WATER POLLUTION CONTROL RESEARCH SERIES • 13O30 ELY O8/7i~9


                                     REC-R2-7I-9


                                     DWR NO.I74 -12
         BIO-ENGINEERING ASPECTS OF AGRICULTURAL DRAINAGE
               SAN JOAQUIN VALLEY, CALIFORNIA
     THE EFFECTS OF AGRICULTURAL WASTE

     WATER TREATMENT ON ALGAL BIO ASS AY

                    RESPONSE
                     AUGUST 1971
                                         '."•M
                                         S^BHRfe.
                                    K?:  >^j*
ENVIRONMENTAL PROTECTION AGENCY«RESEARCH AND MONITORING

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       BIO-ENGINEERING ASPECTS OF AGRICULTURAL DRAINAGE
                SAN JOAQUIN VALLEY, CALIFORNIA
The Bio-Engineering Aspects of Agricultural Drainage reports
describe the results of a unique interagency study of the
occurrence of nitrogen and nitrogen removal treatment of
subsurface agricultural wastewaters of the San Joaquin
Valley, California.

The three principal agencies involved in the study are the
Water Quality Office of the Environmental Protection Agency,
the United States Bureau of Reclamation, and the California
Department of Water Resources.

Inquiries pertaining to the Bio-Engineering Aspects of
Agricultural Drainage reports should be directed to the
author agency, but may be directed to any one of the three
principal agencies.
                          THE REPORTS
It is planned that a series of twelve reports will be issued
describing the results of the interagency study.

There will be a summary report covering all phases of the
study.

A group of four reports will be prepared on the phase of the
study related to predictions of subsurface agricultural
wastewater quality — one report by each of the three
agencies, and a summary of the three reports.

A group of three reports will be prepared to include:  (1) the
techniques to remove nitrogen in drainage effluent during
transport, (2) the possibilities of reducing nitrogen in
drainage water by on-farm practices, and  (3) desalination of
subsurface agricultural waste waters.

This report,   "THE EFFECTS OF AGRICULTURAL WASTE WATER TREAT-
MENT ON ALGAL BIQASSAY RESPONSE," has been prepared with
three other reports on the treatment methods studied and on
the biostimulatory testing of the treatment plant effluent.
A summary of the three basic reports will also be prepared.

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   BIO-ENGINEERING ASPECTS  OF AGRICULTURAL DRAINAGE
            SAN JOAQUIN VALLEY,  CALIFORNIA
  THE EFFECTS OF  AGRICULTURAL WASTE WATER TREATMENT
              ON ALGAL  BIOASSAY RESPONSE
                   Prepared by the

            Environmental  Protection Agency
         William D.  Ruckelshaus, Administrator
     The agricultural  drainage study was conducted
                under  the direction of:
 Paul De Falco,  Jr.,  Regional Administrator,   Region 9
            ENVIRONMENTAL PROTECTION AGENCY
100 California Street,  San Francisco, California  94111
 Robert J. Pafford,  Jr.,  Regional Director,   Region 2
          UNITED  STATES BUREAU OF RECLAMATION
  2800 Cottage Way,  Sacramento, California   95825
           John  R.  Teerink, Deputy Director
       CALIFORNIA DEPARTMENT OF WATER RESOURCES
   1416 Ninth  Street,  Sacramento, California   95814
                       REC-R2-71-9
                     DWR No.  174-12
                    13030 ELY 08/71-9
                       August 1971
   For nale by tb.a Superintendent ot Documents, U.S. Government Printing Office, Washington, D.C. 2M02 - Price $1.00

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                REVIEW NOTICE
This report has been reviewed by the U.S. Bureau
of Reclamation and the California Department of
Water Resources, and has been approved for
publication.  Approval does not signify that the
contents necessarily reflect the views and policies
of the Bureau of Reclamation, or the California
Department of Water Resources.

The mention of trade names or commercial products
does not constitute endorsement or recommendation
for use by either of the two federal agencies
or the California Department of Water Resources.
                  .ii

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                           ABSTRACT
     Laboratory bioassay experiments were performed to test
the effect on algal growth of agricultural waste water before
and after the waste water had been subjected to two different
nitrogen removal processes.  The waste waters were added in
various percentages to San Joaquin River Delta water for bio-
assay.  The algal growth throughout time was monitored by
chlorophyll fluorescence techniques.  The fluorescence meas-
urements showed logarithmic growth similar to the type usually
observed in the Delta water over the vernal growth period.

     The laboratory experiments gave positive statistical
evidence that the untreated agricultural waste water would
promote substantial algal growth above that of the San Joaquin
River controls.  Both nitrogen removal processes were equally
effective in lowering the algal growth to that of the Delta
water controls as long as the nitrate-nitrogen level in each
removal system had been lowered to approximately 2 mg N/l
or less.
Key words:  Algal blooms - control, bioassay - algal,
            chlorophyll, denitrification, fluorometry, tile
            drainage.
                             iii

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                          BACKGROUND
     This report is one of a series which presents the
findings of intensive interagency investigations of practical
means to control the nitrate concentration in subsurface
agricultural waste water prior to its discharge into other
water.  The primary participants in the program are the Water
Quality Office of the Environmental Protection Agency, the
United States Bureau of Reclamation, and the California
Department of Water Resources, but several other agencies also
are cooperating in the program.  These three agencies initiated
the program because they are responsible for providing a
system for disposing of subsurface agricultural waste water
from the San Joaquin Valley of California and protecting
water quality in California's water bodies.  Other agencies
cooperated in the program by providing particular knowledge
pertaining to specific parts of the overall task.

     The need to ultimately provide subsurface drainage
for large areas of agricultural land in the western and
southern San Joaquin Valley has been recognized for some time.
In 1954, the Bureau of Reclamation included a drain in its
feasibility report of the San Luis Unit.  In 1957, the
California Department of Water Resources initiated an
investigation to assess the extent of salinity and high
ground water problems and to develop plans for drainage
and export facilities.  The Burns-Porter Act, in I960,
authorized San Joaquin Valley drainage facilities as part
of the State Water Facilities.

     The authorizing legislation for the San Luis Unit of
the Bureau of Reclamation's Central Valley Project, Public
Law 86-488, passed in June 1960, included drainage facilities
to serve project lands.  This Act required that the Secretary
of Interior either provide for constructing the San Luis
Drain to the Delta or receive satisfactory assurance that
the State of California would provide a master drain for the
San Joaquin Valley that would adequately serve the San Luis
Unit.

     Investigations by the Bureau of Reclamation and the
Department of Water Resources revealed that serious drainage
problems already exist and that areas requiring subsurface
drainage would probably exceed 1,000,000 acres by the year
2020.  Disposal of the drainage into the Sacramento-San Joaquin
Delta near Antioch, California, was found to be the least
costly alternative plan.

Preliminary data indicated the drainage water would be
relatively high in nitrogen.  The then Federal Water Quality

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Administration conducted a study to determine the effect of
discharging such drainage water on the quality of water in
the San Francisco Bay and Delta.  Upon completion of this
study in 1967, the Administration's report concluded that
the nitrogen content of untreated drainage waters could have
significant adverse effects upon the fish and recreation
values of the receiving waters.  The report recommended a
three-year research program to establish the economic
feasibility of nitrate-nitrogen removal.


     As a consequence, the three agencies formed the
Interagency Agricultural Waste Water Study Group and developed
a three-year cooperative research program which assigned
specific areas of responsibility to each of the agencies.
The scope of the investigation included an inventory of
nitrogen conditions in the potential drainage areas, possible
control of nitrates at the source, prediction of drainage
quality, changes in nitrogen in transit, and methods of
nitrogen removal from drain waters including biological-
chemical processes and desalination.
                          VI

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                   TABLE OF CONTENTS
ABSTRACT	     ill
BACKGROUND	     v
CHAPTER I - CONCLUSIONS	     1
CHAPTER II - INTRODUCTION	     3
CHAPTER III - METHODS AND  PROCEDURES	     5

  Water Collection	     5
  Experimental Procedure	     5
  Experimental Design	     8

CHAPTER IV - RESULTS	     9

  Chemical Analyses	     9

    San Joaquin River Water	     9
    Agricultural  Drain  Waste Water	     9

  Algal Bioassay  Responses	    13

    Extracted Fluorescence Experiments	    13
    Direct Fluorescence Experiments	    17
    San Joaquin River Nitrate and Chlorophyll	    25

CHAPTER V - DISCUSSION	    29
APPENDIX A:  RESULTS  OF THE INDIVIDUAL EXPERIMENTS   33
APPENDIX B:  ALGAL SPECIES AND NUMBERS	    43
APPENDIX C:  CELL COUNT AND CHLOROPHYLL
             CONCENTRATION	    45
APPENDIX D:  BIOASSAY NITROGEN RESPONSES	    47
ACKNOWLEDGEMENTS	    53
LIST  OF REFERENCES	    54
PUBLICATIONS	    55
                             VI1

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                         FIGURES
Number                                               Page

 1     Seasonal Chlorophyll Variations of San Joaquin
       River Water	  4
 2     Flasks in Culture Box.
 3     Growth Responses Measured by Culture
       Chlorophyll a	  19

Al     Algal Growth  Response of San Joaquin River
       Water and Mixed Samples	  41

A2     Algal Growth  Response of San Joaquin River
       Water with Added Agricultural Waste Water	  42

Cl     Cell Count vs Chlorophyll a_ Concentrations,
       Initial  and Terminal Measurements	  46

Dl     Bioassay Decrease  in Nitrate-Nitrogen  as  a
       Function of Chlorophyll Increases	  50

D2     Bioassay Chlorophyll Maximum as  a  Function
       of Total Inorganic Nitrogen (NO3-N plus
       N02-N)	  52
                             viii

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                          TABLES
Number                                                Page

 1     Chemical Analysis of San Joaquin River Water
       Collected in the Vicinity of Antioch ............  10

 2     Chemical Analyses of Agricultural Tile Drain
       Waste Water Utilized in the Nutrient
       Re-addition Experiments .........................  11

 3     Chemical Analysis of Agricultural Tile Drain
       Waste Water Utilized in the Comparative
       Nitrogen Removal Process Experiments ............  12

 4     Maximum Extracted Fluorescence of Agricultural
       Tile Drain Waste Water Bioassays Before and
       After Inorganic Phosphorus Additions ............  14

 5     Maximum Extracted Fluorescence of Agricultural
       Tile Drain Waste Water Bioassays with Nitrogen
       Additions .......................................  16

 6     Normalized Algal Bioassay Response of Treated
       and Untreated Agricultural Waste Water ..........  21

 7     Normalized Algal Bioassay Response of the
       Treated Agricultural Waste Water ................  23

 8     Normalized Summary of Growth Rate Responses,
                                                         24
 9     Bioassay Comparison of the Nutrient Removal
       Efficiency of Algal Pond and Bacterial Filter
       Systems ..................... , ...................  26

10     Seasonal Chlorophyll and Nitrate Variations in
       the San Joaquin River at Antioch Bridge .........  27

Al     Bioassay Chlorophyll Increases ..................  34

A2     Bioassay Chlorophyll Maxima .....................  37

Bl     The Proportional Counts of Algae Found
       Initially and at the Termination of the
       Bioassay Experiments ............................  44

Dl     Nitrate Concentrations in Test Waters Before
       and After Bioassay ..............................  48
                              ix.

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                    CHAPTER I - CONCLUSIONS
1.   San Joaquin River algal bioassay responses were related
    to the nitrate and algal content.  Summer and early fall
    bioassay samples had a high initial algal growth and
    a low nitrate concentration; little growth occurred in
    the laboratory.

    Winter and spring samples had a low initial algal
    population and a high nitrate concentration; large algal
    growth occurred during the course of the bioassays.

2.   Mixtures of San Joaquin River water and untreated
    agricultural drainage consistently stimulated algal
    growth, regardless of the season.

3.   The bioassay responses of mixtures of treated agricultural
    drainage with San Joaquin River water showed that nitrogen
    removal from agricultural drainage is definitely effect-
    ive in reducing biostimulation.

4.   Effluents from algal pond and anaerobic denitrification
    treatment systems produced statistically equal bioassay
    responses when compared on the basis of the nitrate and
    nitrite remaining in the effluent.  In general, algal
    bioassay responses varied inversely with the efficiency
    of nitrogen removal.  High terminal chlorophyll bioassay
    concentrations were associated with inefficient removal
    of nitrogen from the waste water.

5.   When inorganic nitrogen was below 2 mg/1 in treated
    drainage, algal growth responses of the mixture of
    treated drainage and San Joaquin River water produced
    responses similar to those of San Joaquin River controls.

6.   Algal growth responses observed in untreated agricultural
    waste water were reproduced when nitrate-nitrogen was
    re-added to the treated waste waters bringing the levels
    back to those found in the original waste water.
                            -1-

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                   CHAPTER II - INTRODUCTION
     Algal bioassay methods were used to evaluate nutrient
removal processes under study at the Interagency Agricultural
Waste Water Treatment Center  (IAWWTC), Firebaugh, California.
This study was conducted as part of a project to reduce algal
growth when agricultural waste water is mixed with San Joaquin
Delta v/ater.  Upon adding waste water or nitrogen and phospho-
rus to San Joaquin River water, analyses were made comparing
bioassay algal growth responses in treated and untreated
agricultural waste water.  The experimental growth responses
were similar to algal growth observed in the San Joaquin
River  (Figure 1).  The proposed agricultural tile drain will
empty into the San Joaquin River near Antioch,  The first
part of this report is devoted to the chemical analyses of
both the San Joaquin River water collected near Antioch and
the treated and untreated agricultural waste water.  This
section is followed by the algal bioassay responses to
nutrient chemical addition and to agricultural waste water.

     Much of the data has been normalized to overcome the
variable growth response of the control medium, the
San Joaquin River water.  Normalization consisted of dividing
all of the data from each bioassay by the bioassay value
for the San Joaquin River water for that date.  In this way,
results can be logically compared or summarized when the
responses for the basic medium are equalized for all bioassays
This normalization technique was applied to the waste water
treated chlorophyll growth responses and growth rates
(ufc, day  ) and to the algal pond and bacterial filter
bioassay efficiency comparisons.  The final part of the
main report relates seasonal variations in chlorophyll
and nitrate concentrations in the San Joaquin River water.

     The detailed analyses of all the experiments utilizing
bacterial filter, algal pond, and untreated agricultural
waste water are given in the appendices along with sections
reporting bioassay algal species changes, and bioassay
correlations between cell count/ chlorophyll, and nitrate
concentrations.
                                -3-

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  40
  30
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                                         '8
                      »
          Samples  taken in 1966

             atAntioch Bridge


          1 - 1 - 1 - 1 -
       JFMAMJJASO


                            	MONTHS	



    Figure (.Seasonal  Chlorophyll  Variations in  San Joaquin  River

                               Water
                AGRICULTURAL WASTE WATER STUDIES

                  SAN JOAQUIN VALLEYV CALIFORNIA



                   ENVIRONMENTAL PROTECTION AGENCY

                              REGION  IX

                       SAN  FRANCISCO,CALIFORNIA
                           -4-

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             CHAPTER III - METHODS AND PROCEDURES
WATER COLLECTION

     The agricultural waste water was collected  in
polyethylene containers on the afternoon prior to the
beginning of each bioassay, while the San Joaquin River
water was collected early the next day.  Personnel  from
the Interagency Agricultural Waste Water Treatment  Center
stopped at Antioch, California to collect San Joaquin River
water on their way to the EPA laboratory at Alameda,
California.  Usually the Antioch samples were collected
approximately four hours before the bioassay was started.
The algal pond waste water was prefiltered through  GFA
glass filter pads at Firebaugh immediately after collection,
reducing algal concentration to that found in the bacterial
filter and anaerobic pond waters.  This filtration  also
corresponded to the algal harvesting from the pond  waters
which is an integral part of the algal stripping process.
EXPERIMENTAL PROCEDURE

     Untreated agricultural drainage, algal pond, anaerobic
pond, or bacterial filter waters were added separately to
San Joaquin River water in dilutions ranging from 1 percent to
50 percent of the total sample volume.  These percentage addi-
tions were chosen because they approximated the probable future
concentrations of waste water in the San Joaquin River.
Undiluted waste water was used in some initial experiments to
test for specific nutrient deficiencies in these waters.  Each
sample for bioassay was prepared in triplicate.  A sample
of 300 ml was placed in a 500 ml Erlenmeyer flask which was
then plugged with a foam rubber stopper.  An aliquot was
saved for nitrate analysis.

     In another series of bioassays, inorganic nutrients
(KN03 or KH2P04) were added to the San Joaquin River water
to determine the results of nitrate or phosphate, alone or
combined, on the algal growth.  Before any assay was
started, chlorophyll a concentrations were measured.

     Incubation of the cultures was in a 20° C + 1° C
constant temperature walk-in refrigerator.  The flasks were
put on plate glass shelves with cool-white fluorescent lights
mounted one inch below each shelf (Figure 2).  Approximately
600 ft-candles of light reached the bottom of the flasks.
Flasks were incubated from 5 to 15 days until algae reached
maximum growth.   A blower and  a system or ducts forced
                           -5-

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Figure 2.  Flasks in Culture  Box
                  -6-

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air constantly through the space between the fluorescent
tubes and the plate glass and through the area above the
glass shelves.   (Without this forced-air system, the
fluorescent tubes cause a slight warming of the plate
glass shelves.)  Since the blower was running continuously,
the air temperature was kept nearly constant.

     In the first series of bioassays (12/13/68-4/4/69),
algal growth was monitored by extracted chlorophyll
fluorescence.  The cultures were subsampled on the day they
were set up and on alternate days thereafter.  Fifty-milliliter
subsamples were taken from each culture.  Following filtration
through GFC glass fiber pads, they were macerated in a cell
homogenizer, and chlorophyll was extracted by 90 percent
acetone.  The method is described in detail by Bain (1969).

     Algal growth in the second series of bioassays (6/18/69 -
11/17/69) was measured daily, except for weekends, by direct
fluorescence of a small subsample (  10 ml) taken from each
flask being incubated.  The foam rubber stoppers were
replaced by ones of hard rubber and the flasks were shaken.
Readings were taken on a Turner Fluorometer equipped with
a Corning CS 5-60 primary filter and a Corning CS 2-60
secondary filter.  Normally the 30x scale was used.  The
subsamples were discarded after fluorescence readings since
the flasks contained enough volume for many readings with-
out appreciable change in the surface-to-volume ratio.  The
readings were taken until algal fluorescence had reached a
plateau or had started to decline, which usually'occurred in
5 to 7 days.  At the termination of the experiment, the water
from the replicate flasks was combined and used for NC>3
determinations.

     Direct fluorescence readings were converted to chlorophyll
concentrations for each sample.  These readings were
followed by filtration and 90 percent-acetone extraction of larger
volumes of water from 1-liter flasks containing the same
media as the smaller flasks.  Extracted samples were measured
for optical densities at specific wavelengths following
chlorophyll determination procedures recommended by Strickland
and Parsons  (1965).  Analyses for nitrate - nitrogen,
nitrite-nitrogen, organic nitrogen, ammonia, inorganic
phosphorus, and total phosphorus followed the procedures
listed in the FWPCA Methods for Chemical Analyses of Water
and Wastes,  (1969)".
                                 -7-

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EXPERIMENTAL DESIGN

     The experimental design was factorial, permitting an
analysis of variance of the data followed by a multiple
range test, if the variance was significant.  The multiple
range test used was that of Student-Neuman-Keuls  (Steel and
Torrie, 1960).  Three growth parameters were derived from
the flask culture growth curves:.   (1) increase in chlorophyll
concentration, (2) maximum chlorophyll concentration, and
(3) maximum observed growth rate
                          -8-

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                        CHAPTER IV - RESULTS
CHEMICAL ANALYSES

San Joaquin River Water

     Chemical analyses for the San Joaquin River water used
as the basic medium in the algal bioassays are given in
Table 1.  The collection dates correspond to bioassay
experiment dates and cover a period of one year, from
December 1968 through November 1969.

     Changes in most of the measured parameters appear
moderate.  Total P has a value of 0.65 mg/1 for June, but
all other samples have concentrations between 0.07-0.16 mg
P/l.  Changes in P04-P and organic N concentrations are
small.  The highest total inorganic N values  (NH3-N plus
NC-3-N) occur in the January and February samples, 1.10 and
0.95 mg N/l respectively, with low values of slightly
more than 0.10 mg N/l present during the summer months.
The highest total inorganic N to P04-P ratio was approximately
11:1 in the January 4 sample; the lowest was 2:1 in July
and August.

Agricultural Drain Waste Water

     The chemical analyses of agricultural waste water
samples added to San Joaquin River water are listed in
Tables 2 and 3.  The determinations include NO3-N, NC-2-N,
PC-4-P, and soluble organic nitrogen.  Any NH3-N in the
samples was reported as part of the soluble organic nitrogen.
On the basis of many determinations, the NH3-N level is
0.10 mg N/l or less in the algal pond and untreated agricultural
tile drain waste water and less than 1.5 mg N/l in the bacterial
filter water (Sword, 1970).

     Untreated agricultural tile drainage is high in
NO3-N concentrations, 9.2 mg N/l to 22.5 mg N/l.  Nitrate-
nitrogen concentrations also vary in the treated agricultural
waste water because samples showed different levels of
nitrogen removal.  The highest effluent nitrate value was
5.1 mg N/l produced by bacterial filter No. 19 on August 18,
1969.  The measurement 9.2 mg NC-3-N in the original untreated
waste water indicated that less than half of the nitrate
was removed.  Some of the treated samples had trace
concentrations of nitrate  {<0.10 mg N/l) showing a high
efficiency of NC-3-N removal.
                                 -9-

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TABLE 1. - CHEMICAL ANALYSIS OF SAN JOAQUIN RIVER  WATER COLLECTED  IN THE VICINITY OF ANTIOCH
    1968                                            1969
Chemical
Alkalinity
CaC05,tng/l
P04-P,mgP/l
Total P,ragP/l
Org N,mg N/l
NH5,rog N/l
N03,mg N/l
Total inorganic
nitrogen, mg N/l
12/13 1/4 1/28 2/14 5/10 4/4 6/18 7/24 8/18 9/7 9/29 10/20 11/17
68 - 59 - 46 54 68 118 68 69 78
0.07 0.07 .02 0.08 0.07 0.07 0.07X 0.07 0.07 0.06 0.4 O.OS 0.06
0.07 - 0.09 0.11 0.08 - 0.65 0.12 0.13 0.10 0.12 0.14 0.16
0.54 0.46 0.47 - 0.37 0.51 0.42 0.26 0.35 0.41 0.32
0.15 0.17 0.28 0.08 0.15 0.12 0.09 0.10 <0.08 0.09 CO. 08 0.08 0.09
0.43 0.61 0.82 0.95 0.53 0.22 0.18 0.04 0.05 0.08 - 0.09 0.22
0.58 0.78 1.10 1.13 0.68 0.34 0.27 0.14 0.13 0.17 (0.08) 0.17 0.31
i
H
O
1

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        TABLE 2. - CHEMICAL ANALYSES OF AGRICULTURAL TILE DRAIN WASTE WATER
                 UTILIZED IN THE NUTRIENT RE-ADDITION EXPERIMENTS
Chemical
Date Anal yses
mc/1

12/13/69 NOg-N
Sol .Org-N

N05-N
N02-N
1/4/69 Sol. Org-N
P04-P
Total -P


N03-N
1/28/69 NOg-N
Sol. Org-N
P04-P

NOs-N
2/14/69 IK02-N
Sol .Org-N
P04-P

NOS-N
3/10/69 NOg-N
Sol .Org-N
P04-P
Untreated
Agricultural
Waste Water

20.3
^. 0.001
0.40

17.9
< 0.001
0.27
0.05
-£0.02


17.0
0.004
0.39
<0.02

19.6
0.005
0.36
0.10

18.6
0.01
0.25
0.04
Treated Agri

cultural Waste Water3

Bacterial Filter
No. 14
1.3
1.0
1.94
No. 11 No.
0.2 0.
-£0.001 0.
0.39 0.
No. 19
3.0
3.7
0.48
13 No. 14
6 0.4
01 0.04
70 0.89
-£0.02 -^0.02 <0.02
^0.02 <0.
02 <0.02
Algal "Pond Bacterial Filter

1.2
5.4
1.25
0.05
No.
1.1
14 No. 19
1.2
2.22 3.7
0.57 0.59
-£0.02 -3D. 02
No. 19 No. 20
3.2 0.36
2.80 1.44
1.60 1.48
0.03 0.12

8.0
0.04
1.17
0.02





No. 19 No. 20
2.7 <0.10
2.26 0.12
0.60 0.66
-£0.02 <0.02
a.  Identification numbers refer to pilot test unit  effluent   not  to  bioassay
    samples.  Some of these units were not identified by  numbers.
                                       -11-

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 TABLE 5.  - CHEMICAL  ANALYSIS OF AGRICULTURAL TILE DRAIN WASTE WATER UTILIZED  IN
               THE COMPARATIVE NITROGEN  REMOVAL PROCESS EXPERIMENTS
Chemical Untreated Treated Agricultural Waste Water
Date Analyses Agricultural
_ (me/1) Waste Water Aleal Pond Bacterial Filter
NQ5-N
NOg-N
6/18/69 Soi.Org-N
POi-P
NQ3-N
7/24/69 NOg-N
Sol.Org-N
POi-P
NOs-N
8/18/69 NOg-N
Sol.Org-N
P04-P
NOs-N
W69 Sol**g:!j
P04-N
NOs-N
9/29/69 NOg-N
Sol.Org-N
POA-P
NOs-N
10/20/69 NOg-N
Sol.Org-N
POd-P
NOs-N
11/17/69 NOg-N
Sol.Org-N
POd-P
11.0
0.002
0.50
0.16
10.0
0.006
0.25
0.19
9.2
0.02
0.04
0.12
11.5
0.01
0.04
0.07
16.5
0.01
0.4
0.08
16.5
0.01
0.4
0.2
22.5
0.004
0.4
0.15
-£0.10

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     Nitrite concentrations were unusually high in some
bacterial filter and algal pond samples taken before
June 18, 1969.  For example, all of the bacterial filter
and algal pond samples collected on January 28, 1968,
contained more than 2 mg NO^-N/l and little more than 1 mg
N03-N/1.  The agricultural tile drain waste water from which
the algal pond and bacterial filter samples were derived
contained 17.0 mg N03-N/1 but only 0.004 mg N02-N/1 indicating
that some of the effluent nitrite came from the biological
reduction of nitrate.

     Inorganic PO.-P concentrations were usually less than
0.2 mg P/l in the untreated agricultural waste waters and
occasionally below the sensitivity of the analytical method
used.
ALGAL BIOASSAY RESPONSES

Extracted Fluorescence Experiments

     a)   Inorganic Phosphorus Additions:

         The bioassay response of raw and treated agricultural
         tile.drain waste water, with and without phosphorus
         additions, is shown in Table 4.  These extracted
         fluorescence values are each the mean of two
         replicate samples.  Since these data are reported
         in extracted fluorescence units it should be noted
         that algal cellular chlorophyll concentration and
         extracted fluorescence are highly correlated
         (correlation coefficient = 0.99).  The fluorescence
         value  in units is approximately 0.5 times the
         concentration of chlorophyll a in micrograras per
         liter.

         The two samples of raw agricultural waste water
         (12/13/68 and 1/4/69)  had a marked response to
         inorganic phosphorus additions.  Extracted
         fluorescence of the 12/13/68 sample increased from
         220 to 1660 fluorescence units and from 330 to 750
         fluorescence units in the 1/4/69 sample.

         The bacterial filter effluent exhibited a lower
         response than the raw waste water.  The 1/4/69
         samples for bacterial filter Nos. 11, 13, and 14
         showed almost no growth relative to that of the
         original waste water (1, 2, and 1 extracted
         fluorescence units, respectively).
                              -13-

-------
               TABLE 4.  - MAXIMUM EXTRACTED FLOURESCENCE OF AGRICULTURAL TILE DRAIN WASTE
                         WATER BIQASSAYS BEFORE AND AFTER INORGANIC PHOSPHORUS ADDITIONS
Sample
100% Concentration
Untreated
Agricultural
Waste Water
Bacterial Filter
Effluent
No. 11
No. 13
No. 14
No. 14
Mb. 19
Date
12-13-68
1-4-69

1-4-69
1-4-69
12-13-68
1-4-69
12-13-68
Total
Soluble
mj N/l
20.70
18.17

0.59
1.31
4.24
1.33
7.18
Before P Addition
xng P04-P/1
0.2
<0.05

<0.02
<0.02
<0.02
<0.02
<0.02
Extracted
Flourescence
220
330

1
2
200
1
56
After P Addition
itg P04-P/1
1.2
<1.05*

<0.22 +
<0.22
<0.22
<0.22
<0.22
Esctracted
Flourescence
1660
750

1
10
232
33
600
*  At least 1.00 ng PO.-P/l
+  At least 0.20 mg PO4-P/1

-------
    Additions of phosphorus to the bacterial filter
    effluent brought dramatic algal response only in
    the No. 19 sample of 12/13/68  (Table 4), where
    fluorescence increased from 56 to 600 units.  This
    filter had the highest remaining concentration of
    total soluble nitrogen (7.18 mg N/l) of any listed
    for 12/13/68 or 1/4/69.  Phosphorous limited samples
    (N:P>300) gave the highest growth responses upon
    the additions of phosphorus.

b)  Nitrate-Nitrogen Additions:

    Nitrate was added to effluent from algal ponds and
    bacterial filters to determine whether nitrate
    re-addition would increase algal growth to values
    comparable to those of untreated waste water (Table 5).

    For all of the responses listed, algal growth in
    the treated controls was less than that in raw
    waste water.  The addition of nitrate-nitrogen to
    the bacterial filter and algal pond samples usually
    increased algal chlorophyll fluorescence above the
    level found in the original bacterial filter and
    algal pond effluents.  For example, the 10 percent algal
    pond sample of 1/28/69 had a fluorescent bioassay
    response of 262 units.  Nitrogen additions increased
    the fluorescence to 370 and 344 units but did not
    increase fluorescent values to those of the original
    untreated waste water.  The sample containing 10 per-
    cent untreated agricultural waste water had a direct
    fluorescence of 392 units.

    Based on the ratios of nitrogen-to-phosphorus, it
    was noted that nitrogen did not appear to be lacking
    in the cultures.  Phosphorous values include only
    inorganic phosphorous values, and not organic
    phosphorus which could have been used by the algae.
    Increases in fluorescence did not occur after each
    nitrogen re-addition to the treated waste waters.
    In the 100 percent  bacterial filter samples, as shown by
    the bacterial filter No.  19 samples of 3/10/69,
    algal bioassay responses, both with and without
    nitrogen additions, were very low and almost equal
    to each other.  One probable reason for the lack of
    response was the few algae contained in the 100 percent
    bacterial filter effluent.
                              -15-

-------
                                             TABIJS s. - MAXIMUM Ejowcam FLOORESCBCE CF AGRJCUIOTRAL THE DRAIN
                                                               WATER BICASSAYS WITH NITROGEN ADDITICNS

SAMPLE AND DATE

1-28-69
Agricultural Waste Water
Treated AP (e)
Agricultural. BF(f)-14
Waste Water BF-19
2-14^69
Agricultural Waste Water
Treated AP-19
Agricultural AP-19
Haste Water AP-20
AP-20
3-10-69
Agricultural Waste Water
AP
Treated BF-19
Agricultural BF-19
Vfeste Mater BF-20
BF-20
% of (x)
DPAIN


10%
10%
10%
10%
10%
10%
30%
10%
30%
10%
100%
10%
30%
100%
30%
100%

P04-P/1


<0.02
0.02
<0.02
0.02
0.08
0.08
0.06
0.08
0.09
0.07
0.04
0.06
<0.06
0.02
<0.06
<0.02
Before NC^-N Addition

Total Solids
ng N/l

2.73
1.78
1.38
1.54
3.01
1.78
3.07
1.34
1.78
2.50
18.86
1.54
2.14
5.56
0.74
<0.88
Extracted
Flourescence

392
262
341
346
225
162
115
154
95
400
114
300
260
8
200
2
After 1/2 NO3-N Addition (d)

Total Solids
rag N/l

— —
10.28
9.88
10.04
__
11.58
12.87
11.14
11.58
,
	
10.84
11.44
14.86
10.06
10.18
Extracted
Flourescence

—
370
395
356
— — _
200
115
199
185
	
	
355
390
4
230
30
After 1 NC^-N Addition

Total Solids
mg N/l

— —
18.78
18.38
18.54
•.„•
21.38
22.67
20.94
21.38
__
___
20.14
20.74
24.16
19.34
19.48
Extracted
Flourescence

___
344
380
392
__
180
115
215
245
-__
	
440
380
7
205
1
on
 I
     **' Percentage of drain waste water of total sample equals drain water plus San Joaquin River water.
     }d;The nitrogen was re-added to the treated waste water at concentrations equal to or half of that found in the tile drainage.
     (e'AP = Algal Pond
          = Bacterial Filter

-------
Direct Fluorescence Experiments

     a)  Cell Mass Changes;

         The measured experimental cell mass changes were
         the increases in chlorophyll and maximum chlorophyll
         concentrations.  These were derived by conversion
         of direct fluorescence to chlorophyll a concentra-
         tion for the three replicates per sampTe type.
         Both variables were used because the samples for
         comparison did not initially contain equal con-
         centrations of algae; the amount depended mainly on
         the dilution of the San Joaquin River water.  The
         test samples contained some algae, but most of the
         algae were present in the San Joaquin River water.

         The chemical analyses of the waste water used in the
         direct fluorescence experiments are given in
         Table 3.  The waste water was added to San Joaquin
         River water on each experiment date.  The analyses
         for the river water areishown in Table 1.

         The complete experimental results from the algal
         growth comparisons in Appendix A show increases
         in chlorophyll and maximum chlorophyll concentrations.

     b}  Summary of Cell Mass Changes;

         In five out of seven experiments, the untreated
         agricultural tile drain waste water consistently
         gave growth responses above those obtained for the
         San Joaquin River control (0 percent concentration).
         Usually the 10 percent and 20 percent concentrations
         gave equal responses.  However, the 1 percent addition
         of treated agricultural waste water rarely gave growth
         responses above those of the San Joaquin River control,

         The results of the 10 percent and 20 percent bacterial
         filter and algal pond additions can be understood by
         relating the growth response to effluent nitrogen.
         For example, in samples taken on August 18, 1969,
         (Table 3), inorganic nitrogen in algal ponds
         Nos. 14 and 15 was 3.61 and 1.78 mg N/l, respectively,
         and 7.44 mg N/l in bacterial filter No. 19.  In
         contrast, bacterial filter No. 11 had only 0.50 mg
         N/l of inorganic nitrogen.  None of the samples
         containing filter No. 11 effluent exceededjthe
         control in algal response, whereas all of the other
                             -17-

-------
    samples gave growth  responses  above  the controls
    at  one or both  of  the  10 percent  or  20 percent
    additions  (Table Al).

    A few of the responses could not  be  explained on
    the basis of the nitrogen  content of the  treated
    effluents.  Both of  the treated agricultural
    waste water samples  of July 24, 1969 had  a  low
    total inorganic nitrogen content  (Table 3).   The
    algal pond effluent  contained  0.17 mg N/l while
    the bacterial  filter contained 0.10  mg N/l.   Yet
    algal growth responses of  the  20  percent  bacterial
    filter samples  exceeded responses from the  algal  pond
    (Appendix A)« Another  example  of  responses  not
    clarified by the inorganic nitrogen  concentrations
    is  shown by effluent samples  from filters Nos.  10
    and 15 of October  20,  1969.   Sample  No.  15  had  a
    total inorganic nitrogen content  of  1.45  mg N/l,
    while sample No. 10  totaled <0.15 mg N/l  (Table 3).
    Bioassay chlorophyll increases for the two
    bacterial filters  effluents,  however, were  equal
    in  the 10 percent  additions.   Sample No.  10 exceeded
    No. 15 in the  20 percent additions by 20.4  to 12.6  mg
    chlorophyll a_ per  liter (chl  a/1) .

    Bacterial filter effluent  contained  up to 1.5 mg
    NH3-N/1 during  the summer  months  but normally
    contained  less  than  1.0 mg KH3-N  during  other
    periods  (Sword, 1970). Since ammonia was analyzed
    as  part of the  soluble organic nitrogen,  it was not
    included in  the total  inorganic nitrogen  values.

    In Figure  3,  contrary  to  expectation, the bacterial
    filter growth  response drops  below that  of the
    algal pond effluent  response.   For the  same
    concentrations, the  algal  pond and agricultural
    tile drain waste water flasks show growth increasing
    monatonically  with waste  water concentration.

c)  Normalized Bioassay Chlorophyll Cell Mass Responses:

    The variable  algal growth in  San  Joaquin River
    water  (0%, without addition  of waste water) indicated
    that in  order to  summarize the results,  normalization
    of the  data  was more logical  than utilization of  the
    absolute  response  values.  Normalization was
    accomplished by dividing  the  results for each
    experiment by maximum bioassay growth response of
    the control  water used.  Furthermore, bacterial
    filter and algal  pond values  were not separated
    but placed into four categories based on their
    original  N03-N plus N02-N concentrations:

-------
   50
    40
ID
CL
E

2   30
o>
o
    20
X
rx
o

-------
0.13-0.17, 0.46-1.00, 1.45-1.86, and 2.94-7.44 mg
N/l.  These categories were chosen arbitrarily
for statistical evaluation.  The untreated
agricultural tile drain waste water responses were
reported together.  Total inorganic nitrogen in
the tile drain ranged from 9.22 to 22.50 mg N/l
(Table 3).  San Joaquin River water contained
from 0.13 to 0.27 mg N/l  (Table 1).  For comparison
purposes, the normalized responses for the San Joaquin
River water are 1.00.  Agricultural tile drain
waste water comparisons are included in Table 6 but
not in Table 7.  The ranges and means of inorganic
nitrogen in the original samples are listed under
each category.

It is best to utilize the mean value for the
inorganic nitrogen in the river water and the waste
water to calculate the mixed sample inorganic
nitrogen.  Using this procedure, a sample made by
adding 10 percent treated waste water containing
2.94-7.44 mg N/l to San Joaquin River water would
have a final inorganic nitrogen concentration of
approximately 0.59 mg N/l.  Utilizing the mean
values of the San Joaquin River water and of the
waste waters, the mean inorganic organic nitrogen
value of treated waste water is 4.12 mg N/l.  Therefore,
100 ml of this water would contain 0.412 mg N,
and 900 ml of San Joaquin River water  (0.20 mg
N/l, mean value) would contain 0.18 mg N.  Adding
0.412 to 0.18 gives a concentration of 0.59 mg N/l
for the mixture.  Other samples can be calculated
in the same manner, i.e. a 20 percent addition of the
same waste water to San Joaquin River water would
contain 0.98 mg N/l.

Another feature of Table 6 is the bracketing and
underlining of values which do not differ signifi-
cantly from each other at the 95 percent confidence
level.  For the 1 percent additions, therefore, the
values 1.00 through 3.20 are connected by underlining.
This means that these responses are statistically
equal, regardless of their absolute value.  The
number 6.63 is significantly higher in value than
the others.

Table 6 shows that the 10% and 20% additions of
the treated agricultural waste water containing
2.94 - 7.44 mg N/l were the only samples exceeding
San Joaquin River water in algal growth.
                    -20-

-------
                 TABLE 6. - NORMALIZED AIGAL BIOASSAY RESPONSE^) CF TREATED AND UNTREATED AGRICULTURAL WASTE WATERln)
                                               mg N/l of NOa-N and NO?-N in Sample;  Range and Mean
Normalized
Response (g)
Percent
Addition
1%
10%
20%
Combined 1%, 10%,
and 20% Additions
San Joaquin River Treated Agricultural Waste Water
Agricultural Waste Water
0.13-0.27 0.10-0.17 0.46-1.00 1.45-1.86 2.94-7.44 9.22-22.50
0.20 0.13 0.66 1.65 4.12 13.86
, 1.00 1.81 1.04 1.29 3.20 , 6.63
,1.00 2.70 1.88 3.91, 13.31 27.89
,1.00 5.94 3.94 9.36, ,20.98 29.55,
, 1.00 3.34 2.29 4.85 , 12.50 20.95

(g) Normalized Response = /«a*mun chlorophyll increases in treated water	
                 e^      VMaxomum chlorophyll increases in San Joaquin River water

(h) Samples connected by underlining do not differ in the 95% confidence level

-------
   Untreated  tile  drain waste water  gave growth. re~
   sponses  above the  undiluted  San Joaquin River
   water  at all percentage  additions.   Growth
   response was above the treated tile drainage
   for  all  additions  except the 20 percent addition,
   where  response  equaled that  of the  2.94-7.44 mg/1
   nitrogen category.

   Table  7  analyzes the data from the  Table 6 column
    "Treated Agricultural Waste  Water."  For example,
   the  1%,  10%, and 20% data in the  0.10-0.17 mg/1
   category in Table  6,  (1.81,  2.70, and 5.94)  are
   located  on the  first horizontal data line in
   Table  7.  The number 1.00 is again  included in
   the  analyses, because of the normalization technique.
   When compared to Table 6, a  finer distinction in
   the  responses is possible in Table  7 because of the
   smaller  variances  associated with the treated
   waste  water as  compared  to the untreated tile
   drainage.

    Response differences at  the  1% and 10% additions
    in Table 7 are  similar to those  found in Table 6.
    For  the  20% additions  of the treated waste water in
    all  nitrogen categories, however, bioassay
    chlorophyll increases  are greater than either the
    1% additions or the San  Joaquin  River controls.
    This response  is  unexpected  since a 20% addition
    of treated waste water  (containing 0.46-1.00 mg
    N/l) to  San Joaquin River water  would increase
    sample inorganic  nitrogen concentration to an
    average  of 0.36 mg N/l.   Addition of the 0.10-0.17
    mg N/l water would actually  dilute the inorganic
    nitrogen in the San Joaquin  River water.

d)  Normalized Growth Rates, MK:
                             i *"'
    The normalized  maximum growth rate data for two
    experiments are listed in Table  8.   The growth rate
    values are based on two sample readings of the
    sample fluorescences per day, rather than the usual
    one-a-day recordings.   Thus, they gave a better
    estimate of the maximum growth rates in this experi-
    mental series.

    Growth responses for the untreated agricultural
    tile drain waste water were the  only ones exceeding
    the San Joaquin River control (Table 8).  Results
    of all  samples containing untreated  agricultural
    waste water (1%, 5%, 10%, and 20%) fell at  the  upper
    end of  the multiple range values,  although  they
    did not significantly exceed  all percentage  addition


                              -22-

-------
                       TABLE 7. - NORMALIZED ALGAL BIOASSAY RESPONSE^
                         OF THE TREATED AGRICULTURAL WASTE
to
LO
1
NORMALIZED
RESPONSE (8>
NO3-N & NO2-N
in Sample
mg N/l
rH
(fl
ij 0.10-0.17
-p
rH
3
0 M
M-P 0.46-1.00
«
T3 
-------
i
N>
                       TABLE 8.  - NORMALIZED SUMMARY OF GROWTH RATE RESPONSES, g(fb)  J


                                       AP-10
           SJR-0                       BF-10
           AP-20     BF-1      AP-1      BF-5      BF-20      AP-5     TD-1     TD-5     TD-20     TD-10
1.00 1.03 1.04 1.08 1.11
[
[
[
1.12 1.28 1.29 1.33 1.36
1
1
1
        Code:  SJR - San Joaquin River
               AP  - Algal Pond
               BF  - Bacterial Filter
               TD  - Agricultural Tile Drain Waste Water


        j.   The data are from two experiments in which the test  waters were added  to  San Joaquin
            River water  to make 0, 1, 5,  10,  and 20% of total  volume.  Values  connected by under-
            lining do not differ at the 95$ confidence level.

-------
          values of the algal pond and bacterial filter
          water.  The line does not include the tile drain
          1% addition (1.28)  indicating that even this small
          percentage of untreated waste water gives higher
          algal growth rates  than the San Joaquin River
          control.   A substantial overlapping of the multiple
          range confidence lines occurs between the growth
          values of the various samples of the treated waste
          water and the untreated tile drainage.  Furthermore,
          the underlining for the normalized San Joaquin
          River water (1.00)  extends to the algal pond 5%
          addition  response (1.12),  signifying no statistical
          difference in the responses included by the under-
          lining (Table 8).

      e)   Comparative Algal Pond and Bacterial Filter Nutrient
          Removal Efficiencies";

          A comparison was made of the normalized growth
          responses of the two nitrogen removal systems.  The
          only  growth responses utilized had comparable
          concentrations of inorganic nitrogen and the same
          sample dilution for both systems.   In Table 9 the
          normalized response values 6.30 and 6.83 are
          averages  of the ratio of maximum chlorophyll increase
          in the treated waste water samples to that of the
          San Joaquin River water.   The response values
          6.30  and  6.83  are not statistically different
          (P< 0.01) .

San Joaquin River  Nitrate and Chlorophyll

      Table  10  presents  initial  nitrate concentrations and
seasonal  chlorophyll  concentrations both initially and
following  incubation.

      Initial field chlorophylls  for the San Joaquin River
samples were low from November  through May.   They reached a
peak  of 40.9 'y/g chl.  a/1  in  July.   Little growth  occurred during
incubation  in  the  laboratory  for  the samples  taken in the
summer; no  bioassay  response  was  recorded for the July sample
when  NC>3-N  concentrations were  the  lowest of  all  those listed
(0.04 mg NC>3-N/1) .   Highest chlorophyll increases  were
recorded  for the 1/28/69  and  2/14/69  samples.   These  contained
the highest  initial NO.,-N concentrations,  0.74  and 0.95 mg  N/l
respectively.   Most of  the bioassay maximum values for the
San Joaquin  River  water used  in controls  ranged from  30 to
45 jug chl.  a/1.  Maximum algal chlorophyll  values  appear
to b'e equal  for many of the sampling months,  regardless
of whether results were obtained from  field or  laboratory
data.
                              -25-

-------
 TABLE 9.  - BIOASSAY COMPARISON OF  THE  NUTRIENT REMOVAL k
 EFFICIENCY OF ALGAL POND AND BACTERIAL FILTER SYSTEMS


Algal Pond
Bacterial Filter
Number of
Observations
78
72
Normalized
Response i
Mean
6.30
6.83

F Value
0.12 l

Combined normalized data from inorganic nitrogen  content
groupings in which both removal  systems are represented.

The F value would have to be in  excess of 3.91  before  the
normalized response could be considered statistically  dif-
ferent  (F>0.05).
                              -26-

-------
 TABLE 10. - SEASONAL CHLOROPHYLL AND NITRATE VARIATIONS  IN THE
               SAN JOAQUIN RIVER AT ANTtOCH BRIDGE
                              Chi, a/1                NOs-N. rog N/l
 Date           Initial            Maximum               Initial
Sample          (Field)           (Bioassay)             (Field)
1968
11/14
12/13
1969
1/4
1/28
2/14
3/10
4/4
6/18
7/25
8/18
9/9
9/29
10/20
11/17

3.8
E.O

1.3
1.5
1.1
1.8
4.2
28.4
40.9
34.8
31.0
32.0
27.2
20.7

34.9
43.5

38.8
104.1
69.2
45.6
15.2
38.9
40.9
35.3
33.4
35.7
35.5
32.1

-
0.43

0.61
0.74
0.95
0.53
0.22
0.18
0.04
0.05
0.08
-
0.09
0.22
                                -27-

-------
                    CHAPTER V - DISCUSSION
     Bioassays were used to evaluate algal growth-promoting
properties of treated and untreated agricultural waste
water.  Samples were diluted by adding them to San Joaquin
River water at 1%, 10%, and 20% by volume.  The algal growth
bioassays were evaluated by measuring chlorophyll concen-
tration maxima and increases.  San Joaquin River water
collected in the summer contained a high initial
concentration of algae and so growth was minimal, thus termi-
nating with only a small increase in chlorophyll concentration,
Maximum chlorophyll concentration gives a measure of the
total algal mass which can be supported by the sample regard-
less of whether the conversion of nutrients has occurred in
the receiving waters before or after laboratory incubation.
Increase in chlorophyll indicates the amount of algal
mass formed during the bioassay, however, bioassays that
ended with highest increases in chlorophyll also had the
highest maximum concentrations of chlorophyll.  Bioassay
responses can be referred to without specifying either
chlorophyll maxima or increases.

     The agricultural waste water treated by either the
algal pond or anaerobic denitrification systems showed small
algal growth responses in the 1% dilution samples.  In 10% or
20% dilution samples treated waters promoted algal growth
when the removal systems were not performing well.  Treated
waste water with nitrate concentrations at the lower limit
of chemical detectability «0.10 mg N/l) showed growth
responses in the 10% and 20% dilution samples similar to
those of the San Joaquin River controls.

     Instances of substantial algal response occurred when
nitrate concentrations were low, indicating other forms of
available nitrogen: ammonia and nitrite-nitrogen were
present in some samples.  There were also examples of little
growth in spite of the high nitrate-nitrogen content of the
algal pond and bacterial filter samples, suggesting that
these systems might have removed other essential plant
nutrients or added toxic materials to their effluents.

     Samples from the nutrient removal systems almost always
gave growth responses below those of untreated agricultural
tile drain waste water during the same series of algal assays.
This did not necessarily hold true for 1% additions to
San Joaquin River water since such small growth differences
between samples are more difficult to detect than those at
the higher percentage additions.
                                   -29-

-------
     Normalization of the assay values shov:s algal response
of treated waste water above that of the San Joaquin River
control.  These data were also grouped to compare experimental
responses on the basis of the residual effluent N03~N and
NO2-N in the treated tile drain waste water.  Statistical
evaluation indicates that an effluent nitrogen concentration
of less than 2 mg N/l would stimulate algal growth response
equal to that of the San Joaquin River control.  However,
all of the 20% dilutions of treated waste water  (Table 7),
even those containing less than 2 mg N/l, gave responses
above the control.  The 20% dilutions of treated waste water
in the category containing 0.10-0.17 mg N/l were statistically
equivalent to the river water and hence should not increase
the bioassay response.  There could be several explanations
for the anomalous responses in Table 7:

     1.  The response might be due to limitation by nutrients
         other than nitrogen.

     2.  The ammonia in the bacterial denitrification system
         effluent, which was not differentiated  from the
         organic nitrogen by the analysis, probably con-
         tributed significantly to the algal growth.

     3.  Interpretation of algal growth as based solely upon
         waste water inorganic nitrogen content  might not
         take all factors into account since the algal pond
         system obviously would remove algal nutrients other
         than inorganic nitrogen.  Some of these nutrients
         might be potentially limiting in river/waste water
         mixtures.

     Normalization techniques were also used on  growth rate
data.   These normalized growth rates should not  be confused
with the absolute growth rates although the numbers obtained
are similar.  The maximum specific growth rate data  (u^)  is
significant in evaluating residence time effects on algal
growth.  Doubling of the growth rate may more  than double  the
algal mass.  This may be critical in areas of  short residence
time, such  as in  the transport canals and Carquinez Straits.
Observed growth rates during treated waste water bioassays
were not statistically different from San Joaquin water.
Samples containing untreated agricultural waste  water showed
growth  rates above those of the San Joaquin River controls.

     The high and variable nitrate concentration found in the
San Joaquin River water which was used  for  sample dilution
might have  caused observed growth rate differences in
different effluents.  Most samples of river water exceeded
the 0.06 mg N/l saturation coefficient  (Ks) found in
                              -30-

-------
earlier studies  (McGauhey,  et  al.,  1968).


     Since  the detention time  of  Delta water  is  sufficient
to allow present algal crops to reach their peak CBain, et  al.,
1968), nutrient content is  presently more  important  in the
San Joaquin Delta  than is growth  rate.   If sediment  concen-
trations become lower and thereby increase light penetration
in the Delta-San Francisco  Bay System  (Krone,  1966),  the
high bioassay growth rate presently found  in  San Joaquin
River water would  indicate  the possibility of  frequent algal
bloom.


     Initial bioassays were carried out  to determine  the
effects of  inorganic nitrogen  and phosphorus  compound additions
upon the waste water bioassay  responses.   The  addition of
nitrate-nitrogen to the algal  pond or bacterial  filter sample
effluents stimulated algal  responses so  that  they equaled
the untreated tile drain waste water six out of  eight times
in samples of comparable dilution.  Additions  of inorganic
phosphorus to treated waste water effluents gave mixed
responses*  Inadequate algal bioassays were made to provide
a statistical evaluation of the results.   Two  samples of
untreated tile drain water  responded to  the addition  of
PO4-P, as did one  sample out of five of  the bacterial filter
systems when PO^-P was added.


     The concomitant use of effluents from two distinct
methods of nutrient removal in algal bioassays suggested
probable comparison of their efficiency  for nutrient  removal.
Only bioassay data in which both  systems had similar  con-
centrations of inorganic nitrogen were compared.  It  was
found that normalized bioassay responses for both systems are
almost equal and their differences not statistically
significant.  Growth rate data comparisons (Table 10)
indicated no differences in the algal pond and bacterial
filter responses.


     The algal bioassay growth of the San  Joaquin River water
was related to its NO3-N.    Summer and early fall samples
exhibited little or no growth  between the  initial and
terminal values made over a period of a week or more.  This
                                    -31-

-------
lack of algal growth suggests nutrient exhaustion in the
Delta waters sampled during the summer and early fall.  By
contrast, samples from other seasons of the year showed
large differences between initial and terminal chlorophyll
values.  This condition was probably due to available
nitrate.  Most of the maximum bioassay values recorded
were between 30 and 45 /*g chl. a/1.  This would corres-
pond  to  approximately 6 - lOmg algae/1 dry weight.
                              -32-

-------
                        APPENDIX A:
           RESULTS  OF  THE INDIVIDUAL EXPERIMENTS
      Results  from  the  seven  experiments  utilizing waters
collected  from  June  18  to  November 17,  1969,  are listed in
Tables  Al  and A2.  The  data  from each experiment are divided
into  two sections.   One compares all  water  types for a
single  percentage  dilution (Al.a,  A2.a)  and the  other
compares all  of the  percentage  dilutions for  a specific water
type  (Al.b, A2.b) .

      In comparing  the  data,  underlining  was used to  indicate
values  similar  to  each  other at the 95%  confidence level.
This  procedure  was allowable because  the analyses of
variance F values were  highly significant for water  types,
percentages and interaction.  In these experiments
"interaction" means  that increasing the  percentage of test
waters  in  San Joaquin  River  water  did not yield  comparable
increases  or  decreases  in  the algal growth  for the different
test  waters.

      For example, the values  in Tables Al and A2 should be
read  in conjunction  with the  responses for  the July  24
experiments in  Figures  Al  and A2.  Both  figures  show algal
chlorophyll concentration  changes  throughout  the conduct of
the experiment.  It  can  be seen in Figure Al  that initial
chlorophyll values for  the different  water  samples at the
beginning  of  the experiment  are not equal and that both the
control and the 20%  algal pond  flasks had no  growth.
Chlorophyll concentrations decreased  from their  initial
values  for these latter  two  samples.

      In Table Al.a,  the  July  24  experiment  shows  increases in
chlorophyll for the  20%  agricultural  waste  water,  bacterial
filter, and algal pond  samples;  44.0, 18.1  and 0.0 Mg chl. a/,
respectively.  Because each value  is  statistically different,
underlining does not connect  them.  The  maximum  chlorophyll
concentrations for the same samples as shown  in  Table A2.a
are 80.4,  51.5 and 40.9  pg chl.  a/.   All values  are
statistically different  at the  95"% confidence level.

     The algal growth responses  for all  percentage additions
of untreated waste water are  graphed  in  Figure A2.   Increases
in chlorophyll concentrations,  shown  in  Table Al.b,  for the
0, 1, 10 and  20% waste water  additions are  0,0,  13.6, 47.0
and 44.0 jug chl. a/,  respectively.  The  highest  responses,
47.0 and 44.0 jug chl. a/, are statistically equal.   Table
A2.a lists the maximum chlorophyll concentrations  for the
same samples.   The maximum values  for the 10% and 20%
additions,  86.4 and  80.4 vg chl. a/,  are  statistically  equal.
                              -33-

-------
                                     TABLE Al. - BICftSSAY CHLOROPHYLL INCREASES  (M)
       (a.)    PERCENTAGE COMPARISONS
t
u>
\. ug Chi. a/1
\% Additions
Experiment \
Date, 1969 \
1%
6/18 10%
100%
1%
7/24 10%
20%
1%
8/18 10%
20%
1%
9/8 10%
20%
1%
9/29 10%
20%
1%
10/20 10%
20%
1%
11/17 10%
20%
AGRICULTURAL
WASTE WATER
33.4
125.7
0.5
13.6
47.0
44.0
, 6.2
39.6
39.3
, 5.3
45.6
, 50.1
11.1
52.3
46.1
27.5
64.3
63.4
, 23.1
, 36.8
40.9
TREATED AGRICULTURAL WASTE WATER
Bacterial
t 19.9
, 27.3
16.5
, 1.5
, 3.0
18.1
No. 19
~^r
15.8
26.3
5.2
16.9
45.6 ,
No. 15
,~o~
, 7.6
, 15.6
No. 10
, 10.7
, 14.1
20.4
No. 6
2O"
36.5 ,
t 19.8

Filter W




No. 11
0.5
, 0.5
3.8
Covered Pond
3.8
, 2.4
, 2.4
No. 18
0.3
5.8 ,
15.8 j
No. 15
9.1,
10.1 ,
12.6


Algal Pond 
14.6 ,
32.6 ,
61.4
No. 4
TT5,
1.5 ,
0.0
No. 15 No.14
~O~ ~CT[
5.2 , 8.5
i i
12.2 19.7
No. 6 No. 19
1.6 0.9 ,
3.8 , 31.9
1.4 , 36.5
No. 5
1.9^
39.7
34.6
No. 6
17.6
35.7
58.2
No.14
TO",
16.6
26.7 ,

        Values  connected by underlining do not differ at the 95 percent confidence level.

        The Firebaugh unit numbers are given when available.

-------
                           TABLE Al. - BIOASSAY CHLOROPHYLL INCREASES   (continued)
      (b.)    WATER TYPE COMPARISONS
EXPERIMENT
DATE, 1969


6/18



7/24





8/18






Q/Q
y/o

WATER TYPE

Agricultural Waste Water
Algal Pond

Bacterial Filter

Agricultural Waste Water
Algal Pond No. 4
Bacterial Filter

Agricultural Waste Water
Algal Pond No. 14

Algal Pond No. 15
Bacterial Filter No. 11
Bacterial Filter No. 19

Agricultural Waste Water
Algal Pond No. 6
Algal Pond No. 19
Bacterial Fi 1 t:p.r
Covered Pond
Bacterial Filter


0%
10.6
, 10.6
0%
, 10.6

0%
oTo
, 0.0
, 0.0

0.5
0.5

0.5
0.5
0.5

, 2.4
, 2.4
, 2.4
, 2.4
, 2.4

PERCENTAGE
1%
(jug Chi.
33.4
14.6 ,
100%
16.5 ,

1%
T376
1.5
1.5

6.2 j
4.4 ,
,
0.9
0.5
3.8 ,

5.3^
1.6
0.9 ,
3.8
5.2 ,

ADDITIONS
10%
a/1)
125.7
32.6
1%
19.9

10%
, 47.0
1.5
3.0 ,

4 39.6
8.5
i
5.2 ,
0.5
15.8

, 45.6
3.8
, 31.1
2.4
16.9


100%
0.5
61.4
10%
27.3

20%
44.0 ,
0.0 ,
18.1

39.3 ,
19.7

12.2
3.8 ,
26.3

50.1 ,
1.4 ,
36.5 ,
2.4 ,
45.6

I
U)
Ul

-------
                                  TABLE Al.  - BIOASSAY CHLOROPHYLL INCREASES -  (continued)


       (b.)   WATER TYPE COMPARISONS - (continued)
EXPERIMENT
DATE, 1969
9/29
10/20
11/17
WATER TYPE
Agricultural Waste Water
Algal Pond No. 5
Bacterial Filter No. 15
Bacterial Filter No. 18
Agricultural Waste Water
Algal Pond No. 6
Bacterial Filter No. 10
Bacterial Filter No. 15
Agricultural Waste Water
Algal Pond No. 14

Bacterial Filter No. 6
PERCENTAGE ADDITIONS
1%
11.1
i 1.9
0.8
0.3
0%
8.3
8.3
i 8.3
. 8.3
10.5
10.5
0%
10.5
0%
3.7
3.7 ,
3.7
,3.7

1%
27.5
17.6
10.7 »
*
9.1
23.1
t 18.3

10%
36.5
10%
Chi. a/1)
52.3
39.7
7.6
5.8 t

10%
, 64.3
35.7
14.1
i
10.1
L36.8
16.6 ,

20%
, 19.8

20%
46.1
34.6
15.6
15.8
20%
63.4^
58.2
20.4
12.6 t
40.9 ,
26.7
1%
20.8 i

to
Oi
I

-------
                                  TABLE A2.  - BIOASSAY CHLOROPHYLL MAXIMA



       (a.)    PERCENTAGE COMPARISONS
Njg Chi. a/1
i Additions
experiment
Date, 1969
6/18
7/24
8/18
9/8
9/29
\
\
1%
10%
100%
1%
10%
20%
1%
10%
20%
1%
10%
20%
1%
10%
20%
AGRICULTURAL
WASTE WATER
60.1
155.6
, 24.2
51.5
86.4
80.4
Agricultural
Waste Water
,39.4
72.8
74.3
, 32.6
74.1
,78.9
43.5
83.5
75.3
TREATED AGRICULTURAL WASTE
Bacterial Filter (Q)
, 42.5
48.5
28.6 ,
, 42.5
, 42.5
51.5
Algal
Pond
No. 14
39T4~
L46.2
, 60.6
Bacterial
35.7
45.5
76.6 ,
No. 15
35.0
,38.2
, 45.6




Bacterial
Filter
No. 19
^779-
48.5 ,
59.1 ,
Filter
Covered Pond
31.1
i 31.9
,34.1
No. 18
t32TT-
36.3 ,
43.8 ,




Algal
Pond
No. 15
"3673"
i 40.2
47.8
No. 6
31.1
33.4 i
31.9 ,
No. 5
32TT ,
76.2
66.5
WATER
Algal Pond ®b
41.7 j
63.6
152.3
No. 4
1275",
40.9 .
40.9
Bacterial
Filter
No. 11
35TT".
32.6 i
35.7
Algal Pond
No. 19
31.9 i
60.5
65.0
w
^J
I
       (p\
          Values connected by underlining do not differ at the  95 percent confidence  level.

              Firebaugh unit nunbers are given when available.

-------
                             TABLE A2. - BIOASSAY CHLOROPHYLL MAXIMA   (continued)


           (a.)   PERCENTAGE COMPARISONS    (continued)
\ lug Chi. a/1
\% Additions
Experiment X
Date, 1969 \
10/20
11/17
1%
10%
20%
1%
10%
20%
AGRICULTURAL TREATED AGRICULTURAL WASTE WATER
WASTE V3ATER
Bacterial Filter (N) Algal. Pond 
-------
                             TABLE A2.  - BIQASSAY CHLOROPHYLL MAXIMA   (continued)




        (b.)   WRITER TYPE CCMPARISCNS
EXPERIMENT
DATE, 1969
6/18
7/24
8/18
9/8
WATER TYPE

Agricultural Waste Water
Bacterial Filter
Algal Pond
Agricultural Waste Water
Bacterial Filter
Algal Pond No. 4
Agricultural Waste Water
Bacterial Filter No. 11
Bacterial Filter No. 19
Algal Pond No. 15
Algal Pond No. 14
Agricultural Waste Water
Bacterial Filter Covered Pond
Bacterial Filter
Algal Pond No. 6
Algal Pond No. 19
PERCENTAGE ADDITIONS
0%
38.9
, 38.9
, 38.9

0%
40.9
, 40.9
, 40.9

34.9
34.9
34.9
36.4
36.4

33.9
34.1
34.9
34.1
33.9
1%
(pg Chi. a
60.1
42.5 ,
,
41.7 ,

1%
51.5
42.5
42.5

39.4 ,
35.7
37.9 ,
36.4
39.4 ,

32.6 ,
31.1
35.7 ,
31.1
31.9 ,
10%
A)
155.6
48.5
63.6
10%
, 86.4
40.9
42.5 ,

, 72.8
32.6
48.5
40. 2 j
46.2
, 74.1
31.9
45.5
33.4
, 60.5
100%
24.2
28.6
152.3
20%
80.4 ,
40.9 ,
51.5
74.3 ,
35.7 ,
59.1
47.8
60.6
78.9 ,
34.1 ,
76.6
31.9 ,
65.0 ,

vo

-------
                              TABLE A2. - BIOASSAY CHLOROPHYLL MAXIMA   (continued)



        (b.)   WATER TYPE COMPARISONS -  (continued)
EXPERIMENT
DATE, 1969
9/29
10/20
11/17
WATER TYPE
Agricultural Waste Water
Bacterial Filter No. 15
Bacterial Filter No. 18
Algal Pond No. 5
Agricultural Waste Water
Bacterial Filter No. 10
Bacterial Filter No. 15
Algal Pond No. 6
Agricultural Waste Water
Algal Pond No. 14
Bacterial Filter No. 6

1%
43.5
, 35.0
32.1
32.1
0%
35.5
, 35.7
, 35.5
35.0
30.6
30.6
0%
30.6
PERCENTAGE
0%
(jug Chi
35.7
35.7
, 35.7
35.7
1%
54.6
36.5 ,
,
35.5
43.5
44.5
, 39.7

1%
( 42.2

ADDITIONS
10%
. a/1)
83.5
38.2 ,
36.3 ,
71.2
10%
i 90.7
39.9
36.5,
i
60.9
L58.2
38.1 ,

20%
41.8


20%
75.3
45.6
43.8
66.5
20%
88.8 ,
45.0
39.7
i
82.8
62.4 t
48.1
10%
58.6
I
*»
o

-------
   100
    80
v
I
a
O
ir
O
    0%  100% San Joaquin River Water
              0.04 mg- N03 - N/L
   20
-O  20% Agricultural Watte Water
               2.03 mg.N03- N/L

-a  20% Algal Pond Water
               0 06 mg.N03- N/L
-A  20% Bacterial Filter Water

             < 0-04 mg
-------
  loo -
•r
Q
o
Q
. >
 *
X
D-
x

•Q
0%

1%

10%
100% Son Joaquin  River Water

Agricultural Waste Water

Agricultural Waste  Water
       -O 20%  Agricultural Waste Water


               -July ,24,1969-
                            234
                         ELAPSED TIME.DAYS


      Figure A2  Algal Growth  Response of San Joaquin  River

                  Water with Added Agricultural Waste Water.
                 AGRICULTURAL  WASTE WATER  STUDIES

                   SAN JOAQUIN VALLEY, CALIFORNIA


                    ENVIRONMENTAL PROTECTION  AGENCY
                              REGION IX
                       SAN FRANClSCO.CALIFORNlA
                                -42-

-------
                          APPENDIX B:
                   ALGAL SPECIES AND NUMBERS
     Algae were identified and counted at the beginning and
end of several experiments.  Personnel from the California
Department of Water Resources identified and counted algae
in Firebaugh water collected on September 9, 1969.  Propor-
tional counts of the algal species were made by algal groups
(Table Bl).  The proportional counts listed in Table Bl show
that diatoms comprised more than 74% of the species at the
beginning and end of the experiment in San Joaquin River
waters, in the bacterial filter, and in the pond water addi-
tions, with or without supplemental inorganic nitrogen.

     The addition of phosphorus to any sample shifted the
population percentages from diatoms to green algae.
Inorganic nitrogen and phosphorus were also added to some
water samples.
                            -43-

-------
                           TABLE 81.- THE PROPORTIONAL  COUNTS OF ALGAE FOUND INITIALLY AND AT THE TERMINATION OF THE BIOASSAY EXPERIMENTS
                              San Joaquin River
Algal Pond
Bacterial Filter
Agricultural
Waste  Water
\ Percent of
X Total
N. No.
Alcal Grou^V.
Greens
Blue-greens
Diatoms
Flagellates
Initial
6
-
86
8
Terminal
6
15
75
6
6.7 wo
N/l
10
1
86
3
6.7 mg N/l r 256
6.7 mg P/l AP
56 2
3
23 89
21 6
556 AP
6.7 mg N/l
15
1
74
10
556 AP
6.7 mg N/l
3.3 me P/l
69
21
6
4
556 AP
6.7 mg N/l
13.2 me P/l
68
3
21
8
256
BF
1
13
84
Z
556 BF
6.7 mg N/l
3.3 TIE P/l
51
39
7
3
556 BF 1056
6.7 mg N/l
6.7 me P/l
53 12
12
28 83
7 5
556
13.2 mg P/l
55
1
35
9
r.  These are additions in waste water concentrations.

-------
                          APPENDIX C:
           CELL COUNT AND CHLOROPHYLL CONCENTRATION
     Algal cells were counted and chlorophyll concentrations
were measured at the beginning and end of four experiments
conducted on June 18, 1969; July 24, 1969; August 18, 1969;
and November 17, 1969.  Replicate flasks were combined and
a single count made of the algal cell concentrations numbers
in the combined samples.  The correlation coefficient between
the algal numbers and the sample chlorophyll concentration
is 0.69, and is highly significant statistically (P<0.01).
The cell count versus chlorophyll data are shown in Figure
Cl.
                             -45-

-------
CTl

 I
                           a>

                           2
                           a>
                           a.
                              io2
                           2
                           U

                           s
X
0.
o
a.
o
_i
r
L)
                              10
                                                                                   logy; 0.4094 logx + 0.2388
                                10*        Z      J    4  S  6 r 8 9|03         2      S   4  5  6 7 8 9,04        2     3



                                                               CELLS PER  MILLIMETER



                                Figure  Cl.  Call Count vs  Chlorophyll a Concentrations, Initial and Terminal Measurements.

-------
                           APPENDIX D
                   BIOASSAY NITROGEN RESPONSES
      Nitrate determinations were made of each concentration
 at the beginning and end of all experiments except the first
 two (June 18 and July 24).   It was expected that algal
 growth would decrease the  N03~N concentrations at the end of
 the experiment period.   This supposition proved true; most
 samples had a terminal  nitrate concentration of 0.10 mg N/l
 or less (Table Dl).

      Higher terminal NO3~N concentrations were found when the
 initial concentration of nitrate was  high.  For example,
 in the 20% agricultural waste water flasks of August 18,  the
 NO3-N was  1.88 mg N/l at the beginning and 1.00 mg N/l at
 the end.   Nitrate-nitrogen decreased  0.88 mg/1 for this
 particular sample.   In  the  10% agricultural waste water
 sample of  September  29th the maximum  decrease in NO3-N was
 1.32  mg/1.

      Slight increases in nitrate also occurred during some
 experiments.   For example,  NO3-N for  all concentrations
 increased  in the  anaerobic  covered pond of September 8th
 although  there was no detectable N03~N in the original
 covered pond sample  (Table  3).   There was however 1.13 mg/1
 of organic  nitrogen  found  in the sample.   Since no discern-
 able  growth of algae occurred using this water,  N03-N
 increases may have resulted from bacterial oxidation of the
 organic nitrogen.  In some  of the experiments an increase in
 nitrate may have  resulted from oxidation of nitrite to
 nitrate.  Many of the samples listed  in Table 2  had a high
 concentration of  nitrite-nitrogen.

     The increase in chlorophyll and  the  decrease  in NO3-N
 are plotted in Figure Dl.   The  correlation coefficient
 (r =  0.82)  between the  two  variables  is highly  significant,
 the regression line  having  the  equation:

        •^g  N/l =  11.6x  (ng  chl.  a/1)  -  15.0

This formula  gives a  ratio  of nitrate-nitrogen decrease to
chlorophyll increase  of approximately 11:1.   The ratio  closely
agrees with the organic nitrogen-chlorophyll  ratio  of  Yentsch
and Vaccaro  (1958) for nitrogen  enriched  cultures,  (7:1 to
10:1) and the ratio  found by Manny  (1969) which was  6:1 to
12:1.  These ratios referred to  fixed organic nitrogen while
ratios used in  this project were based  on the assumption
that the nitrate-nitrogen was metabolized into an organic
form.
                              -47-

-------
TABLE Dl.  NITRATE CONCENTRATIONS  IN TEST WATERS BEFORE AND AFTER BIOASSAY



                                                        N05-N
Experiment Dates- 8/18/69 to  8/25/69    Beginning
End
Decrease
San Joaquin River 100%
Agricultural
Waste Water
Algal
Pond No. 14
Hieh-rate
Algal
Pond No. 15
Low-rate
Bacterial
Filter No. 11
High-rate
Bacterial
Filter No. 19
low Rate
Experiment Dates
San Joaquin Ri
Agricultural
Waste Water
Algal
Pond No. 6
Alsal
Pond No. 19
Anaerobic
Covered
Pond
Bacterial
Filter
1%
10%
20%
1%
10%
20%
1%
10%
20%
1%
10%
20%
1%
10%
20%
- 9/8/69 to 9/16/69
ver 100%
\%
10%
20%
1%
1056
20%
1%
10%
2056
1%
10%
20%
1%
10%
20%
0.05
0.14
0.96
1.88
0.06
0.18
0.32
0.08
0.36
0.66
0.05
0.09
0.14
0.10
0.56
1.06

0.08
0.16
0.80
2.30
0.08
0.07
0.08
0.08
0.30
0.64
0.06
0.06
0.05
0.06
0.06
0.05
0,10
0.08
0.30
1.00
0.08
0.08
0.08
0.10
0.08
0.08
0.10
0.08
0.08
0.10
0.09
0.11

0.05
0.07
0.52
1.60
0.11
0.12
0.07
0.10
0.09
0.16
0.08
0.07
0.08
0.07
0.05
0.10
0
0.06
0.66
0.88
0
0.10
0.24
0
0.28
0.58
0
0.10
0.04
0
0.47
0.95

0.03
0.09
0.28
0.70
0
0
0.01
0
0.21
0.48
0
0
0
o 1
0.01
0 1
                                      -48-

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TABLE Dl.  NITRATE CONCENTRATIONS IN TEST WATERS BEFORE AND AFTER BIOASSAY - (continued)
                                                         mg/1 NOS-N
      Experiment Dates - 9/29/69 to 10/6/69  Beginning
End
Decrease
San Joaquin River
Agricultural
Waste Water
Algal
Pond No. 5
Bacterial
Filter
No. 15
Bacterial
Filter
N°r 18
100$
1%
10$
20$
1%
10$
20$
1%
10$
20$
1$
10$
20$
0.03
0.18
2.1
3.8
0.05
0.47
0.84
0.03
0.08
0.20
0.03
0.08
0.11
0.03
0.03
0.78
2.7
0.03
0.03
0.14
0.04
0.04
0.03
0.03
0.03
0.03
0
0.15
1.32
1.1
0.02
0.45
0.70
0*
0.04
0.17
0
0.05
0.08
Experiment Dates - 10/20/69 to 10/27/69
San Joaquin River
Agricultural
Waste Water
Algal
Pond No. 6
Bacterial
Filter
No. 10
Bacterial
Filter
No. 15
100$
1$
10$
20$
1$
10$
20$
1$
10$
20$
1$
10$
20$
0.12
0.30
1.6
5.6
0.14
0.30
0.51
0.10
0.12
0.12
0.11
0.14
0.18
0.19
0.47
0.75
3.0
0.03
0.05
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0
0
0.85
0.6
0.11
0.27
0.48
0.07
0.09
0.09
0.08
0.11
0.15
Experiment Dates - 11/17/69 to 11/24/69
San Joaquin River
Agricultural
Waste Water
Algal
Pond No. 14
Bacterial
Filter No. 6
100$
1$
10$
£0$
1$
10$
20$
1$
10$
E0$
0.22
0.45
3.2
4.1
0.22
0.29
0.37
0.38
1.5
3.2
0.03
0.04
2.4
3.7
0.04
0.03
0.03
0.04
1.0
0.19
0.41
0.80
0.4
0.18
0.26
0.34
0.34
0.50
                                          -49-

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    1.5
UJ
en
UJ
oc
=   i.o
E
o
UJ
o
O
ir
 I
la
cc
H

Z
0.5
                                              Y-O.OII6 X-O.OJ5
                20       40        60        80        too       120

                 CHLOROPHYLL o ,(Mlcrogroms per Liter) INCREASE
                                                                       140
      Figure D I. Bioassoy  Decrease in Nitrate-Nitrogen as .a Function of Chlorophyll
                 Increases..
                              -50-

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     Figure D2 shows the log of the chlorophyll  concentration
maximums as a function of the log of  the original  NO^-N  con-
tained in the cultures.  The correlation is highly significant
(r = 0.77) , the equation of the line  being:
     log  (jLig chl. a/) = 0.2003  (log /*..g N/l)  +  1.195


The ratio of total initial inorganic nitrogen  to maximum
chlorophyll in the culture is approximately  16:1.
                                    -51-

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                iO
Ui
K>
I
             o»
             01
             CX
 E
 o

 o
 L
 o
                10'
X
a.
o
£T
O
—i
X
o

S
z>
S

X
<
2
                10'
                                  logy-0.2003  log C X x !03) + M95
o   o
     ID'2                           10-'                             10°

                       INITIAL TOTAL INORGANIC NITROGEN, (Milligrams per Liter)


     Figure 0 2. Bio assay Chlorophyll Maximum as a Function of Total Inorganic Nitrogen ( NO 3- N plus N02-N ),
                                                                                                               10'

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                        ACKNOWLEDGEMENTS
 The following list includes the personnel actively involved
 in the algal bioassay project.


            Firebaugh Project under the Direction of

 Percy St.  Amant	Engineer/ Environmental
                                    Protection Agency
 Louis A.  Beck	Engineer, California
                                    Dept.  of Water Resources
 Donald Swain	Engineer/ U.S. Bureau of
                                    Reclamation

                    Consultants to  the Project

 Dr.  Perry  L.  McCarty	 .Stanford University,
                                    Palo Alto
 Dr.  William J.  Oswald	University of California,
                                    Berkeley
 Dr.  Clarence G.  Golueke	University of California,
                                    Berkeley

           Algal  Bioassays  of Agricultural Waste Water
                         Conducted by

 Dr.  Milton G. Tunzi	Limnologist,  Environmental
                                    Protection  Agency

                           Assisted by

 Albert Katko	Aquatic  Biologist,  EPA
 Richard C.  Bain/ Jr	Sanitary Engineer,  EPA
 James E. Thoits	Biological  Technician,  EPA
 Ella M. McGehee	Physical Science Aide,  EPA
 Dorothy M.  White	Physical Science Aide,  EPA
 Margaret Y. Chu	Chemist,  EPA
 Mustafa M.  Salma	 . .Chemist,  EPA
 David R. Wood	Supervisory Chemist,  EPA
 Kathleen Shimmin	Supervisory Microbiologist,
                                   EPA
 Gary Varney	Biologist, California
                                   Dept.  of Water Resources
                      Report Prepared by

Dr. Milton G. Tunzi	Limnologist, Environmental
                                   Protection Agency


                                   -53-

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                LIST OF REFERENCES
Bain, R.C., Jr.  1969.  Algal Growth Assessments by
     Fluorescence Techniques:  Proceedings of the
     Eutrophication—Biostimulation Assessment Workshop,
     Federal Water Quality Administration, Corvallis,
     Oregon.

Bain, R.C., Jr., H.E. Pintler, A. Katko, and R.F. Minnehan.
     1968.  Nutrients and Biological Response:  San Joaquin
     Master Drain, Appendix, Part C, Federal Water Quality
     Administration, Southwest Region, San Francisco,  117p.

FWPCA Methods for Chemical Analysis of Water and Wastes.
     November 1969.  Federal Water Quality Administration,
     Analytical Quality Control Laboratory, Cincinnati,
     Ohio, 280p.

Krone, R.B.  1966.  Predicted Suspended Inflows to the
     San Francisco Bay System, Prepared for the Federal Water
     Pollution Control Administration, Southwest Region,
     San Francisco.

Manny, B.A.  1969.  The Relationship between Organic Nitrogen
     and the Carotenoid to Chlorophyll A Ratio in Five
     Freshwater Phytoplankton Species:  Limnology and
     Oceanography, V. 14, p. 69-79.

McGaughey, P.H., G.A. Rohlick, E.A Pearson, M. Tunzi,
     A. Adinarayana, and E.J. Middlebrooks.  1968.
     Eutrophication of Surface Waters-Lake Tahoe:  Bioassay
     of Nutrient Sources.  LTAC, FWPCA Progress Report for
     Grant No. WPD 48-01  (R 1), May.

Provisional Algal Assay Procedures.  1969.  Joint Industry-
     Government Task Force on Eutrophication, P.O. Box 3011,
     Grand Central Station, New York, 43p.

Sword, B. R.  1970.   Denitrification of Agricultural Tile
     Drainage in Anaerobic Filters and Ponds, in this series.

Yentsch, C.S. and R.F. Vaccaro.  1958.  Phytoplankton Nitrogen
     in the Oceans:  Limnology and Oceanography, V. 3,
     p. 443-448.
                             -54-

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                         PUBLICATIONS

          SAN  JOAQUIN  PROJECT,  FIREBAUGH,  CALIFORNIA
 1968
 "Is Treatment  of Agricultural Waste Water Possible?"
     Louis A.  Beck and Percy P. St. Amant, Jr.  Presented
     at Fourth International Water Quality Symposium, San
     Francisco, California, August 14,  1968; published
     in the proceedings of the meeting.
1969

"Biological Denitrification of Wastewaters by Addition of
Organic Materials"
     Perry L. McCarty, Beck, and St. Amant, Jr.  Presented
     at the 24th Annual Purdue Industrial Waste Conference,
     Purdue University, Lafayette, Indiana.  May 6, 1969,

"Comparison of Nitrate Removal Methods"
     Beck, St. Amant, and Thomas A. Tamblyn.  Presented at
     Water Pollution Control Federation Meeting, Dallas,
     Texas.  October 9, 1969,

"Effect of Surface/Volume Relationship, COg Addition, Aeration,
and Mixing on Nitrate Utilization by Scenedesmus Cultures
in Subsurface Agricultural Waste Waters"
     Randall L. Brown and James F. Arthur.  Proceedings
     of the Eutrophication-Biostimulation Assessment
     Workshop, Berkeley, California.  June 19-21, 1969.
"Nitrate Removal Studies at the iriteragency Agricultural
Waste Water Treatment Center, Firebaugh, California"
     St. Amant and Beck.  Presented at 1969 Conference,
     California Water Pollution Control Association, Anaheim,
     California, and published in the proceedings of the
     meeting.  May 9, 1969.

"The Anaerobic Filter for the Denitrification of Agricultural
Subsurface Drainage"
     Tamblyn and B. R. Sword.  Presented at the 24th
     Annual Purdue Industrial Waste Conference, Lafayette,
     Indiana.  May 5-8, 1969.
                               -55-

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                 PUBLICATIONS (Continued)
1969 (Continued)
"Research on Methods of Removing Excess Plant Nutrients
from Water"
     St. Amant, Beck.  Presented at 158th National Meeting
     and Chemical Exposition, American Chemical Society,
     New York, New York.  September 8, 1969.

"Nutrients in Agricultural Tile Drainage"
     W. H. Pierce, Beck, and L. R. Glandon.  Presented
     at the 1969 Winter Meeting of the American Society of
     Agricultural Engineers, Chicago, Illinois.  December
     9-12, 1969.

"Treatment of High Nitrate Waters"
     St. Amant, McCarty.  Presented at Annual Conference,
     American Water Works Association, San Diego, California,.
     May 21, 1969.  American Water Works Association Journal,
     Vol. 61, No. 12.  December 1969, pp. 659-662.


The following papers  were presented at the National Fall
Meeting of the American Geophysical Union, Hydrology Section,
San Francisco, California.  December 15-18, 1969.  They are
published in Collected Papers Regarding Nitrates in Agri-
cultural Waste Water.  USDI, FWQA, #13030 ELY December 1969.


"The Effects of Nitrogen Removal on the Algal Growth
Potential of San Joaquin Valley Agricultural Tile Drainage
Effluents"
     Brown, Richard C. Bain, Jr., and Milton G. Tunzi.

"Harvesting of Algae Grown in Agricultural Wastewaters"
     Bruce A. Butterfield and James R. Jones.

"Monitoring Nutrients and Pesticides in Subsurface Agricultural
Drainage"
     Glandon and Beck.

"Combined Nutrient  Removal  and  Transport  System  for Tile
Drainage  from  the San Joaquin Valley"
     Joel C. Goldman, Arthur, William J.  Oswald, and Beck.

"Desalination  of  Irrigation  Return Waters"
     Bryan  R.  Sword.
                               -56-

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                 PUBLICATIONS (Continued)
"Bacterial Denitrification of Agricultural Tile Drainage"
     Tamblyn,  McCarty,  and St.  Amant.

"Algal Nutrient Responses  in Agricultural Wastewater"
     Arthur,  Brown, Butterfield,  and Goldman.
                              -57-

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1

5
Afcfxsioti fifumbcr
~ Subject Fii'ld & Croup
05D
SELECTED WATER RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
Organization
Environmental Protection Agency, Region IX
              Water Quality  Office
              100 California Street,  San Francisco  CA  94111
    Title
              The Effects of Agricultural Waste Water Treatment  on Algal
              Bioassay Response
J Q Authors)
TUNZI, Milton G.
16

21
Project Designation
13030 ELY
Available
from:
                                          Environmental Protection Agency
                                          Region IX, 100 California  Street
                                          San Francisco  CA  94111
 22
    Citation
           Agricultural Waste Water Studies
           Report No. 13030 ELY  8/17-9
           Pages:   59, Figures;   8, Tables;  14, References
    Descriptors (Starred First)
23
           *  Eutrophication
           *  Bioassay
           *  Denitrification  ,  Nitrates,  Nitrogen
           *  Fluarometry
           *  Tile Drains
 25
   Identifiers (Starred First)

          *  Algal Blooms  -  control, bioassay - algal,  chlorophyll,
             denitrifica-tion,  fluorometry, tile drainage
 27
   Abs'ract
          Laboratory bioassay  experiments were performed  to  test the effect
     on algal growth of  agricultural waste water befote and  after the waste
     water had been subjected  to two different nitrogen removal processes.
     The waste waters were  added in various percentages to San Joaquin River
     Delta water* for bioassay.'  The algal growth throughout  time was monitored
     by chlorophyll fluorescence techniques.  The fluorescence measurements
     showed logarithmic  growth similar to the type usually observed in the
     Delta Water over the vernal growth period.

          The laboratory experiments gave positive statistical evidence
     that' the untreated  agricultural waste water would promote substantial
     algal growth above  that of the San Joaquin River controls.  Both
     nitrogen removal processes were equally effective in lowering the
     alga] growth to that of the Delta water controls -as  long  as the
     nitrates-nitrogen level in each removal system had been  lowered to
     approximately 2 mg  N/l, or less.
Abstractor
                          Institution
 V»f«:IOI (REV. JULY IS69I
 WRSIC
                                            I: WATER, RESOURCES SC'ENTIFIC IN FORM* TICN
                                             .. U.S. DEPARTMCNT OF THE INTERIOR
                                              WASHING TOW.' D, C. 90240
  ft U, S, GOVERNMENT PRINTING OFFICE :  1972-514-l48/««

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