                         A - Effluent
                                        S.E. - Standard Error of the Mean
                                                 Experiment Number
                                                                              ± one S.E.
                                                     Figure 3

-------
Ol
                                  CORRECTED  VALUES : TOTAL  SOLIDS
                                  (RESIDUE ON EVAPORATION) (mg/l )
                          o>
                          o
                          ti   2
                         X
  o - Raw Sewage
  • - Algal Culture
  * - Effluent
S.E. - Standard Error of the Mean
                                             234
                                         Experiment Number
                                    ± one S.E.
                                               Figure 4

-------
      TOTAL  INORGANIC  PHOSPHORUS  (mg/l!
    i.o
   0,8
O> 0.6
E
o>
o
II
X
0.4
   0.2
            o - Raw Sewage
            • - Algal Culture
            a - Effluent
          S.E. - Standard Error of the Mean
           1234
                 Experiment  Number
                                              5? ± one S.E.
                         Figure  5
                           547

-------
          TOTAL KJELDAHL NITROGEN (mg/I)
	  2
"v
o»
E
o>
JO

II
X
  o- Raw  Sewage

  • - Algal Culture
  *- Effluent
S.E.- Standard Error of the Mean
                                        5

                                        I
              1234

                   Experiment  Number
                                        X± one S,E.
                          Figure 6

-------
              COLIFORM MPN  / 100ml
    10
            ° - Raw Sewage
            • - Algal Culture
            * - Effluent
          S.E.- Standard Error of the Mean
o
II
X   4
                      Experiment  Number
                          Figure  7
                                                  X  one S.E.
                             543

-------
            ENTEROCOCCI  MPN /  100 ml




           o - Row Sewage

           • - Algal Culture

           «. - Effluent

         S.E. - Standard Error of the Mean
I  3
 o>
 o
                      234



                    Experiment Number
X ± one S.E.
                           Figure  8
                             544

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s

Organism
Salmonella
Shigella
V. parahaemolyticus
V. alginolyticus


Raw sewage
0/4
0/4
0/4
0/4
LOCATION
Algal culture
0/4
0/4
0/4
0/4
IN SYSTEM
Effluent
0/4
0/4
0/4
3/4

Brine shrimf
0/4
0/4
0/4
1/4
               Table 1:  NUMBER OF SAMPLES YIELDING POSITIVE TESTS/NUMBER  OF SAMPLES TESTED

-------
         PRINCIPLES OF SEWAGE TREATMENT THROUGH
               UTILIZATION IN FISH PONDS *

                          by

                     Erno Donaszy **

                  LITERATURE SURVEY

More and more planners are suggesting that sewage could
be used as food in commercial fish ponds.  However,
these same ideas were expressed by fisheries managers in
the hopes of increasing fish production.  Certain waste
waters (sewage) replace organic fertilization and
increase the fish food supplies and in turn the natural
 Field of the fish pond.  The disposal of waste water
 sewage) through irrigation is in an advanced state.
We have already examined irrigation plants and the
guiding-^principles for these plants are being developed.
Balogh   gave an account of putting waste water to use.
He writes that "The effluent from waste water spray-
treatment plants as well as from other treatments contain
a certain amount of soluble nutrients.*1  Commercial fish
ponds were established, in Germany, for the utilization
of such water.  Small organisms utilize the remaining
nutrients.  They in turn serve as fish food thus
increasing the fish yield of the pond.  However, such
clarified (pre-treated) waste waters are poor in oxygen.
Therefore the ponds are not fed solely with treated waste
water but with a mixture of fresh and waste water in a
1:3 ratio.
    dealt 5with this question in 1953 when following
Imhoff's ? lead we began investigating the problems of
carp fisheries.  Imhoff dealt with carp Cyprinus carpio
** National Agricultural Quality Control Institute,
   Budapest, Hungary.
*  Original title;Donaszy, Erno. 1965.  A halastavas
   szennyviztisztitas elvi kerdesel.  Hidrologiai Kozlony.
   4:173-177.
   Translated from Hungarian by G. Teleki, 1971.
   Fisheries, Humboldt State University.  Present address:
   Box 429, Ontario Ministry of Natural Resources,
   Port Dover, Ontario, Canada,  Special assistance by
   S. Teleki, Toronto, Canada.
                            545

-------
and tench Tinea yulgaris fisheries in sewage ponds,  i.e.
ponds built for the purpose of sewage disposal.  Imhoff
suggested that in the situation similar to the above a
five fold dilution should be used.  If no diluent is
used only Foxinus foxinus and Gasterosteus aculeatus etc.
can be brecHIn existing ponds fish production is the
primary objective.

Prerequisites of waste water utilization are:  The oxygen
content of the lake water must remain normal;  the
aquatic flora must be controlled since extremely large
populations consume the lake nutrients (primarily Og) at
an accelerated rate resulting in sudden die offs of
organisms;  only fish with low oxygen requirements (carp
Cyprenus carpio and tench Tinea vulgaris) should be
stocked.The waste water should be cleared of mud and
diluted with natural fresh water.  Waste water should not
be introduced into lakes fed solely by rain water.  The
dissolved oxygen may not fall below 3mg/l.  One should be
able to shut off the waste water supply at any given time,
e.g., when harvesting the fish or  drying and disinfecting
the pond bottom.

According to Imhoff, a  one hectare (2.4-7 acres) fish
pond would be sufficient to purify sewage of 2000 people.
In the case of a  5 fold dilution,  with water depth of at
least i meter, 5m2 of pond area are necessary per person
as opposed to 20mz in the case of  the 1 hectare pond.
Imhoff says that  natural bacteriological purification of
the waste water is better than artificial sewage treat-
ment.  However, Imhoff  is concerned  primarily^with  sewage
treatment and not fish  breeding.   Woynarovich  using some
of Imhoff's data  discusses the establishment of a sewage
fishery in Hungary.

1.   He feels that the  one hectare per  2000 inhabitants
     should not be limiting  sewage treatment to that
     small  an area.

2    He feels that the  question  of the  diluent  can  be
     solved even  if  sufficient  fresh water  cannot
     be obtained.   Since  the  specific gravity  of  sewage
     is greater than water the undiluted  sewage would
      sink  to the  pond bottom, begin  decaying,  upsetting
     the  lake's biological  balance.   Woynarovich  also
      states that  the diluent  can be  one-part recycled
     water if the lake water is  too  warm  to be added
     to the effluent.   These  problems,  states
     Woynarovich, can only be solved by practical
      experimentation.

                             547

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              THE EXAMINATION OF FISH PONDS
                SUPPLIED WITH WASTE WATER

In Hungary there are no fisheries established specifi-
cally for sewage water purification.  The personnel of
the National Health Institute examined a number of
commercial fish ponds and small natural lakes.  They
introduced an effluent into these waters2to determine
if the sewage presented a health problem >;>.  Based on
24-hour examinations of the sewage-supplied fish ponds
in Balatonfoldvan on June 19 and 20, 1962, Csanady and
co-workers2 established the following:  The pond
contained 250 inhabitant-equivalents of sewage per 1
1 Katasztralis Hold (KH or cadastral yoke = 1.412 acres).
This quantity of sewage could be effectively decomposed
by the pond's organisms.  The dilution was only 2:2 yet
there were no adverse effects noted in the open water.
However, at the effluent outfall an aerobic condition
prevailed.  Oxygen in the open pond waters was abundant
especially during mid-day.  The biologically processed
sewage of the Balatonfoldvan sewage plant is pumped into
these ponds.  The Balatonfoldvan hydrologists stated that
the biological purification treatment was quite ineffi-
cient.  A double layered sedimenter would insure the
sedimentation of parasite eggs.  In the opinion of the
plant managers, purification of waste water in the sewage
plant can only be considered a good mechanical but only
partial biological purification.  The decomposition is
actually completed in the pond.

Csanady and Gregacs^ have conducted investigations of the
sewage plant of Balatonfoldvan and several lakes into
which sewage was introduced.  They found that partially
purified household sewage did not appreciably disrupt
the lake's ecosystem.  However, decaying sewage was harm-
ful to aquatic life even if greatly diluted.  The bacter-
ioligical influence of the sewage may be beneficial.
Even though these ponds were not established  for sewage
treatment, they are large enough to have sewage intro-
duced into them without detrimental effects.  No experi-
mental data concerning the most economical quantity of
sewage  purified per given lake area by a given fish
population was obtained.  There is, then, an  abvious need
for such  experimentation to  determine these quantitative
figures.

To examine  this problem theoretically, one must study the
technical biological  aspects of the  commercial fisheries.
                             548

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      THE PHYSICAL ASPECTS OF COMMERCIAL FISHERIES

The commercial fisheries are a series of artificial
lakes in which all breeding operations can be carried
out.  These operations are:  spawning;  raising of young;
fish production (for human consumption);  feeding and
wintering.  The lakes are classified according to the
function they serve, i.e. the spawning lake, breeding
lake and production lake, etc.  Continuous sewage clean-
ing can be done in the production lakes, but not in the
fish storage lake because of crowded conditions.  The
high dissolved oxygen levels required in the storage
waters can only be obtained by a continuous fresh water
flow or aeration.  Introducing sewage into the storage
ponds would only deplete the oxygen supply.

The water level in the breeding lakes is low and the
spawning lakes are operating only intermittently.  It is
possible to raise young fish in lakes which have second-
ary, purified, diluted sewage added.  The sewage could
increase the plankton population thus increasing the
fingerling's food supply.  In an active commercial
fishery the production lakes are most important, the
spawning lakes second.

Different lake types such as mountain lakes (with valley
dams),flatland lakes (with dikes), or intermediate types
are suitable for sewage purification, but the technical
approach will have to differ.  Lakes with valley dams
seem to be the most economical because the water supply
can also serve as a diluent.  Sewage is purified much
faster flowing through the series of valley lakes.  The
nutrient rich effluent fertilizes a number of lakes as
opposed to the single pond method mentioned earlier.

Controlling the size of the fish population in these
lakes is an important operation.  Population size is
related to natural food supply.  Both are increased with
the introduction of sewage into a lake.  The feeding
program (additional feeding) of the fish has to be
adjusted accordingly.  To do this one has to know the
level of the natural food supply and the fish yield
obtained without additional feeding.  The time of "fish
harvest" will determine how long the sewage can be pumped
into the lake sector and when it can be reintroduced.
Considering the technical and practical aspects of such
fisheries, their success depends on the synchronous
planning and effort of the hydrologist  and  fishery manager,
                            549

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In ray opinion, a fishery sewage treatment plant not
prepared in this manner is unacceptable since vital con-
ditions will invariably be overlooked, e.g.,pond water
capacity, fish density, harvesting time, periodic drain-
ing and dry draining schedule, and refilling schedules.
Also, these conditions should be fixed and stipulated in
the water rights permit.

      BIOLOGICAL POTENTIAL OF COMMERCIAL FISHERIES

When introducing sewage into a body of water, one has to
consider the biological as well as physical conditions
present.  The open water, aerobically respiring, popu-
lations (algae, protozoan, plankton and aerobic bacteria)
of production lakes will aerobically decompose the
introduced sewage.  As Woynarovich" pointed out, one has
to achieve a rapid mixing of the sewage with the diluent
water or recycled lake water to prevent the sewage's
settling to the bottom.  The sewage must remain available
for decomposition by the organisms in the water.

In numerous lakes, methods similar to "agitated bottom"
sewage plant procedures could be used to increase
decomposition.  This method involves stirring up the
bottom sediments (mud), bringing the anaerobic organisms
into contact with the effluent thus assisting in its
decomposition.

Water quality is closely related to a lake's biological
potential.  In many lakes, especially in western Hungary,
because of eutrophic conditions, the oxygen utilization
is high (10-20 mg/1 and higher).  At the same time, blue-
green algaes such as Microcystis. Anabaena and Aohanizo-
menon abound.  They store nitrogen and produce oxygen.
There is, under normal conditions, an excess of
dissolved oxygen.  In some regions the wind disperses
these masses of organisms vertically or deposits piles
of them on the shores.  During calm hot summer days and
according to some during atmospheric changes, these algae
begin dying off.  The oxygen balance is upset and within
hours there is a fish kill.  The introduction of sewage
obviously enhances the possibility of such occurrences,
therefore we must continually be prepared to avert such
dangers.  Research is needed to solve this problem, so
that the optimum productivity of a water, in terms of
sewage treatment and fish production, can be achieved.

Another biological problem is the presence of substances
in the sewage which adversely affect the flavor of the


                            550

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fish or render the fish toxic for consumption.  The
presence of flavor spoiling substances can be tolerated
in low concentrations if we produce fish for industrial
uses only.  There must, however, be clean water, clearing
sections of the lake where the fish can rid themselves
of the toxic substances.

The above discussion shows that the problem of combining
large scale sewage treatment with commercial fisheries
can be solved.  Obviously it has great advantages with
respect to fish-production and waste utilization.  I must
re-emphasize that the planning and execution of such a
project is a multifaceted task requiring great foresight.
We have to distinguish between two possibilities:

1.   The introduction of sewage into a lake with such
     quantities of water that after a short while sewage
     can be detected in only a small area.  This is not
     commercial fishery (fish-pond) sewage treatment,
     but simply placement of sewage into a lake.

2.   The close association of a sewage treatment
     commercial fishery with a sewage treatment plant.
     The most efficient and economical treatment of
     mechanically cleaned sewage would be decomposition
     by the microorganisms in the water.  This is a
     balanced procedure of fish production and sewage
     treatment.

               SEWAGE TREATMENT FISHERIES

The sewage treatment potential can be utilized efficiently
only if the fishery is established for the primary
purpose of sewage treatment and fish production secondary.
These two goals must be maintained in harmony with each-
other.  An important differentiation here is that the
objective of sewage placement is not solely the fertili-
zation of the fish-pond but the treatment of sewage as
well as its utilization.  Although it is possible to
convert a primarily fish producing fishery into a sewage
treatment fishery, as long as certain alterations of the
fishery management and the sewage treatment plant have
not taken place, we face obstacles which will interfere
with either the raising of fish or the treatment of
sewage.

Let me describe a commercial fishery established for
the purpose of sewage treatment.  The lake represents
an extension of the sewage plant, the fishery managers
have to adjust to the sewage plant management.  Such

                            557

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fisheries  consist  of a  series of ponds some of which  can
always accept  sewage.   The area of the ponds and its
daily  and  seasonal fluctuations are correlated with the
amount of  sewage added  daily.  This fishery has clearing
sections (as described  earlier) where marketable fish are
raised.  Fingerlings could also be raised for other
fishery needs.  In planning such a fishery the following
points must be  considered:

1.   The structure of the lake.

2.   The structure of the sewage plant.

3.   Water supply,  sewage supply, sewage conveyance
     and sewage distribution.

4.   Production plan and continuity of sewage treatment.

5.   Supply of fish  (fingerlings).

6.   Winter operation of harvested (empty) and stocked
     lakes.

7.   Fish health.

8.   Public health aspects.

9»   A one-year operational plan, outlined.

What are alternatives to incomplete (seasonal) opera-
tions?  In such instances the lakes may be smaller and
the number of lake sections will depend on the following:

1.   If no diluent is used particular sections should
     receive (evenly spaced) repeated monthly doses of
     sewage.

2.   If a diluent water is used the sewage input
     should be adjusted to a constant ratio with the
     water.

Toxic substances in the sewage must be 50^ below their
toxicity level.  In the presence of flavor spoiling sub-
stances one should keep the fish in the ponds for only
one season whereupon they are transferred to clean water
and allowed to regain their natural flavor.

The period of sewage input has to be determined experi-
mentally according to lake size, sewage quantity and
                            552

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biological conditions of the water.  (The clearing factor
is a value which varies from season to season),

     SEWAGE INTRODUCTION INTO COMMERCIAL FISHERIES

The primary purpose of such lakes if fish production.
The purpose of seivage introduction Is fertilization.
The sewage input must be controlled at all times by
diversion into other lakes or disposal systems.  One
should also be able to channel the sewage into several
lake sections.  Sewage introduction has to cease two
weeks prior to fish harvest (both summer and fall) and
when the lake bed is dry or during disinfection periods.

For our climatic conditions we must establish the lake
area a) before stocking and b) after stocking, required
for the amount of sewage to be pumped in, i.e. the amount
of sewage which can be introduced per unit of lake area,
considering water depth and the season so that ^-mesosa-
probial conditions already exist at the time of stocking.
Furthermore, we have to establish the ratio of sewage to
water to maintain the ^9-mesosaprobial conditions.

One has to equip the lakes with a sewage by-pass and
distribution canal system.  The sewage input ducts must
be separated so as to assure even distribution.  The
number of inlets should be variable according to sewage
quantity.  Even distribution is desirable especially
when no diluent water is used.  If sewage concentrate
(sedimented sewage) is used, it is desirable to apply
calcium oxide prior to stocking and after the fish
harvest.

Checking the water quality and saprobic conditions
regularly by chemical and microscopic examinations is
important.  This is a part of lake management.  If algae
become over abundant the causative agent (compound) has
to be determined and depending on the extent of the
bloom one may have to stop sewage introduction.  In
lakes of this type one can raise yearling and two year
old carp and tench and possibly other grazing species.
Experimentation will determine whether other useful
species can be raised.

Fish to be sold for consumption must be kept in clean
water lakes for a month (or at least 2-3 weeks) prior
to harvest.  Specific regulations should be established
by the animal health service.  Fish may be raised
without compliance to such regulations if they are used
for other purposes, e.g. breeding stock.

                            553

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        EXPERIMENTAL SEWAGE TREATMENT FISHERIES

All our colleagues agree that planning of sewage treat-
ment fisheries will encounter difficulties as long as
experiments cannot be conducted.  They are essential in
gaining experience (knowledge).  Aside from the above
described tasks we shall summarize the factors which
we have considered in the establishment of an experi-
mental sewage treatment fishery.  When choosing the site
we have to consider the sewage plant and fish production
aspect equally.  The fishery should consist of a number
of pond sections (series).  It is desirable that the
experimental ponds be in a star configuration as at
Szarvas, Hungary.  In these 16 small ponds of equal
size the number of possible variations and duplications
is sufficiently large.  To obtain dependable results a
minimum of 4 variations in duplicate are required.  A
water supply must be available to allow experimentation
with and without diluent.

The experiments have to be oriented to the following
problems:

1.   Degree of sewage clearing efficiency with
     different dilutions and without dilution.

2.   Determination of fish population density at
     different levels of sewage concentration.

3.   Fish yield without additional feeding.

4.   Experiments with additional feeding of different
     population levels.

5.   Determination of sewage introduction methods.

6.   Rate of weight gain (average weight) of
     different aged fish when fertilization with
     sewage only.

7.   Determining how to inhibit the effect of flavor
     spoiling substances.

Apart from the experimental operations, one also has
to establish a laboratory where continuous checks on the
microbial and chemical processes of sewage decomposition
and animal and public health examinations can be made.

As a result of the investigations into fishery sewage
treatment many new aspects of this concept have been

                            554

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elucidated.  The microbial activity in lakes is now
better understood.  Decomposing sewage with algae gained
prominence among biological treatment methods.  The
proposed experimental sewage treatment fishery would
make domestic experimentation possible.

                        SUMMARY

After urgent requests from planning engineers and
fishing specialists, the (governmental) Sewage Branch
put the question of sewage treatment fisheries on its
agenda.  The examination of our small natural lakes into
which sewage is introduced has commenced.  The planner
has to consider the technical and biological properties
of such fish producing lakes first.  We have to distin-
guish between sewage treatment fisheries and the
commercial fisheries into which sewage is introduced.
In the former, the sewage plant and the fishery
constitute a coordinated operation, sewage treatment
being the primary objective and fish breeding the
secondary one.  In the latter, fish breeding is the
primary and sewage treatment the secondary objective.
The questions raised can only be solved satisfactorily
through trials conducted in experimental sewage treatment
fisheries.  Such experimental operations would also
afford opportunity for needed algae research.  The
utilization of algaes in sewage treatment offer great
possibilities.

                    LITERATURE CITED

1.  Balogh, J.  1963.  Utilization of wastewaters.
    Termeszettudomanyi Kozlony.  7(7):3l6-3l£ and
    7(10):461-463.

2.  Csanady, M., M. Gregacs, S. Mahunka and A. Lengyel.
    No date. 24 hour in depth public health examination
    of the Balalon foldvar sewage treatment fisheries.
    Egeszsegtudomany.  194(3):145-156

3.  Csanady, M., and M. Gregacs.  1964.  Examination
    of public health questions, regarding sewage
    treatment, in fisheries.  Hidrologial Kozlony. 2.

4.  Donaszy E.  19 5#.  Waste water and fishponds.
    Halaszat 5(1):2.

5.  Imhoff, K.  1951.  Handbook of city water system 14.
    Auf1.  Munchen.

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6.  Woynarovich, E.  1959.  Can the sewage treatment
    fishery be established in Hungary?  Halaszat, 6(4):64.
                            556

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PART 8   APPENDICES

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PROGRAM COMMITTEE

George H. Allen, Ph.D. - Chairman
Professor of Fisheries
School of Natural Resources
California State University, Humboldt
Arcata, California  95521

Richard E. Thomas - Co-chairman
Research Soil Scientist
Robert S. Kerr Water Research Laboratory
Environmental Protection Agency
P.O. Box 1198
Ada, Oklahoma  74820

Leland J. McCabe, M.P.H., B.S.C.E.
Chief, Criteria Development Branch
Water Supply Research Laboratory
National Environmental Research Center
Environmental Protection Agency
Cincinnati, Ohio  45268

Kenneth 0. Allen, Ph.D.
Fisheries Biologist
Fish Farming Experimental  Station
Bureau of Sport Fisheries  and Wildlife
P.O. Box  860
Stuttgart, Arkansas   72160

John Marsh, P.E.
President, Engineering  Enterprises
P.O.  Box E
Norman,  Oklahoma   73069
                                 557

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LOCAL ARRANGEMENTS COMMITTEE
Charles D. Newton, P.E. - Chairman
Chief, Water Quality Services
Oklahoma State Department of Health
Northeast Tenth and Stonewall
Oklahoma City, Oklahoma  73105
                                      Charles  Lawrence, P.E. , Ph.D.
                                      Assoc. Professor of Environ-
                                      mental Health
                                      Univ. of Oklahoma Health
                                      Sciences Center
                                      P.O. Box 26901
R. LeRoy Carpenter, M.D. - Co-chairman Oklahoma City, Oklahoma  73190
Commissioner of Health
Oklahoma State Department of Health
Northeast Tenth and Stonewall
Oklahoma City, Oklahoma  73105
Mark S. Coleman, Director
Water Quality Monitoring and
Research Division
Water Quality Services
Oklahoma State Department of Health
Northeast Tenth and Stonewall
Oklahoma City, Oklahoma  73105

Dave Dougall, M.E.S.
Administrative Assistant
Water Quality Services
Oklahoma State Department of Health
Northeast Tenth and Stonewall
Oklahoma City, Oklahoma  73105

Reginald Frank, Ph.D.
Research Grants Coordinator
Water Quality Service
Oklahoma State Department of Health
Northeast Tenth and Stonewall
Oklahoma City, Oklahoma  73105

Bill Griffith
Associated Milk Producers, Inc.
1700 North Sooner Road
Oklahoma City, Oklahoma  73141

Ron Jarman
Assistant Chief, Management Fish
Division
Oklahoma Dept. of Wildlife
Conservation
1801 Lincoln
Oklahoma City, Oklahoma  73105
Jim Pollard, Supervisor
Environmental Control, Air
and Water
Oklahoma Gas & Electric Co.
P.O. Box 321
Oklahoma City, Oklahoma 73101

Jim Russell
Aquaculture Industry, Inc.
2116 West 24th Avenue
Stillwater, Oklahoma 74074

Lester Settle, P.E.
Settle, Dougall & Spear,
Engineers
Colcord Building
Oklahoma City, Oklahoma 73102

Michael R. Spear, P.E.
Settle, Dougall & Spear,
Engineers
Colcord Building
Oklahoma City, Oklahoma 73102
                                      PUBLICITY

                                      Toni Morrow
                                      Public  Information
                                      Oklahoma State Dept. of Health
                                      Northeast Tenth and Stonewall
                                      Oklahoma City, Oklahoma 73105
                                558

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REGISTRANTS
Ronald K. Abe
Associate Professor
Fort Valley State College
FVSC Box 1494
Fort Valley, Georgia 31030

Kenneth 0. Allen, Ph.D.
Fish Farming Experimental Station
Box 860
Stuttgart, Arkansas 72160

Jack Ashman
OEIMC
East Central State College
Ada, Oklahoma 74820

Carl Astley
Graduate Student
School of Environmental Health
641 Northeast 15th
Oklahoma City, Oklahoma 73101

Robert S. Ayers
Soil & Water Specialist
Agriculture Extension
University of California
Davis, California 95616

Dr. Dan  Badger
Professor
Agricultural Economics
Oklahoma State University
Stillwater, Oklahoma  74074

Bill  Bailey
Game  & Fish Biologist III
Arkansas Game  &  Fish  Commission
P.O.  Box 178
 Lonoke,  Arkansas 72086

 John  M.  Baker
 Asst.  Head,  Civil Engineering
 Benham-Blair
 6323  N.  Grand  Blvd.
Oklahoma City, Oklahoma 73118
James V.  Basilico
Chief, Process Development
Branch
Environmental Protection Agency
3702 D. WSM
Washington, D.C.  20460

Robert K. Bastian
Resident EPA Rep.
U.S. Environmental Protection
Agency
P.O. Bldg. - 268 Market Street
Muskegon, Michigan 49440

John W. Baxter
Assistant Engineer
Water Quality Service
Oklahoma State Dept.  of Health
Northeast Tenth and Stonewall
Oklahoma City, Oklahoma 73105

Herbert Beauchamp
Supervisor, Virology Section
Oklahoma State Dept.  of Health
P.O. Box 53551
Oklahoma City, Oklahoma 73105

Thomas E.  Berry
Box 528
Stillwater, Oklahoma 74074

E.  E.  Blanchard
Civil  Engineer
Oklahoma Gas  & Electric Co.
P.O.  Box 321
Oklahoma City, Oklahoma 73101

Fred C.  Boswell
Professor, Agron  - Soils Res.
University of Georgia
Georgia  Station
Experiment,  Georgia  30212
                                 559

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W. V. Bowennan
Product Development Engineer
Manitoba Government
Dept. of Industry & Commerce
210  - 185 Carlton
Winnipeg, Manitoba, Canada R3C-1P3

David Bowman
Department of Zoology
Oklahoma State University
Stillwater, Oklahoma 74074

Homer Buck
Aquatic Biologist
Illinois National Historical Survey
R.R. #1
Kinmundy, Illinois 62854
Dr. Robert L. Bunch
Chief, Treat. Proc. Div.  Branch
AWTRI, Environmental Protection
Agency
NERC 4676 Columbia Parkway
Cincinnati, Ohio 45268

Donald R. Bunn
Assistant City Engineer
City of Fayetteville
P.O. Box "F"
Fayetteville, Arkansas 72701

Nathan C. Burbank, Jr., Sc.D.
Prof, of Environmental Health &
Sanitary Engineering
School of Public Health
University of Hawaii
1960 East-West Road
Honolulu, Hawaii 96822

Krisen Euros
Research Engineer
Black, Crow & Eidsness
P.O. Box 7133
Christiansted, U.S. Virgin Is,

Allen Butchbaker
Ag. Engineering Dept.
Oklahoma State University
Stillwater, Oklahoma  74074
     Charles P. Carlson
     Fishery Biologist
     Bureau of Sport Fisheries
     & Wildlife
     Box  292
     Stuttgart, Arkansas  72160

     Clarence A. Carlson
     Assoc. Prof, of Fishery  Biology
     Colorado State University
     301  Aylesworth Hall
     Fort Collins, Colorado 80521

     John W. Carter, Sr.
     Dir. of Res. Protection  & Dev.
     Grand River Dam Authority
     P.O. Drawer G
     Vinita, Oklahoma  74301

     Preston E. Carter
     Chief of Water Pollution Sec.
     Oklahoma City-County Health
     Department
     921  N.E. 23rd Street
     Oklahoma City, Oklahoma  73105

     Jim  Carver
     City Engineer
     City of Duncan -  Box 969
     Duncan, Oklahoma  73533

     H. A. Caves
     Sanitarian
     Garvin County Health Dept.
     Box  695
     Pauls Valley, Oklahoma  73075

     Harry Chichester,  Dom.  Insp.  I
     Oklahoma State Dept. of  Health
     Northeast Tenth  and  Stonewall
     Oklahoma City, Oklahoma  73105
      Lester J.  Clarke
00820 Consulting Engineer
      316 Wildewood Terrace
      Oklahoma City, Oklahoma 73105
                                560

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Jeannette Cook
OEIMC
East Central State College
Ada, Oklahoma 74820

Dr. Berniece N. Crockett
Research - Education
Oklahoma Water, Inc.
625 Fidelity Plaza
Oklahoma City, Oklahoma 73102

Robert E. Cullen
Engineer
Dept. of Pollution Control
Box 53504
Oklahoma City, Oklahoma 73105

Dr. Dudley D. Culley, Jr.
Associate Professor
Louisiana State University
249 Ag. Center
Baton Rouge, Louisiana 70803

Dr. Runyan Deere
State Health Education Leader
Arkansas Cooperative  Extension
Service
P.O. Box 391
Little Rock, Arkansas 72203

Robert Delano
Assistant Chemist
Oklahoma State  Department of  Health
Northeast Tenth and  Stonewall
Oklahoma City,  Oklahoma  73105

Don Denton,  Lab Supervisor
South  Okla.  City Jr.  College
7777 South May  Avenue
Oklahoma City,  Oklahoma  73135

William  G.  Dodd
 Industrial  Specialist
Southeastern State College
Durant,  Oklahoma 74701
William R. Duffer,  Ph.D.
Research Aquatic Biologist
Environmental Protection  Agency
Box 1198
Ada, Oklahoma 74820

Ray Duffy
Utility Superintendent
City of Frederick
P.O. Box 399
Frederick, Oklahoma 73542

Judy Duncan
Sanitarian
Water Quality Service
Oklahoma State Dept. of Health
Northeast Tenth and Stonewall
Oklahoma City, Oklahoma 73105

Mike L. Packer
Graduate Student
Oklahoma State University
2422 S. Hudson Place
Tulsa, Oklahoma 74114

Dwain Farley
Environmental Program Spec. 1
Oklahoma State Dept. of Health
Northeast Tenth and Stonewall
Oklahoma City, Oklahoma 73105

John Feddes
Agricultural Engineer
Pollution Control  Branch
Parliament Buildings
Victoria, British  Columbia
Canada

Stephen A. Flickinger
Assistant Professor
Dept. of  Fishery & Wildlife
Biology
Colorado  State  University
Fort  Collins, Colorado 80521
                                 567

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 Ted H. Forester
 Sanitary Engineer III
 Missouri Clean Water Commission
 Box 154
 Jefferson City, Missouri 65101

 Pedrito A. Francois
 Director, Environmental  Health
 Virgin Islands Health Dept.
 Box 543
 Charlotte Amalie,  Virgin Islands

 Reginald H.  Frank. Ph.D.
 Research Grants Coordinator
 Water Quality  Service
 Oklahoma State Dept. of  Health
 Northeast Tenth and  Stonewall
 Oklahoma City,  Oklahoma  73105

 Dr.  B. A.  Friedman
 Union Carbide  Corporation
 Linde Division
 Box  44
 Tonawanda, New York  14150

 Westal W.  Fuchs  (Fox)
 State Soil Scientist
 Soil Conservation Service
 Farm Road  & Brumley
 Stillwater, Oklahoma 74074

 Everett  Gartrell
Mayor
 City of  Weatherford
 P.O.  Box 133
 Weatherford, Oklahoma 73096

 Glen Gebhard
 Department of  Zoology
Oklahoma State University
 Stillwater, Oklahoma 74074

 Bernie Gipson
Chemist
Oklahoma State Dept.  of Health
Northeast  Tenth and Stonewall
Oklahoma City,  Oklahoma 73105
    Harry Hansen
    Environmental Manager
    U.S. Pollution Control, Inc.
    2000 Classen Center
    Suite 200 South
    Oklahoma City, Oklahoma 73106

    Eldon T. Head
    City Manager
    City of Duncan
00801  Box 969
    Duncan, Oklahoma 73533

    Elliott S. Hechtman
    President, Aquarium Farms, Inc.
    Box 1015
    Fremont, Nebraska 68025

    Phil Henderson
    Biologist
    Oklahoma State Dept.  of Health
    Northeast Tenth and Stonewall
    Oklahoma City, Oklahoma 73105

    Scott Henderson
    Joe Hogan State Fish  Hatchery
    Arkansas Game & Fish  Commission
    P.O.  Box 178
    Lonoke,  Arkansas 72086

    Jim Holland
    Assn.  of S.  Central Okla.  Govts.
    802 Main Street
    Duncan,  Oklahoma 73533

    Dr. Charles  C.  Hortenstine
    Associate Professor
    Soil  Science Dept.
    University of Florida
    106 Newell Hall
    Gainesville,  Florida 32611

    David  G.  Hughes
    Warm Water Fish  Culturist
    Zoology  Department
    Oklahoma State University
    Stillwater,  Oklahoma 74074
                               552

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Andrew H. Hulsey
Joe Hogan State Fish Hatchery
P.O. Box 178
Lonoke, Arkansas 72086

E. E. "Gene" Humes
Sanitary Engineer
Williams Bros. Waste Control, Inc.
321 S. Boston
Tulsa, Oklahoma 74103

James V. Rusted
Graduate Assistant
Penn State University
108 Ag. Engineering
University Park, Pa. 16802

Brad Jackson
Biologist II
Oklahoma State Dept. of Health
Northeast Tenth and Stonewall
Oklahoma City, Oklahoma 73105

Lee W.  Jacobs
Assistant Professor
Dept.  of Crop & Soil Sciences
Michigan State University
East  Lansing, Michigan 48824

Col. Howard R. Jarrell, Assoc.  Dir.
Okla.  Water Resources Research  Inst.
Oklahoma State University
Stillwater, Oklahoma  74074

Lawrence M. Jones
Environmental Engineer
Western Electric
7725  West Reno
Oklahoma City, Oklahoma  73125

Timothy Joyner
Manager, Aquaculture  Program
Northwest Fisheries  Center
 2725  Montlake Blvd.  E
Seattle, Washington  98112
C. H. Lawrence, P.E.,  Ph.D.
Assoc. Professor of Environ-
mental Health
Univ. of Okla. Health  Sciences
Center
641 Northeast 15th Street
Oklahoma City, Oklahoma 73105

Patterson Lay
Oklahoma City Public Works Dept.
200 North Walker
Oklahoma City, Oklahoma 73102

Ronald D. Lees
Research Engineer
Hercules Incorporated
900 Green Bank Road
Wilmington, Delaware 19809

Erne  Lewis
Architect
Kramer, Chin  & Mayo
1917  First Avenue
Seattle, Washington 98101

Dr. Ronald F.  Lewis
Research Microbiologist
Environmental Protection Agency
Robert A. Taft  Laboratory
4676  Columbia Parkway
Cincinnati, Ohio 45226

Harold  Loyacano
Asst. Professor Zoology
Clemson University
Ent.  &  Econ., Zoology
Clemson,  South Carolina  29631

Robert  A. Mah
Assoc.  Professor Public  Health
U.C.L.A.
 School  of Public Health
 Los Angles,  California 90024

Harold  K. Malone
 Asst. Chief,  Laboratory  Services
Oklahoma State Dept.  of  Health
 Box 53551
 Oklahoma City, Oklahoma  73105
                                 563

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Harry Manges
Associate Professor
Agricultural Engineering
Kansas State University
Manhattan, Kansas 66506

John Marsh
Engineering Enterprises
P.O. Box E
Norman, Oklahoma 73069

Leland J. McCabe, Chief
Criteria Development Branch
Water Supply Research Laboratory
Environmental Protection Agency
Cincinnati, Ohio 45268

Donald L. Mehlburger, President
Mehlburger Engineers, Inc.
1218 W. 3rd Street
Little Rock, Arkansas 72201

James W. Merna
Fisheries Research Biologist
Mich. Dept. of Natural Resources
Inst. for Fisheries Research
Museums Annex
Ann Arbor, Michigan 48104

Dennis Messmer, Ph.D.
Assoc. Professor Micro.
Southwestern State College
Dept. of Biological Sciences
Weatherford, Oklahoma 73096

Ray A. Mill
Planner
Oklahoma Dept. of Pollution Control
Box 53504
Oklahoma City, Oklahoma 73105

Aaron Mitchum
Microbiologist II, Diag. Bact.
Oklahoma State Dept. of Health
Northeast Tenth and Stonewall
Oklahoma City, Oklahoma 73105
Bill Moyer
Program Director
Oklahoma Municipal League
4040 Lincoln Boulevard
Oklahoma City, Oklahoma 73105

Charles D. Newton
Chief, Water Quality Services
Oklahoma State Dept. of Health
Northeast Tenth and Stonewall
Oklahoma City, Oklahoma 73105

John G. Nickum
Assistant Leader
U.S. Dept. of Interior
Fish and Wildlife Service
Bureau of Sport Fisheries &
Wildlife
New York Cooperative Fishery
Unit
Cornell University
Ithaca, New York 14850

W. Harold Ornes
Biologist
University of Florida
IFAS Exp. Sta. Res. Center
3205 S.W. 70th Avenue
Fort Lauderdale, Fla. 33314

Mario M. Pamatmat
Associate Professor
Auburn University
Dept. of Fisheries & Allied
Aquacultures
School of Agriculture &
Aquaculture
Experiment Station System
Auburn, Alabama 36830

Lloyd Parham, Director
Milk & Interstate Carrier Div.
Oklahoma State Dept. of Health
Northeast Tenth and Stonewall
Oklahoma City, Oklahoma 73105
                               564

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John T. Patton
Prof, of Chemical Engineering
Michigan Tech. University
Houghton, Michigan 49931

Dr. Thomas D. Peace
Director
Okla. Dept. of Pollution Control
Box 53504
Oklahoma City, Oklahoma 73105

Howard S. Peavy
Chief Engineer
Okla. Dept. of Pollution Control
Northeast Tenth and Stonewall
Box 53504
Oklahoma City, Oklahoma 73105

Ron Peterson
Planning Engineer
Okla. Dept. of Pollution Control
Box 53504
Oklahoma City, Oklahoma 73105

Jim Pollard, Supervisor
Environmental Control, Air and Water
Oklahoma Gas & Electric Company
321 North Harvey
P.O. Box 321
Oklahoma City, Oklahoma 73101

Loyd F. Pummill, Deputy Com. for
Environmental Health Services
Oklahoma State Dept. of Health
Northeast Tenth & Stonewall
Oklahoma City, Oklahoma 73105

Robert E. Reid
Councilman, Town of Brookhaven
Brookhaven National Laboratory
Upton, New York 11973

Marcel C. Reynolds
Staff Member
Sandia Laboratories
Albuquerque, New Mexico 87115
Clifford Risley,  Jr.
Director, Research and
Development
U.S. Environmental Protection
Agency
1 North Wacker Drive
Chicago, Illinois 60606

Dr. James M. Robertson
Associate Professor
University of Oklahoma
202 West Boyd
Norman, Oklahoma  73069

H. Randall Robinette
Asst. Professor
Mississippi State University
P.O. Drawer LW
Mississippi State, Miss.  39762

Mickey Rowe
OEIMC
East Central State College
Ada, Oklahoma 74820

Ara Roy
Microbiologist IV
Supervisor, Sanitary  Bacterio-
logy
Oklahoma State Dept.  of  Health
Box 53551
Oklahoma City, Oklahoma  73105

Jim Russell
2116 West 24th Avenue
Stillwater, Oklahoma  74074

Patrick J. Ryan
Chief, Environmental  Affairs
Oklahoma Gas & Electric  Co.
321 North Harvey
P.O. Box 321
Oklahoma City, Oklahoma  73101
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William R. Schtnieding, Ph.D.
Chief, Laboratory Services
Oklahoma State Dept. of Health
Box 53551
Oklahoma City, Oklahoma 73105

Delbert Schwab
Ext.  Irrigation Spec.
Oklahoma State University
Room  216 - Ag. Hall
Stillwater, Oklahoma 74074

Kenneth R. Settle
Sanitarian I
Oklahoma State Dept. of Health
Northeast Tenth and Stonewall
Oklahoma City, Oklahoma 73105

Lester L. Settle
Settle, Dougall & Spear, Engineers
Colcord Building
Oklahoma City, Oklahoma 73105

Manse R. Sharp, Jr.
Banyon Engineering & Management Co.
1315 East First National Center
Oklahoma City, Oklahoma 73102

Thomas R. Sharp
Graduate Student - Fisheries
Humboldt State University
Box 51 WRS
Trinidad, California 95570

W. W. Shepherd
Chemist
Oklahoma State Dept. of Health
Northeast Tenth and Stonewall
Oklahoma City, Oklahoma 73105

Kenneth Shirley
Department of Zoology
Oklahoma State University
Stillwater, Oklahoma 74074
 Dewey  L.  Shroyer
 Director,  Grounds Maintenance
 Dept.
 Texas  Tech. University
 Box 4169
 Lubbock,  Texas 79409

Max Slifer
Watersaver Company, Inc.
P.O. Box  16465
Denver, Colorado 80216

Maxwell M. Small
Special Projects Manager
Brookhaven National Laboratory
Building  318
Upton, New York 11973

K. E.  Sorrells, Chief Chemist
Arkansas  Dept. of Pollution
Control
8001 National Drive
Little Rock, Arkansas 72209

Richard D. Spall
Asst. Professor
College of Veterinary Medicine
Oklahoma  State University
Stillwater, Oklahoma 74074

Michael R. Spear
Settle, Dougall & Spear,
Engineers
Colcord Building
Oklahoma City, Oklahoma 73102

John Spine Hi
Research Chemist
National Marine Fishery Service
2725 Montlake Bldg. E
Seattle, Washington 98112

Dr. Robert C. Summerfelt
Department of Zoology
Oklahoma  State University
Stillwater, Oklahoma 74074
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David L. Sutton, Ph.D.
Agronomist
University of Florida
IFAS, Exp. Sta. Res. Center
3205 S.W. 70th Avenue
Fort Lauderdale, Florida 33314

Benny F. Swafford
Hydraulic Engineer
U.S. Corps of Engineers
Forrestal Building
Washington, D.C. 20314

Tom Switalski
Sales Engineer
Certain-Teed Products
Pipe & Plastics Group
Box 86D
Valley Forge, Pa. 19482

Dr. Denver Talley
Assistant Director
Department of Pollution Control
Box 53504
Oklahoma City, Oklahoma 73105

Howard A. Tanner
Professor and Director
Michigan State University
109 National Resources Bldg.
East Lansing, Michigan 48824

Richard E. Thomas
Research Soil Scientist
Environmental Protection Agency
Box 1198
Ada, Oklahoma 74820

Larry H. Thompson
Assistant Director
Okla. Economic Development Assn.
Box 668
Beaver, Oklahoma 73932
Dr. Gary H. Toenniessen
Asst. Director, National &
Environmental Science
Rockefeller Foundation
111 West 50th Street
New York, New York 10020

Ronald L. Thune
Graduate Student
Western Illinois University
Dept. of Biology
Macomb, Illinois 61455

Dr. Thomas C. Tucker
Water Resources Research Center
University of Arizona
Tucson, Arizona 85721

Dr. James M. Vaughn
Virologist
Woods Hole Oceanographic Inst.
Department of Biology
Woods Hole, Massachusetts 02543

W. Frank Wade
Associate Professor
Southeastern State College
Biology Department
Durant, Oklahoma 74701

George R. Waller
President & Managing Director
Midcontinent Environmental
  Center Association
Box 201
Tulsa, Oklahoma 74102

G. W. Wallingford
Dept. of Agronomy
Kansas State University
Waters Hall
Manhattan, Kansas 66506

Jack N. Walter
Graduate Assistant
Penn State University
233 Ag. Engineering
University Park, Pa. 16802
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Charles W. Ware, Engineer
Assn. of So. Central Okla. Govts.
802 Main Street
Duncan, Oklahoma 73533

Thomas P. Wasbotten
Sanitary Engineer
Mich. Dept.  of Natural Resources
Municipal Wastewater Division
120 Maplewood Drive
East Lansing, Michigan 48823

William R. Whiting
President
Sea Ranch, Inc.
Route 2, Box 604
Sheridan, Arkansas 72150

David Wilds
Fish Fanner
Wilds Fish Farm
Route 2, Box 55
Yukon, Oklahoma 73099

George Wilkinson
Administrator
City of Weatherford
Box 569
Weatherford, Oklahoma 73096

Ted A. Williamson
Senior Engineer
Oklahoma State Dept. of Health
Northeast Tenth and Stonewall
Oklahoma City, Oklahoma 73105

Gary Wolgamott, Ph.D.
Assoc. Professor Micro.
Southwestern State College
Department of Biological Sciences
Weatherford, Oklahoma 73096

Kenneth J. Ziegler
Civil Engineer
Missouri