EPA-909/9-74-004
                      WORKSHOP

                        ON

       SAMPLING,  MONITORING AND ANALYSIS

                        OF

              WATER AND WASTEWATER
                 March 6-12, 1974
                 Honolulu, Hawaii
                     1
                             \
                             ul
 U.S.  Environmental Protection Agency/ Region IX
            San Francisco CA  94111
                  June 1974

-------
EPA-909/9-74-004
                    U.S. ef*A~NEie LfBNARY
                    Denver Federal Center
                    Building 25, Ent. E-3
                    P.O. Box 25227
                    Denver, CO 80225-0227
                       VJORKSHOP

                          ON

       SAMPLING,  MONITORING AND  ANALYSIS

                          OF

               WATER AND WASTEWATER
                      LIBRARY, NFIC - DENVER
                   ENVIRONMENTAL PROTECTION AGENCY
                  March 6-12,  1974
                  Honolulu,  Hawaii
                                \
                                UJ
                                a
  U S. Environmental Protection Agency, Region IX
              San Francisco CA  94111
                    June  1974

-------
                        INTRODUCTION
     On March 6-12, 1974 the U.S. Environmental Protection
Agency conducted a Workshop on Sampling, Monitoring and
Analysis of Water and Wastewater.  The training course was
sponsored by the Environmental Protection Agency, Region IX
in cooperation with the National Field Investigation Center
(Denver, Colorado), and the State of Hawaii Department of
Health, the University of Hawaii, and the Hawaii Water
Pollution Control Association.

     Over 85 persons attended the 5-day training course which
was held on the campus of the University of Hawaii.  Attendees
included: State of Hawaii public health officials, State
and local laboratory and field personnel as well as technical
personnel from American Samoa, consulting engineers, and
University of Hawaii students.

     The workshop provided information and training in both
the standard and new techniques currently employed in the
sampling, monitoring and analysis of water and wastewater.

     This document is a compilation of papers presented
during that workshop.  Also included is a list of the sev-
eral publications which were disseminated as part of the
training course.  In most cases, copies can be obtained
through the Government Printing Office.

-------
                        CONTENTS
Introduction
     Speakers
     Agenda
     Registration Form
     Attendees
Federal Requirements for Sample Handling, Analyses,
     Quality Assurance	Shimmin
Compliance Monitoring	Wills


Flow Measurement	Hathaway and Walz


Organics Sampler and Field Extraction Procedures	Walz


Comparative Sampling Results: Some Examples	Kumagai


An Outline on the Bacteriology of Water	Shimmin


Analytical Methods for Metal and Pesticide Analysis...Young

                i
304 (g)  Water Quality Guidelines	Young


Chemical Analysis  for Demand, Nutrient and Oil
     and Grease	Young


Selected Field and Laboratory Biology Methods	Tunzi


Elements of a Quality Assurance Program	Shimmin

-------
 QaM__                   WORKSHOP ON
 SAMPLING, MONITORING AND ANALYSIS OF WATER AND WASTEWATER

                       March 6-12, 1974
                           SPEAKERS
 Paul DeFalco, Jr.
   Administrator
 Henri P- Minette
   Deputy Director of Health
 Ho Young
   Chemist
 Kathleen Shimmin
   Microbiologist
 Milton Tunzi
   Biologist
 Helen Johnson
   Biological Aid


 Carroll Wills
   Enforcement Specialist
James  Hathaway
  Sanitary  Engineer
                                  EPA, Region IX
                                  San Francisco CA  94111
 State of Hawaii  Dept.  of  Health
 Honolulu HI   96801
                                  EPA.  Region IX
                                  San Francisco CA  94111
 EPA,  Region IX
 San Francisco CA  94111
 EPA,  Region IX
 San Francisco CA  94111
 EPA,  Region IX
 San Francisco CA  94111
 EPA,  Nat'l  Field.Investigation-Ctr.
 Denver CO   80225
EPA, Nat 11 Field Inventigation Ctr.
Denver CO  80225
Laurence Walz
  Physical Science Technician
EPA, Nat'l Field Investigation Ctr.
Denver CO  80225
James Kumagai
  Sanitary Engineer
Sunn, Low, Tom and Kara,  Inc.
Honolulu,  HI  96813

-------
                        WORKSHOP ON
SAMPLING, MONITORING AND ANALYSIS OF WATER AND WASTEWATER

                      March 6-12, 1974
                           AGENDA
March 6, 1974

 8:30
 9:00
10:00
10 :15
12:00
 1:30
 2:15
 2:30

 3:30
 3:45-4:45
Wednesday

Registration
Introduction  (DeFalco, Minette)

Federal Requirements for Sampling, Sample
  Handling, Analyses, Quality Assurance,
  WQ Act  (Shimmin)
Coffee Break
Survey Planning - Site Selection  (Tunzi)
Lunch
Parameter Selection  (Tunzi)
Coffee Break
Monitoring and Flow Measurement  (Hathaway and
  Walz)
Compliance Monitoring  (Wills)
Comparison of Sampling Methods in Waste
  Effluents  (Kumagai)
March 7, 1974

 8:30

10:00
10:15
12:00
 1:30-5:00
Thursday

Monitoring and Flow Measurement  (Hathaway and
  Walz)
Coffee Break
Instrumentation  (Walz)
Lunch
Field Exercise (Johnson, Shimmin, Tunzi)
March 8, 1974

 8:30
 9 :00

10:15
10:30

12:00
Friday

Discussion of Field Exercise -questions
Chemistry  (Young)
  Federal Register-304 g Guidelines
Coffee Break
Bacteriology  (Shimmin)
  Quality Assurance
Lunch

-------
                     AGENDA - Continued
March 8, 1974

 1:30
 2:30
 2:45-4:30
                  Friday  (continued)

                  Statistics
                  Coffee Break
                  Biostimulation, Toxicity  (Tunzi)
March 11, 1974
 8:30
 9
 9
  :00
  :30
12:00
 1:30
 3:00
 3:15-5:00
Monday  (LABORATORY)

Registration  (New Attendees)
Introduction, Federal Requirements  (Shiininin)
Bacteriology  (Shimmin, Johnson) .
Lunch
Chemistry  (Young)
  Demand, Nutrient, Oil and Grease
Coffee Break
Chemistry  (Young)
  Demand, Nutrient, Oil and Grease
March 12, 1974

 8:00

10:00
10:15
12:00
 1:30
 2:30
 2:45-5:00
                  Tuesday

                  Heavy Metals  (Young)
                    Pesticides
                  Coffee Break
                  Bioassays  (Tunzi)
                  Lunch
                  Biological Methods Manual  (Tunzi)
                  Coffee Break
                  Quality Assurance  (Shimmin)

-------
            U.S. ENVIRONMENTAL  PROTECTION AGENCY
                     IN  COOPERATION  WITH
THE STATE OF HAWAII  DEPARTMENT  OF HEALTH, THE UNIVERSITY OF HAWAII
    AND THE HAWAII WATER  POLLUTION  CONTROL ASSOCIATION
        Workshop on Sampling, Monitoring,  and Analysis
                  of Water  and  Wastewater

                  March  6,7,8,11,12,  1974


                     REGISTRATION  FORM



Name:	


Address:
Employer:


Address:
Occupation Title:

-------
                        WORKSHOP ON
SAMPLING, MONITORING AND ANALYSIS OF WATER AND WASTEWATER

                      March 6-12, 1974
                         ATTENDEES
AKAZAWA, Eugene T.
 Environmental Health Specialist
AKI, Paul F.
 Acting Chief, Pollution Investi-
 gation and Enforcement Branch

ALLEN, William
 Environmental Engineer
ANAMIZU, Thomas M.
 Environmental Health Specialist
BEENE, Janice C.
 Specialist
BONNET, William A.
 Project Engineer

CHANG, Bei-Jiann
 Graduate Assistant
CHASE, Robert
 Environmental Health Specialist
State Dept. of Health
 Lihue, HI
State Dept. of Health
 Honolulu, HI
R.M. Towill Corp.
 Honolulu, HI
State Dept. of Health
 Honolulu, HI
University of Hawaii
 Honolulu, HI

Austin, Smith & Assoc.,  Inc.
 Honolulu, HI
University of Hawaii
 Honolulu, HI
Dept. of Health
 Wailuku, Maui
CHEN, Ben
 Design Engineer
R.M. Towill Corp.
 Honolulu, HI
CHOW, Randy
 Chemist
State Dept.  of Health
 Honolulu,  HI
                                -1-

-------
                        WORKSHOP ON
SAMPLING, MONITORING AND ANALYSIS OF WATER AND  WASTEWATER

                      March  6-12, 1974
                           AGENDA
March 6, 1974

 8:30
 9 :00
10:00
10:15
12:00
 1:30
 2:15
 2:30

 3:30
 3:45-4:45
Wednesday

Registration
Introduction  (DeFalco, Minette)

Federal Requirements for Sampling, Sample
  Handling, Analyses, Quality Assurance,
  WQ Act  (Shimmin)
Coffee Break
Survey Planning - Site Selection  (Tunzi)
Lunch
Parameter Selection  (Tunzi)
Coffee Break
Monitoring and Flow Measurement  (Hathaway and
  Walz)
Compliance Monitoring  (Wills)
Comparison of Sampling Methods in Waste
  Effluents  (Kumagai)
March 7, 1974

 8:30

10:00
10:15
12:00
 1:30-5:00
Thursday

Monitoring and Flow Measurement  (Hathaway and
  Walz)
Coffee Break
Instrumentation  (Walz)
Lunch
Field Exercise (Johnson, Shimmin, Tunzi)
March 8, 1974

 8:30
 9 :00

10:15
10: 30

12:00
Friday

Discussion of Field Exercise -questions
Chemistry  (Young)
  Federal Register-304 g Guidelines
Coffee Break
Bacteriology  (Shimmin)
  Quality Assurance
Lunch

-------
                     AGENDA - Continued
March 8, 1974

 1:30
 2:30
 2:45-4:30
Friday (continued)

Statistics
Coffee Break
Biostimulation, Toxicity (Tunzi)
March 11, 1974

 8:30
 9:00
 9:30
12:00
 1:30

 3:00
 3:15-5:00
Monday (LABORATORY)

Registration (New Attendees)
Introduction, Federal Requirements  (Shimmin)
Bacteriology (Shimmin, Johnson) .
Lunch
Chemistry (Young)
  Demand, Nutrient, Oil and Grease
Coffee Break
Chemistry (Young)
  Demand, Nutrient, Oil and Grease
March 12, 1974

 8:00

10:00
10:15
12:00
 1:30
 2:30
 2:45-5:00
Tuesday

Heavy Metals (Young)
  Pesticides
Coffee Break
Bioassays  (Tunzi)
Lunch
Biological Methods Manual  (Tunzi)
Coffee Break
Quality Assurance  (Shimmin)

-------
ATTENDEES  (Continued)
FUJIMOTO, Stanley
 Graduate Student
GEE, Henry K.
 Associate Res.
GLENN, Gail 0.
 Biological Technician
GOO, Reginald
 Chemist
HARUNO, Jerry
 Environmental Health Specialist
HASHIMOTO, William Y.
 Environmental Health Specialist
HAYASHI, Robert G.
 Water Microbiologist IV
HENDRICKS, Katherine L.
 Environmental Health Specialist II
HIGUCHI, Gerald T.
 Environmental Health Specialist II
HIRATA, Shiro
 Environmental Health Specialist
 'OKI, Daniel
 Environmental Health Specialist III
University of Hawaii
 Honolulu, HI
V.H. Water Resources Res.Ct
 Honolulu, HI
U.S. Navy, Pacific Div.
 Navy Facilities
 Honolulu, HI
State Dept. of Health
 Honolulu, HI
State Dept.  of Health
 Honolulu, HI
State Dept.  of Health
 Kealekekua, HI
Honolulu Board of Water
 Supply
 Honolulu, HI
State Dept.  of Health
 Honolulu, HI
State Dept. of Health
 Honolulu, HI
State Dept. of Health
 Honolulu, HI
State Dept. of Health
 Honolulu, HI
                                  -2-

-------
ATTENDEES  (Continued)
HORIGAN, Laura L.
 Laboratory Director
ISHIKAWA, Gordon N.
 Environmental Scientist
KANSAKO, Sidney I.
 Sanitary Chemist
R.M. Towill Corp,
 Honolulu, HI
Tripler Army Medical Ctr.
 Honolulu, HI
City Hall, BWS (Sewers)
KARELITZ, Charles W,
 Chemist III
KAWAMOTO, Ted
 Environmental Health Specialist
KAWANISHI, Glenn
 Environmental Health Specialist
KILLIN, Robert D.
 Preventive Med NCO
KONNO, Stanley
 Graduate Student
State Dept. of Health
 Honolulu, HI
State Dept. of Health
 Honolulu, HI
State Dept. of Health
 Hilo, HI
Tripler Army Med Ctr
 Honolulu, HI
University of Hawaii
 Honolulu, HI
KROCK, Hans J.
 Sanitary Engineer
KUBOTA, Edwin Y.
 Reg. Sanitarian IV
KUMAGAI, James S.
KUNIOKA, Robert T.
 Microbiologist
Sunn, Low, Tom & Hara, Inc.
 Honolulu, HI
State Dept. of Health
 Honolulu, HI

Sunn, Low, Tom & Hara, Inc.
 Honolulu, HI
State Dept.  of Health
 Honolulu,  HI
                               -3-

-------
ATTENDEES -  (Continued)
KURAMOTO, Edward
 Environmental Health Specialist III
LAFITAGA, Pasesa
 District Sanitarian
LEE, Bobby
 Analytical Chemist
LEE, Daiwun
 Environmental Health Specialist III
LEE, David
 Sanitary Chemist
LINDSAY, Stephen A.
 Biological Tech/Chemist
LOW, Lionel
 Laboratory Director
McCARTER, Mildred L.
 Chemist III
MIYAMOTO, Howard
 Environmental Health Specialist I
MONCRIEF, Robert
 Marine Biologist
'WRANAKA, Harry
 Sanitary Chemist
State Dept. of Health
 Honolulu, HI
Environmental Health Branch
 Pago  Pago, American Samoa
Brewer Chemical Corp.
 Honolulu, HI
State Dept. of Health
 Honolulu, HI
Co. of Hawaii, Dept of
 Public Works
 Hilo, HI
U.S.  Navy
 Nav Fac. Eng.  Cmd.
 Pearl Harbor,  HI
Sunn, Low, Tom & Kara,  Inc.
 Honolulu, HI
State Dept.  of Health
 Honolulu, HI
State Dept.  of Health
 Honolulu, HI
Dept. of Defense (Navy)
 Pac. Div. NAFFACENGCOM
 Makalapa, HI
County of Kauai
 Lihue,  Kauai
                             -4-

-------
ATTENDEES -  (Continued)
NAKANISHI, Ernest N.
 Sr. Chemist
NAKAMURA, Sidney S.
 Lab Assistant
NAKASONE, Samuel S.
 Chemist
NG, Earl W.M.
 Graduate Student
ONO, Wayne
 Chemist III
OUMI, Charles
 Environmental Health Specialist
POROTESANO, Ati
 Lab Technician
PRIOR, Timothy"
 Environmental Health Specialist
RADCLIFF, Lester
 Microbiologist IV
RICHARDSON, George C,
 Civil Engineer IV
SHIGETANI, Mike
 Engineer
Brewer Analytical Lab
 Honolulu, HI
BWS - Sewers
 City Hall
Board of Water Supply
 City Hall
University of Hawaii
 Honolulu, HI
State Dept. of Health
 Honolulu, HI
State Dept. of Health
 Honolulu. HI
L.B.J. Medical Ctr.
 Pago  Pago, American Samoa
State Dept. of Health
 Honolulu, HI
State Dept. of Health
 Honolulu, HI
City & County of Honolulu
 Board of Water Supply
 Div of Sewers
 Honolulu, HI
Board of Water Supply
 Honolulu, HI
SHIMABUKURO, Seichi
 Chemist
Federal Government,  PWC
 Honolulu, HI
                               -5-

-------
ATTENDEES -  (Continued)
SHIRATORI, Bernard
 Environmental Health Specialist
SHISHIDO, Kazuo
 Environmental Health Specialist
SIMAO, Anthony M.
 Microbiologist
SURFACE, Stephen W.
 Chemist
TILLMAN, R. Bruce
 Environmental Health Specialist III
TOKUNAGA, Edward H.
 Chemist
TOMITA, Wayne
 Student
State Dept. of Health
 Honolulu, HI
State Dept. of Health
 Honolulu, HI
State Dept.  of Health (Labs)
 Hilo, HI
U.S. Navy
 Pac. Diy-,  Nav.  Fac.  Eng.
 Honolulu,  HI
State Dept.  of Health
 Honolulu, HI
Board of Water Supply
 Honolulu, HI
University of Hawaii
 Honolulu, HI
TSUTSUI, Roy
 Research Assistant
UYEMA, George M.
 Civil Engineer V
VICTOR, David
 Student
WAKATSUKI, Helen H.
 Chemist V
University of Hawaii
 Honolulu, HI
Board of Water Supply
 D/S C&C of Hon.
 Honolulu, HI
University of Hawaii
 Honolulu, HI
State Dept. of Health
 Honolulu, HI
                               -6-

-------
ATTENDEES -  (Continued)
WARD, Lawrence J.
 Preventive Med NCO
WILBERTS, Carson E.
 Environmental Health Specialist
WONG, Darryl E.
 Grad. Assistant W.R.R.C.
YAMAMURA, Paul
 Microbiologist III
YAMANE, George
 Environmental Health Specialist
YIM, Spencer
 Graduate Student
YOSHINAGA, Glenn
 Student
YOSHINUTSU, Ushijima
 Sanitary Chemist
Tripler Army Med Ctr.
 Health & Env Service
 APO 96438
State Dept.  of Health
 Honolulu, HI
University of Hawaii
 Honolulu, HI
DOH Lab Branch
 W'Ku,  Maui
State Dept. of Health
 Honolulu, HI
University of Hawaii
 Honolulu, HI
University of Hawaii
 Honolulu, HI
C&C BWS Sewers
 City Hall
 Honolulu, HI

-------
                         PUBLICATIONS
     The following is a list of publications disseminated to
participants of the Workshop on Sampling, Monitoring and
Analysis of Water and Wastewater.  These publications can be
obtained through the Government Printing Office, or by con-
tacting the appropriate EPA office, as indicated.

Abbreviated List of Publications and Guideline Documents
  Dealing with Monitoring Quality Assurance.  EPA, Quality
  Assurance Division, Office of Monitoring Systems, Washington,
  D.C. 20460.  January 1974.

Analytical Quality Control in Water and Wastewater Laboratories
  EPA, National Environmental Research Center - Cincinnati,
  Analytical Quality Control Laboratory, Cincinnati, Ohio
  45268.   (Technology Transfer, GPO-1972-479-971) June 1972.

Biological Field and Laboratory Methods; for measuring the
  quality of surface waters and effluents.  EPA, National
  Envxronmental Research Center - Cincinnati, Office of
  Research and Development, Cincinnati, Ohio 45268, (EPA-670/
  4-73-001) July 1974.

Methods for Chemical Analysis of Water and Wastes.  EPA,
  National Environmental Research Center - Cincinnati, Analyt-
  ical Quality Control Laboratory, Cincinnati, Ohio 45268,
  (GPO-5501-0067) 1971.

Monitoring Industrial Wastewater.  EPA, Office of Technology
  Transfer, Washington, D.C. 20460, August 1973.
     Two "Technology Transfer" audio-visual instruction units
were presented as part of the March 11, 1974 lecture on
"Demand, Nutrient, and Oil and Grease."

     1) "Determination of Grease and Oil"
         58 slides, 15 minute tape, script
         (# XT-56)

     2) "Determination of Total Organic Carbon"
         59 slides, 13 minute tape, script
         (# XT-59)

Both instructional units are available on loan from either the
EPA, National Training Center, Cincinnati Ohio 45268, or the
£i A, Region IX, Air and Water Division, Manpower Training and
Development, San Francisco California 94111.

-------
                FEDERAL REQUIREMENTS


                        FOR

      SAMPLE HANDLING, ANALYSES, QUALITY ASSURANCE
                         By

                  Kathleen Shimmin
                  EPA, Region IX
                  San Francisco CA
Presented at the Workshop on Sampling, Monitoring and Analysis
  u£ Water and Wastewater, March 6-12, 1974, Honolulu HI.

-------
           FEDERAL REQUIREMENTS FOR MONITORING
The following sections of PL92-500 apply to monitoring:
Sec. 106
State Program Grants
Monitor water quality within State
Determine validity of data and update data annually
Specific requirements of Appendix A
Sec. 304

     304b
Effluent Guidelines

Identify constitutents and chemical, physical and
  biological character of the effluent

Analyze according to methodology guidelines
  published in the Federal Register, 10/6/73

Requires information on point source discharges
  including specific requirements on monitoring
  and reporting
Sec. 305
Water Quality Inventory
Annual report on quality of all navigable water
   to be submitted by each State
Sec. 308

     308a
     308c
Inspections, Monitoring, Entry

Install, use, maintain monitoring equipment or
  methods according to EPA-prescribed guidelines
Sample according to method, location, and
  frequency required by EPA

State should set up procedures for inspecting
  and monitoring point source discharges.  These
  are to be submitted to EPA for approval.
Sec. 402
National Pollutant Discharge Elimination
  System (NPDES) - Permits
After permit issued, inspection and monitoring is
  required of permittee - self-monitoring
Necessary for State to have system of judging

-------
  validity and adequacy of self-monitoring data
  reported.  One way is to have periodic  analyses
  of effluent by another laboratory.
California - writing into permit that data must be
  supplied by certified lab.
              -2-

-------
              CHAIN-OF-CUSTODY PROCEDURES
These procedures have been adequate  for EPA, Region IX,
Surveillance and Analysis Division, Microbiology Section.
1) Take sample and write on  label: sampler; witness; time;
date; location or identifying location number.

2)  Safeguard sample at all  times, by either having it within
view or locked up.

3)  Sign when custody transferred,(custody tags have place for
this).

4)  Note in laboratory notebook: name of sampler; name of
analyst; time of sampling and of beginning of analysis.

5)  After bacteriological sample analysis has been initiated
(i.e., sample has been inoculated  into medium) paste label
from sample bottle (label contains information listed in (1)  )
into laboratory notebook assigned  to the particular field
study.

6)  Make a record of all observations in this notebook.
Observations include colony  counts, MPN codes, calculations,
and final density determinations.  Record also biochemical
and serological reactions.

7)  Include temperature records for waterbath in notebook.

8)  Store notebook in locked cabinet.

9)  If the sample were for chemical analyses and could be
stored,  then the sample too  should be stored in a locked
cabinet, together with the chain-of-custody tag, until the
enforcement case is over.
Photographic Documentation

1)  Labels showing sample station location and date can be
included in photograph by having a sign included in the picture

2>  'In notebook, record direction of camera.

3)  If possible, include identifying landmarks in photo and
then take a progressive series of  closeups of problem.

-------
4)  Color film is recommended over black and white film,  unless
photo to be published in black and white.
                           -2-

-------
              COMPLIANCE MONITORING
                        By

                 Carroll G. Wills
                 EPA, NFIC-Denver
                 Denver CO
Presented at the Workshop on Sampling, Monitoring and Analysis
 of Water and Wastewater, March 6-12, 1974, Honolulu HI.

-------
                           COMPLIANCE MONITORING
  I.   Introduction

        Primary responsibility to prevent,  reduce  and  eliminate water
        pollution rests with the States

        Current EPA priorities:   promulgate effluent guidelines and  issue
        permits until  States have authority (8  States  have  program)

        »v65,000 permit applications                          . i

        EPA Goal:  issue all major permits  by December,  1974


 II.   Legal Authority

        Sec. 106 - To obtain grants for  administering  pollution control
        programs, states must have program  to monitor  quality  of  navigable
        waters and groundwaters

        Sec. 304(h) - State program must include monitoring; EPA  promulgate
        guidelines for monitoring for state 402 programs

        Sec. 402(a)(3) - EPA permit program subject to same terms and
        conditions as apply to state permit program


III.   Major Objectives of EPA Compliance Monitoring Strategy

        Document effectiveness of State  agency  monitoring and  enforcement
        program

        Document effectiveness of permittee's self-monitoring  and reporting

        Document violations of NPDES permit conditions and  water  quality
        standards

        Provide evidentiary support to litigation


        EPA strategy recognizes  importance  of permittee self-monitoring
        and reporting system for identifying compliance schedule  and
        effluent limitation violations.

-------
IV   Compliance Monitoring Program Elements
       A.  Facilities Inspections
       B.  Case Preparation Monitoring Investigations
       C.  Review of Self-Monitoring Reports
             a)  Schedules
             b)  Effluent Limits
       D.  Information System
       E.  Quality Assurance
       F.  Ocean Dumping (where applicable)
       G.  Non-filers and False or Fraudulent Information
       H.  Special Actions (Emergency Powers, Citizen Suits, Section 402(h)
           actions)

-------
                  FLOW MEASUREMENT
                         By

                   James Hathaway and
                        and
                   Laurence Walz
                   EPA, NFIC-Denver
                   Denver CO
Presented at the Workshop on Sampling, Monitoring and Analysis
  of Water and Wastewater, March 6-12, 1974, Honolulu HI.

-------
                              FLOW MEASUREMENT
Introduction
     One of the most critical  parameters in a  compliance  monitoring  system
is flow.  Most current regulations and permits are  written  to  allow  either
a net weight of pollutants (kg/day) or net weight of pollutants  per  process
unit manufactured (kg/kkg of product)  to be discharged.   In order  to be
able to verify these regulations flow  measurements  taken  must  be as  accurate
and precise as possible.   The  installation of  a flow device or calibration
of an existing one will require a great deal of effort and  ingenuity as  well
as planning to insure accuracy of  the measurement.
     If an existing flow device is available at the sampling point it is
necessary to determine that the measurements are accurate.   This may be  done
by checking the flow on an instantaneous basis.  For instance, a flume or
wier can be checked by measuring the head on the measuring  structure and
checking the flow tables to determine  if the flow recorder  is  measuring
correctly.  Care should also be given  to make  sure  that the structure is
installed according to the appropriate design  criteria, i.e.,  proper crest
to head ratio, is structure leveled properly,  is head measured in  its proper
place.
                                                            March 7,  1974

-------
                                     -2-

I.   Open channel  flows include  sewers,  open conduits,  ditches,  streams,
                                                 x —
    and rivers.   Flow measurements  in open channels  can  be accomplished
    in the following way:
    A.  Instantaneous flows  are measured  with  1)  velocity meters,
        2) bucket and stop watch, or 3) calculated from  flow equations.
        1.  Velocity meters  are of  three  basic types:
            a.   The Price  meter is  the  most commonly used for larger
                type flows,  i.e., streams and  rivers.
                (1)  The Price  meter can  be used  on  a  wading rod
                     (shallow water depths) or on a  hand line when
                     flows are  measured from a bridge, suspension
                     cable,  boat, or other structure.  A hand line
                     is normally used when water  depth or velocity
                     prohibits  wading.
                (2)  A rating table is  normally furnished with  meters
                     when  purchased.  The curve should be checked to
                     ascertain  whether  the rating was  done for  a hand
                     line  or rod suspension.
                (3)  Measurement is made  by immersing  the meter to  a
                     prescribed depth (0.6 of  the depth  for a d <2  ft,
                     0.2 and a  reading at 0.8  for a  >2 ft the average
                     of the  velocities is  used) and  counting  the
                     revolutions the  wheel  makes  in  a minimum of
                     40 seconds.  The depth  of the water  and  distance
                     from  shore  are also measured.  These  data  are
                     recorded and used in  calculating the  flow.

-------
                             -3-
             Enough measurements  should  be  made  to  completely
             define the stream cross  section.   If the  cross-
             section is defined adequately,  that is measuring
             all  changes in the substrat, no more than 10  percent
             of the total  flow will  be measured  in  any one section.
    b.   Propeller type meters are often  used in  clean  waters  such
        as estuaries and bays.   This  type of meter  should  not be
        used where solids or sediments are  present  because of the
        clogging of exposed bearings.
    c.   Electromagnetic current meters work off of  the principle
        of electromagnetic induction  and therefore  are not subject
        to clogging or interferences.  The  advantage of this  meter
        is that they require very little area  for measurement.
        This means measurements can  be made very close to  pipe
        walls without interference on velocity due  to boundary
        conditions.  Some of these meters will  also measure both
        X and Y components of the current.   These meters are
        direct reading and do not require calibration curves.
2.   Discharge relationships can be established for  sections of open
    channels if the section produces  a  large change in head for a
    small change in flow.  The stage  should be measured and measure-
    ments made of the stream flow at various stages.  After enough
    data points have been collected,  a  rating  curve can be established,
    Flow data can be interpolated from the  curve but extrapolation
    of the curve may often cause the accuracy  of the data  to  be
    questionable.  The stage of the  control section can be measured

-------
                                 -4-

        by installing  a  staff gauge for instantaneous  readings  or
        recording  continuous  stages with a  water level  recorder.
    3.   Small  flows  (less  than 30 gpm)  discharging  from pipes may
        be measured  with a bucket and stopwatch.  When this  approach
        is used, a minimum of three repetitive  measurements  should
        be taken and averaged for the flow  value.
    4.   If the resources are  not  available  for  flow measurement,
        in some instances  the flow may  be calculated.   The following
        are methods  generally used for  such calculations:
        a.  Horizontal or  sloped  open end pipe  (Purdue  method).
        b.  California pipe.
        c.  Manning  equation.
        The accuracy of  these methods is  sometimes  limited because
        of the inability to assign proper constants (friction constant
        and slope).
B.  Continuous recording of flow  conditions is  desirable and should be
    done wherever  possible.  Various flow structures are available to
    give a reliable  control which can be monitored  continuously.   Wiers
    and flumes are among these structures and are discussed  below:
    1.   Wiers  are  most commonly installed for short-term monitoring
        because of the ease of construction and installation.   Rec-
        tangular,  Cipolletti  and  "V"-notch  wiers can be constructed
        out of inexpensive plywood or light metal.  If these wiers
        are properly installed, i.e., head  conditions  right  and leveled,
        they will  produce  reliable and  accurate flow measurements.
        When rags  and  other debris are  present  in the  waste  stream

-------
                             -5-
    clogging may occur at the wier crest and  cause inaccuracies
    The following flow ranges can be measured with these structures:
        a.  Rectangular wiers, .002 to 7,300  ft3/sec.
        b.  "V"-notch wiers, .02 to 4 ft/sec3.
2.  Because of their self-cleaning properties, flumes  are some-
    times installed in conditions where clogging may occur.   Flumes
    generally used are a) Parshall, and b) Palmer Bowles.
    a.  The Parshall flumes are often used in instances when
        continuous-flow measurements are needed for larger
        flows.  The Parshall flume is sometime&more time con-
        suming to set and seal in place, but  proper selection
        of equipment can result in many years of trouble-free
        measurement.  The Parshall must be leveled and all
        approach conditions met.  Recording of the head is taken
        2/3 of the distance on the converging section from the
        throat.  If the downstream flow backs  up and a ripple
        is evident downstream of the wier submergence is taking
        place.  When this occurs it is necessary to measure the
        head on the throat as well as the upstream head.  This
        measurement is necessary to determine the degree of sub-
        mergence for use in the flow equation.
    b.  Palmer-Bowles flumes are easily installed and provide a
        cross-section which can be measured continuously.  This
        type of flume can be blocked or sealed into a pipe or
        channel and will produce errors of less then 3 percent
        in the range of 10 to 90 percent pipe capacities.  The

-------
                     -6-
point of measurement is 1/2 the channel  width upstream
of the flume.

-------
                                       -7-
III.   Flow in pressure conduits  is  more difficult  to measure  and  requires
      more planning than that in open  channels.  Various  meters are  available,
      but because of expense of  installation  most  are  not conducive  to  a
      compliance monitoring program.   Such  meters  include the following:
      A.   Venturi meters.
      B.   Pitot tube.
      C.   Magnetic meter.
      D.   Rotameter.

-------
                              ,8-
III.  Miscellaneous Methods
     A.   Tracer dilution can be used  for large flow systems but
         only provide an instantaneous  measurement.   The expense
         involved and man-hours required for this  type of measure-
         ment is prohibitive in most  cases.   Some  tracers commonly
         used are (1) lithium,  (2}  sodium chloride (3) flourescent
         dyes and (4) radioactive isotopes.
     B.   Tank volumes of batch  discharges can be calculated by
         substraction of levels and then calculating  the area  of
         the structure involved.
     C.   Most facilities meter  the  incoming  water  rate for monthly
         billing.  If the water use is  known, an estimate can
         sometimes be derived by substracting product and process
         losses from this water  use.   At best this  should be
         considered as only  an  estimate.

-------
                  ORGANICS SAMPLER

                         AND

             FIELD EXTRACTION PROCEDURE
                          By

                     Laurence Walz
                     EPA, NFIC-Denver
                     Denver CO
Presented at the Workshop on Sampling, Monitoring and Analysis
  of Water and Wastewater, March 6-12, 1974, Honolulu HI.

-------
             ORGANICS SAMPLER AND FIELD EXTRACTION PROCEDURE

     The flow through the sampler is directed through a 3-way
electric solenoid valve to the resin column, then through the metering
system.  The column consists of a stainless steel tube filled as
follows:
     a)  5 cm polyurethane foam plug
     b)  50 grams of a 50-50 mixture of Amberlite XAD-2 and XAD-7 resins
     c)  10 cm polyurethane foam plug
     d)  glass wool filter
     Water for the liquid composite sample is taken off the system at
the 3-way solenoid valve.  The sample is pumped from the valve to a
19-liter sampling container which had been placed in a refrigerated
cabinet.  Extraction solvent (0.9 liters of freon or methylene chloride)
is initially added to the sampling collection container.  The solvent
and sample are mixed continuously by means of a magnetic stirrer.  The
temperature of the refrigerated cabinet is maintained at 10°C to keep
the extraction solvent from boiling off.  Aliquots of sample (55 ml for
a 72-hr composite and 80 ml  for a 48-hr composite) are collected every
15 minutes.   The time interval  is controlled by a timer.
     Extraction of the solvent from the composite water sample is made
with a modified 1-liter Imhoff cone.  The Imhoff cone should be enlarged
to accommodate a 2-liter sample and a teflon stopcock attached to the
bottom so that the extract can be drained off.
                                                         March  7,  1974

-------
                                   -2-
     TKe sample extraction procedure used is as follows;
     1.  The Imhoff cone is cleaned by thoroughly rtnstng with tap
water followed by a careful rinse down the sides with 100 to 200 ml
of acetone.
     2.  At the end of the sampling period, most of the water is dis-
charged until only the solvent and about 500 to 700 ml  of water are
left.  Measure and record the amount of water poured off.
     3.  Pour remaining solvent-water into the Imhoff cone and allow
the two layers to separate for about one minute.  Empty the organic
layer into a sample container.  Use 20 to 50 ml of solvent to clean
the sides of the Imhoff cone.  This solvent should also be decanted
into the sample container.
     4.  Measure the amount of water left in the cone.
     5.  Add 50 grams of sodium sulfate to the solvent, carefully
to avoid splashing.  After the sodium sulfate is added, cap the
container and shake vigorously for 30 seconds.
     6.  Record the date, sample location, and volume of water in the
composite sample on the bottle containing the solvent.
     7.  Ice the sample for shipment to the NFIC-Denver laboratories
for analysis.
     Grab samples are collected in glass sample containers pre-rinsed
in acetone and methylene chloride.  Samples taken in the above
containers are extracted with a freon or methylene chloride solvent.

-------
                                   -3-
A 2-liter glass separatory funnel is used as the separation container
The sample, extraction procedure, used is as follows:
     1.  Separatory funnel is cleaned by adding 100 ml of solvent,
mixed thoroughly and drained through the stopcock..  This procedure
is done twice.
     2.  The water sample is poured from the sample bottle to the
separatory funnel and 100 ml of solvent used to rinse the sample
bottle.
     3.  The two layers are allowed to separate and the solvent
layer drained into a 250 ml sample bottle.
     4.  The extraction is repeated using 50 ml of solvent.  When
completed, this extract is added to the 250 ml sample bottle.
Add 20 grams of sodium sulfate (20 grams) ice sample for shipment
to NFIC-D laboratory for analysis.

-------
                            SCHEMATIC, Organics Sampler

                                   Flow Diagram
Pump
   i
  By-pass
To liquid
Composite
X	
                        \j
             Solenoid  Valve
           (Timer Controlled)
                                      r
                                    cL
                                   Resin Column
                                          Float Controlled
                                          Constant Head Tank
                                            -f-fVi
                                            I J
! Metering
  Pump
                                                                  Measuring Cup
                                            ^Solonoid Valve
                                                                              Drain

-------
                      SCHEMATICS, Organics Sampler
                            Wiring Diagram
                (Units With New Type Liquid Level Control)
                                                                           Metering
                                                                           Pump
 Counter
Solenoid  °
Valve
            Disconnect
            Switch
            Box
t~' 3
  9

V
                                                             1.
                                                 Liquid
                                                 Level
                                                 Control
                         High
                         Level
7
Low
Level
                                                10
                                              !.J.
                                                                              Power
                                                                              Recent n-. le
                                110vac
                                Line
                         Timer
               i
           Solenoid (water sampler)

-------
    COMPARATIVE SAMPLING RESULTS: SOME EXAMPLES
                        By
                  James S. Kumagai
                  Sunn, Low, Tom and Kara Inc.
                  Honolulu HI
Presented at the Workshop on Sampling, Monitoring and Analysis
  of Watfir and Wastewater, March 6-12, 1974, Honolulu HI.

-------
               COMPARATIVE SAMPLING RESULTS:  SOME EXAMPLES
     Some sampling results are presented here to illustrate the magnitude
of water quality variations in wastewaters and in coastal  waters.   The
results used here as examples were intended either for estimating  the
loads on wastewater treatment works or for evaluating water quality impact
on coastal waters from wastewater discharges.  The fact that water quality
varies over a period of time requires a sampling program broad enough in
scope to cover the spectrum of water quality values.
A.   Program Variables
     The variables considered here are two of the following:
     1.  Number of samples.
     2.  Frequency - how often and when.
     3.  Duration - over what period of time.

B.   Constraints
     1.  Time
     2.  Budget
     3.  Maximizing information gained/unit effort

C.   Guidelines for Sampling Program
     1.  Know purpose of sampling and what statistical parameters  are most
         meaningful: maximum values, average, minimum, frequency distri-
         bution.
     2.  Work with cause-effect relationships: factors which affect water
         quality.
         a.  wastewaters: production practices and rates;  nature of raw
             materials
         b.  receiving waters: surface and subsurface runoff, waste dis-
             charges; mixing, transport, reaction
     3.  Utilize available data on factors affecting water quality.
         a.  stream flow data are available for Hawaii streams
         b.  production rates are available for evaluating industrial
             discharaes

-------
         c.  certain seasonal oceanographic conditions are known from
             experience
     4.  Set up sampling frequency and duration to coincide with variations
         1n the factors causing water quality changes (for example, sea-
         sonal changes).
     5.  Maximize information gained by correlating sampling results to
         causative factors or to quality indicators (for example, coral
         growth, fishes).

D.   Examples
     1.  Municipal raw sewage sampling; required average values
             Situation                              Action
     2.
         Limited Time & Budget
         Limited Manpower

         Result (Source: WQPO, 1971)
                                 Three days, 24-hour composite
                                 Single weekly grab samples over
                                 one year; 90 samples for Pearl
                                 City, 103 samples for Kailua
                             Three Day Composite        One Year Grab
                             (ave mg/1 +_ std dev)    (ave mg/1 +_ std dev)
         Pearl City STP
           BOD
           Suspended Solids
         Kailua STP
           BOD
           Suspended Solids
                          233+85
                          183+13

                          138+51
                          120+34
267+88
282+125

 80+40
151+63
Sugar mill washwaters; required: design suspended solids and
impact on coastal waters (SLTH, 1972-1973)
             Situation
         Limited Time

         Short Term/Long Term
         Variation Required
                                           Action
                                 Composite samples, hourly samples
                                 selected over different seasons
                                 Correlation with cane harvesting
                                 and processing rates over 7-year
                                 periods; determination of con-
                                 centration frequency curve for
                                 design

-------
    Results:

                                     Ton Soil/Ton Net Cane
                                Mean and 95% Confidence Interval
                                       n=24, three days*

    a.  Season

        Dry                                0.12+0.01
        Rainy                              0.19+0.04


     * These results were extended by correlating soil loads to
       trash in field cane to derive estimates of variations from
       day-to-day and year-to-year (see figure).

    b.  Frequency curve, see figure.


3.  Coastal waters (SLTH, 1973)

        Situation                              Action

    Required: natural variation      Monthly sampling over one-year
    in water quality for compari-    concentration frequency curve
    son with sites close to
    sewage outfall

    Result: (see figure)

-------
                                                  70    en   50   <0
                                                                   30
                                                                        ?0
                                                                               10
                                                                                                    0.5   0.? 0.) 005
                                                                                                                     0.01
                        TREQUENCYjDIS
INSTANTANEOUS

          Cascade; Water. S
-------
CUMULATIVE FREQUENAOF SOIL LOADS
  TO WASTEWATER TREATMENT PLANT

-------
1000
 100
  10
                                      I  •   I
                         I   I        I ^
             r—i~»-
                                       ^
                                                 X

                                                   ~&-
           TT
           rorr
                                               -OCL
                                     Or1
                                           I   o°!
                                            rf^
                                        -4-
                                                            i   i
                                                           TT
                                                          ~sr
                                                            j	L i
0.01  0.1  O.S 1  2   5   10  20 30 40 50 CO70 CO  9095  9099
             X OF TIME LESS  THAN OR EQUAL TO INDICATED VALUE
             STATION 1
             -    H33-H.  ug/1
             a   TK.N  ,
                                                            -'-h1
                                                            99;."S'q9"!
                                                                      99.9 99.99
Iftf)
1UU •
10-
1.
0.











t

* , 1 ,
_J







' • . ' '• i
i







1 1
1
















1 !
1 ,













1 '
t

n

"T
i
1












— i — ; —
i i
--



















' : I ' ' '
! ' '













1 1









1 ,
1



~+— — i— A


*£-


/Sf






^












-1




1 	 1








— 1




> 1 i
"
1-
-^



1

1
1 . i
I






t
I


i
i
t
1

n 	 1 	
— 1 	 1 	 1 	 '— 	 -»— 	 <} 	 i —
; 1 I 1 i 1 rO

t
— 1 	




1 •

^
0



o
3



1


0cP


c


HS



	 ^_f_
1


•
D* ' ' '
















•
1 ,,!,!.
1 : '



• 1









_i 	 ! 	 j 	 i 	
| • 1 	




—
;
i


-
On
01 0.1 0.5 1 2 5 10 20 30 40 SO CO 70 SO 9095 S3 S9
X OF TIKE LESS THAN OR EQUAL TO INDICATED VALUE
STATION 1
o P04-P, ug/1
A TCT-P, ug/1


.5"


—



1 —
r.o >"'•
S3.S.

-------
     AN OUTLINE ON THE BACTERIOLOGY OF WATER
                        By

                 Kathleen Shimmin
                 EPA, Region IX
                 San Francisco CA
Presented at the Workshop on Sampling, Monitoring and Analysis
 of Water and Wastewater, March 6-12, 1974, Honolulu HI.

-------
                    BACTERIOLOGY OF WATER


Historically bacteria in water have been of concern as an
indicators of potential disease transmission.  Water-borne
infections can be contracted through ingestion of or contact
with contaminated water and also by ingestion of shellfish
(filter feeders which concentrate particles in water).  Some
reports (Janssen and Meyers, 1968) have indicated that fish
from contaminated waters may be affected by human pathogens
with the concomitant possibility that the fish could be vectors
of human disease.

I.  Diseases Caused by Water-borne Pathogens

    A.  Typhoid fever - an infectious disease beginning with
        intestinal involvement and becoming systemic; fatality
        rate 2% with therapy and 10% in untreated cases;
        carrier state common.

        1.  Organisms - Salmonella typhi.  Other Salmonella sp.
            and Shigella sp. may cause milder forms of
            gastroenteritis.

        2.  History

            a)  Budd - Essay on Typhoid Fever - 1856

            b)  Currently - still appears when water treatment
                systems break down

    B.  Cholera - an acute intestinal disease with rapid onset,
        profuse diarrhea and dehydration.  Death rate may range
        from 5 - 75%.

        1.  Organism - Vibrio cholerae

        2.  History

            a)  Snow's study - Broad Street pump, 1854

            b)  Endemic in India - spreads from there

                   1830 - Pandemic in Europe
                   1817 - 1823; 1883 - 1896, U.S. epidemic
                   1826 - 1837 - Quebec

            c)  1892 - Hamburg - Altona study
                Hamburg got water from Elbe.  Deathrate 134/100,000
                Altona ran Elbe water through sand filter  —>•
                Death rate 23/100,000.

-------
            Encouraged water treatment in Europe and England.

        d)  1970 - Epidemic in Middle East - Turkey,
            Northern Africa (1000's of cases).

C.  Leptospirosis - an acute infectious disease with fever
    and chills; jaundice may occur; fatality rate may
    approach 20%.

    1.  Organism - Leptospira sp. - enters through mucous
        membrane or break in skin.

    2.  Currently of importance in the U. S. and other
        parts of the world wherever people come into
        contact with contaminated waters; e.g., farm
        ponds and streams accessible to infected animals.

        Many animals may carry organism -
            Rodents, cattle, horses, dogs, etc.

        1970 - deaths of seals along California Coast-
        leptospirosis the cause.

    3.  Conditions favoring survival of leptospira - slowly
        flowing or stagnant water; pH slightly alkaline;
        temperature slightly high  (22°C).

D.  Tularemia - an infectious disease of animals and man;
    begins with chills and fever;  usually an ulcer appears
    at site of original infection; fatality, 5% in untreated
    cases.  Disease is spread by arthropod bite, by contact
    with  infectious animals or by  ingestion of contaminated
    food  or water.

    1.  Organism - Franciscella tularensis  (Pasteurella
        tularensis)

    2.  History

        a)  Epidemic in Russia 1937-43

            Disease spread by water in wells and streams

        b)  Has been isolated from water in U.S. (including
            California) and other parts of world.

        Avoid water-borne infection by not drinking untreated
        water in endemic areas.
                          -2-

-------
Tuberculosis - a chronic well-known bacterial disease;
usually pulmonary involvement, but systemic spread may
also occur.

1.  Organisms - Mycobacterium tuberculosis

    Found in discharges from tuberculosis sanitoriums.
    Conventional treatment removes part but not all
    t.b. organisms.
    Chlorination is ncessary - can survive in water a
    long time.
    Cases due to water-borne infection have been
    associated with near-drowning in contaminated water.

Viral diseases

1.  Infectious hepatitis - most cases from direct person-
    person contact, but a low percentage are from water
    or from eating contaminated shellfish.

    One of few viral diseases actually proven to be
    transmitted by contact with contaminated water.

    A number of water-borne epidemics throughout the
    world
       New Delhi in 1950's - over one million people
       were infected.

2.  Poliomyelitis - little substantiated evidence of
    water-borne transmission.

3.  Many potentially pathogenic viruses are found in
    feces and other discharges of man.

    It is possible that studies may give more evidence
    for water-borne infections - those viruses found
    in human sewage include:

             Enteroviruses:  Coxsackie, poliomyelitis,
              ECHO  (enterocytopathogenic human orphan)
             Reoviruses
             Adenoviruses
             Rhinoviruses
             Infectious hepatitis

Diseases can also be caused by protozoa and invertebrate
animals in water.
                     -3-

-------
         Two among many examples:

         1.  Amoebic dysentery - caused by a pathogenic protozoan
             Endamoeba histolytica

         2.  Swimmer's itch - a dermatitis caused by swimming in
             waters contaminated by larvae of schistosomes of
             birds and rodents.

             People have been infected while bathing in lakes
             in the United States.

         A more severe disease, schistosomiasis (blood fluke
         disease), which becomes a chronic infection involving
         the intestinal and urinary tracts, has not been found
         in the United States.

II.  Bacterial Indicators of Pollution - General

     A.  Usually one can't test for pathogens directly.

         1.  Tests too time-consuming.

             Too difficult for routine work in a water laboratory.

             Negative recovery results give a false sense of
             security.

     B.  Characteristics of a good indicator organism

         1.  Applicable to all types of water.

         2.  Present when fecal contamination present and absent
             when fecal contamination not present.

         3.  Persists as long as the most resistant pathogen.

         4.  Can be easily and reliably identified in laboratory.

         5.  Not found as natural inhabitant of unpolluted
             environment.

     C.  Bacterial indicators are used in a variety of circumstances,

         1.  Tests for compliance with bacterial water quality
             standards.

             a)  Drinking water, raw supply and finished water.
                              -4-

-------
              b)   Water for specific purposes:  shellfish culti-
                  vation, recreational waters to be used for
                  primary and secondary body contact.

              c)   Enforcement of standards controlling waste
                  discharge from industries and municipalities.

          2.  Treatment plant effectiveness evaluation.

          3.  Water quality surveys.

              a)   Detecting source and extent of pollution.

          4.  Special studies.

              a)   Epidemiological studies to detect source of
                  pathogenic organisms

              b)   Investigations of problems caused by certain
                  bacteria
                      Sphaerotilus, Clostridia

III.  Coliforms - as Indicators

      A.  History and Description

          1.  In 1885 Escherich isolated from human feces an
              organism which he termed Bacterium coli-commune.
              The current name for the bacterium is Escherichia
              coli.

          2.  Escherich thought the bacterium to be peculiar to
              human feces.  Since his time, however, Escherichia
              coli and other organisms from the coliform group
              have been recovered from polluted and unpolluted
              soils and from vegetation.

          3.  Standard Methods defines the coliform group as
              "gram-negative nonsporulating rods, which ferment
              lactose with the production of gas within 48 hours
              at 35°C".

          4.  The coliform group includes organisms from the
              following genera.

              a)  Escherichia     c) Klebsiella   e) Serratia
              b)  Aerobacter      d) Erwinia
                 (Enterobacter)
                                 -5-

-------
B.  General tests employed for detection of total coliform
    populations

    1.  Requirement and assumptions

        Water quality standards may be written in terms of
        limiting numbers of total coliforms per given volume
        of water.

        It is assumed that the bacteria present in a sample
        can be uniformly dispersed, so that accurate sample
        dilutions may be made.  Either the sample or its
        dilution is inoculated into a given medium and
        incubated.  The resulting growth allows one to
        assess the number of bacterial cells per volume
        in the original sample.

    2.  Tests

        a)  Multiple Dilution Broth Tube Method.

            Replicate 10-fold dilution tubes are inoculated
            and incubated for 24-48 hours at 35°C.
               (Lauryl Tryptose Broth for presumptive test,
                Brilliant Green Bile Lactose Broth for
                confirmed test).
            A positive result is indicated by gas production.
            The numbers of positive tubes and their dilutions
            are noted.  The Most Probable Number  (MPN) of
            coliforms per 100 ml can be calculated by
            consulting tables in Standard Methods.  The
            tables give estimates and confidence intervals
            for the original number of cells per volume in
            the sample.  It should be emphasized that the
            MPN is a statistical estimate, not an actual
            count of the numbers of bacteria.  Confirmed
            coliform results require a minimum of two days
            and a maximum of four.

        b)  Membrane Filter Method.

            Various concentrations of a water sample are
            filtered through a membrane (cellulose acetate)
            filter.  The filter is placed onto m-Endo medium
            and incubated for 24 hours at 35°C.

            It is assumed that each bacterial cell deposited
            onto the filter (and capable of utilizing the
            medium employed - m-Endo,  in this case for total
            coliforms)  will replicate to form a colony-

            The numbers of colonies present at given dilu-
            tions are noted.


                            -6-

-------
            The total number of coliform bacteria per
            100 ml is calculated.  This is an actual
            count, not a statistical estimate.

            Time required for confirmed coliform results
            is 24 hours.

C.  Differentiation of the coliforms:  fecal vs non-fecal

    1.  Requirement

        It is often necessary to distinguish between
        water contaminated with unpolluted soil and that
        contaminated with fecal material.  A positive
        total coliform test does not make this distinction.
        Historically it was felt that Escherichia coli
        was characteristic of fecal contamination and
        Aerobacter aerogenes was typical of non-fecal
        contamination.(This is not necessarily true).

    2.  Tests

        a)  IMViC series.  The letters are a mnemonic
            device to describe:  Indole, Methyl Red,
            Voges-Proskauer, and Citrate.

               i. Indole produced from metabolism of
                  tryptophane, an amino acid.  Reaction
                  is typical for E_. coli, not for A.
                  aerogenes.

              ii. Methyl Red test.  When typical E. coli
                  grow in glucose peptone broth, their
                  fermentation brings the pH to 4.2 - 4.6
                  (methyl red indicator red, positive)
                  and their growth terminates.  The
                  terminal pH for A. aerogenes in a similar
                  culture medium is above 5.6 (methyl red
                  indicator yellow, negative).

            iii.   Voges-Proskauer test.  Typical A.
                  aerogenes growing in glucose-peptone
                  broth produce as a by product acetylmethyl
                  carbinol  (positive test); E.  coli does
                  not (negative test).

             iv.   Citrate test.  Typical Aerogenes can
                  utilize citrate as sole carbon source
                  (positive test); E. coli cannot  (negative
                  test).
                          -7-

-------
        b)  Elevated temperature tests.  Underlying
            assumption for these tests is that organisms
            of fecal origin will grow at elevated tempera-
            tures (45°C), whereas those of non-fecal
            origin won't.  Various media and conditions
            are employed.  All procedures require incuba-
            tion in a water bath (for accurate maintenance
            of temperature).  Incubation should begin
            within 30 minutes of inoculation.

              i.  Eijkman test requires pure culture,
                  lactose broth, 48 hours at 44.5± 0.2°C.

             ii.  EC Broth (the medium currently recommended),
                  does not require pure cultures.  Results
                  are read after incubation for 24 hours
                  at 44.5+ 0.2°C.  Geldreich  (1966) states
                  that the elevated temperature con-
                  firmatory test has an accuracy of
                  correlation between positive coliform
                  tubes and fecal origin  (from warm-blooded
                  animals) of 96%.

            iii.  Boric Acid Lactose Broth gives results
                  similar to those from EC Broth.  Incu-
                  bation is 48 hours.

             iv.  mFC medium may be used with membrane
                  filters.  Results are read after 24 hours
                  incubation at 44.5± 0.2°C.  According
                  to Geldreich  (1966) the accuracy of
                  correlation between positive results
                  by this method and actual fecal origin
                  is 93%.

D.  Applications

    1.  Total coliform test is used especially in evaluating
        potability of drinking water.

    2.  Bacterial standards have been established to
        determine acceptability of a water for a given
        use (eg. , drinking water supply, recreation with
        primary or secondary contact, etc.).  The
        standards are usually expressed in terms of total
        coliform counts and, with increasing frequency,
        may include also fecal coliform levels.
                          -8-

-------
IV.  Fecal Streptococci as Indicators

     A.  Background

         Since coliforms can be found as natural inhabitants
         of unpolluted soil and since their die-off rate in
         seawater is rather rapid, there have been attempts
         to find additional indicator organisms to supplement
         the coliform tests.  Fecal streptococci have been
         investigated in this regard since they are always
         present in the feces of warm-blooded animals, and
         since they are more persistent in seawater than are
         the coliforms.  The ease with which the fecal
         streptococci could be detected and enumerated was
         increased by the use of azide dextrose broth, developed
         in 1950 (Mallmann and Seligmann, 1950).

     B.  Composition of the group

         1.  Standard Methods defines the fecal streptococci
             as theintestinal streptococci from all warm-
             blooded animal fecal wastes.

         2.  Fecal streptococci are gram-positive, spherical,
             chain-forming bacteria, which usually can develop
             at 45°C.  Included are the following groups:
             enterococcus; S_. mitis-salivarius; S_. bo vis;
             §.* equinus; enterococcus biotype.  ~~

     C.  Relationships between fecal streptococci and fecal
         coliforms.

         1.  The fecal streptococci may be compared to fecal
             coliforms, since both originate from fecal sources.
             Ratios between the two groups may vary depending
             upon sources, methods of enumeration, and geo-
             graphical location.

         2.  Generally, an FC/FS ratio of 2-4/1 indicates
             fecal pollution of human origin, whereas a ratio
             of less than 1/1 suggests fecal pollution of
             non-human, animal origin.  The following table
             shows data, compiled by Geldreich  (1966),
             describing the comparative densities of fecal
             streptococci and fecal coliforms in various warm-
             blooded animals, including man:
                               -9-

-------
ESTIMATED PER CAPITA
FROM SOME ANIMALS
CONTRIBUTION OF INDICATOR MICROORGANISMS
Animals
Man
Duck
Sheep
Chicken
Cow
Turkey
Pig

Average indicator Average contri-
density per gram bution per capita
of feces per 24 hr
Avg wt of Fecal Fecal Fecal Fecal
feces/24 hr coliform, strepto- coliform, strepto-
wet wt, g million cocci, million cocci,
million million
150 13.0 3.0 2,000 450
336 33.0 54.0 11,000 18,000
1,130 16.0 38.0 18,000 43,000
182 1.3 3.4 240 620
23,600 0.23 1.3 5,400 31,000
448 0.29 2.8 130 1,300
2,700 3.3 84.0 8,900 230,000
3. Identification of specific groups within the fecal
streptococci may give an indication of certain
characteristics of pollution: recentness and
animal species involved. Organisms of the
S. salivarius group are unique to humans (Kenner
et al, 1960); S. bovis and S. eguinus are usually
not found in humans but predominate in cows , pigs ,
sheep, and horses; enterococcus and enterococcus
biotype groups comprise the predominant flora of
humans and fowl. Presence of S. salivarius
indicates recent pollution, because the organism
has a rapid die-off in surface waters.

Ratio
FC/FS
4.4
0.6
0.4
0.4
0.2
0.1
0.4

    C.   Tests
        1.   Assumptions

            As with the coliform tests, it is assumed that
            uniform dispersal of bacterial cells and that
            accurate dilutions are both possible.

        2.   Multiple Dilution Broth Tube Method
                             -10-

-------
            Replicate, 10-fold-dilution tubes are inoculated
            and incubated 24-48 hours at 35°C.  (azide dextrose
            broth for presumptive test, ethyl violet azide
            broth for the confirmed test - both are listed in
            Standard Methods)

            A positive result is indicated by turbidity of
            the broth.

            By noting the numbers of positive tubes and their
            respective dilutions, one can calculate the MPN
            (see Standard Methods).

            Confirmed results require four days of test time.

            Recent reports  (Buck, 1969) indicate that for
            marine waters the MPN method, using azide dextrose
            and ethyl violet azide broths, should include a
            final microscopic examination to insure that
            streptococci are indeed present in positive tubes.
            Nonstreptococcal growth has been observed in both
            these media following seawater inoculation.

            The filters are placed onto KF medium (which has
            a higher recovery than m-Enterococcus medium) and
            are incubated for 48 hours at 35°C.

V.  Other Bacterial Indicators of Pollution

    A.  Total bacterial counts

        1.  The term "total bacterial count" is fallacious

            a)  Methods which cultivate bacteria in the
                laboratory will recover only those bacteria
                which can grow in the growth conditions
                provided.  It is not possible in the laboratory
                to provide all variations of environment
                simultaneously in the same growth medium.

            b)  Methods which enumerate bacteria directly
                (microscopic counts, turbidity measurements)
                do not distinguish between living and dead
                cells.  Identity of the bacteria remains
                unknown.
                             -11-

-------
    2.  General plate counts can be useful in detecting
        bacterial changes in a water source or treatment
        process.  However, they give no indication of
        fecal origin of pollution and little identifica-
        tion of the bacterial species being cultured.

    3.  Historically plate counts were used to assess
        water quality.

        a)  Robert Koch devised plate-count standards
            for safety of a water source  (limit was  100
            bacteria/ml, using gelatin medium, incubation
            3 days at 20°C).

        b)  Differential temperature counts have been
            used as indicators also.  Duplicate plates
            were inoculated, one incubated at 20°C and
            the other at 37°C.  When count ratios were
            compared, 20°/37° greater than 10 indicated
            non-polluted water; a ratio of one or less
            indicated polluted water.

B.  Testing for miscellaneous indicators

    1.  Clostridium perfringens, a gram-positive, pathogenic,
        sporeforming rod, commonly found  in soil and in
        feces of warm-blooded animals.  Because the  organism
        forms spores, it can exist in soil almost indefi-
        nitely.  Its presence does not necessarily indicate
        presently-polluted water.

    2.  Pseudomonas aeruginosa, a gram-negative, pathogenic
        rod, which may be found in the intestinal tract of
        humans and warm-blooded animals.  Since the  organism
        is not found in large numbers in  all humans, its
        value as an indicator is limited.  (Sutter et al,
        1967) .

    3.  Viruses - may be assessed in the  laboratory.  How-
        ever,  special skills and equipment are required.
        Tests which can be carried out on a routine basis
        are still in the developmental stages.

C.  Direct testing for pathogens in water is possible.

    1.  Applications
                          -12-

-------
             a)  Direct testing is useful in tracing sources
                 for epidemiological studies.

             b)  Direct study of pathogens is also required
                 in special studies for it has been shown that
                 pathogenic bacteria have been present in a
                 water without this danger being reflected by
                 routine coliform tests.  (Greenberg and Ongerth,
                 1966; Seligmann and Reitler, 1965).

             c)  Used in shellfish studies (organism already
                 concentrated by shellfish filtering system).

         2.  Limitations

             a)  A negative finding for a given pathogen does
                 not mean that the water is safe from a public
                 health standpoint.

             b)  Technical skills and equipment required to
                 study pathogens are extensive and may not be
                 found in every water laboratory.

             c)  Since the recovery of the pathogens (which
                 are present, usually, at low levels)  often
                 depends upon concentration techniques,
                 quantified results are not always obtainable.

VI.  Considerations for Bacteriological Testing in Field Studies

     A.  Time of Processing

         1.  Collection and Preservation of Samples, General

             According to recent work done in EPA  laboratories
             in Cincinnati, Ohio and Edison, New Jersey,  if
             there is any delay between collection and processing,
             the samples should be iced (but not frozen).

         2.  Fresh-water Samples

             a)  Properly iced samples should not be held for
                 longer than four to six hours for total coliform
                 analysis.

             b)  Properly iced samples for fecal coliform analysis
                 should be processed within two to four hours.

         3.  Ocean-water Samples
                               -13-

-------
        a)   Samples should be run within one hour.  The
            maximum holding time (for properly-iced samples)
            is two hours.

        b)   Fecal coliform samples should be processed
            within 1/2 hour.  A fecal coliform sample
            preserved for  two to four hours is useful only
            for determination of general range of numbers.

B.  Temperature and Time

    1.  The definition of  the organisms recovered is based
        partially upon the temperature at which they grow.
        Therefore, it is critical that the incubation
        temperature be maintained strictly within the
        designated limits.  Time allowed for growth is
        part of the definition also.

        a)   Total coliforms in multiple tubes, 48 hrs ±
            3 hrs at 35° ± 0.5°C.

            Total coliforms by membrane filter, 22-24 hrs
            at 35° ± 0.5°C.

        b)   Fecal coliforms in multiple tubes, EC medium,
            44.5° ± 0.2°C  for 24 hrs.

            Fecal coliforms by membrane filter, mFC medium,
            44.5° + 0.2°C  for 24 hrs.

            Both these incubations should be in a water
            bath.  Plates  containing membrane filters are
            sealed in plastic bags  (eg. Whirl-pak) to block
            water access.

        c)   Fecal streptococci by multiple tube and by
            membrane filter, 35° + 0.5°C, 48 hrs.

C.  Membrane-Filter Processing vs Multiple-Tube Technique
    (Coliforms)

    1.  Preparation time

        a)   Multiple-tube  media for total and fecal coliforms
            may be prepared in advance.

        b)   Membrane-filter medium  (mEndo)  for total coli-
            forms should not be prepared more than 72 hours
                          -14-

-------
        in advance.  Medium for fecal coliforms  (mFC)
        may be stored for  5  to  7  days.

2.  Membrane-filter technique

    a)  Advantages:  confirmed results within 24 hours;
        test may be done entirely in field  (media is
        not autoclaved); permanent record of results;
        less space necessary than for tubes.

    b)  Limitations:  suitable filtration volume must
        be selected; high  turbidity interferes;  large
        numbers of non-coliform inhibit  appearance of
        coliforms; colonies must be recognized and
        counted; some percentage of counts  should be
        verified by tube testing.

3.  Multiple-tube  technique

    a)  Advantages:  media may  (must) be prepared in
        advance; positive  test  is easy to read;  less
        interference from  turbidity and  large numbers
        of non-coliforms  (than  is the case  with  membrane
        filter technique).

    b)  Limitations:  media  cannot be prepared in field;
        confirmed  results  require a minimum of 48 ho'jrs
        and a maximum,  of  06  hours incubation; a  large
        amount of  storage  anc1  incubator  space is recv:.rcc
         (as compared to that necessary for  membrane
        filters^ .

-------
MOST PROBABLE NUMBER CALCULATION

Replicate ten-fold dilutions of the sample are inoculated into
the appropriate broth and incubated.  Following incubation, the
numbers of positive tubes at each dilution are noted in order.
The 3-number code which is formed may be looked up in MPN
tables for an estimate of the number of bacteria per 100 ml.

The tables are based upon inocula of 10 ml, 1 ml, 0.1 ml sample.
If the concentrations you use are different from these, multi-
plication by the appropriate power of 10 is necessary to put
your results in the correct range.

Examples (5-tube tests)

                                             95%
                                           Confidence
             Results                Code    Limits      MPN/100 ml
                                          Lower  Upper

a.  +++++  ++	   +	             521    23    170       70
    10 ml  1.0 ml  0.1 ml
                   ++	   ++	     522   280   2200      940
    10 ml  1 ml    0.1 ml  0.01 ml

    	  +	   	             010   <. 5      7        2
    10 ml  1 ml    0.1 ml

    	  	   	             000                     <
    10 ml  1 ml    0.1 ml
                                     555                 >2400
    10 ml  1 ml    0.1 ml
                               16

-------
GRAM'S STAIN

1.  Using a clean slide, make a thin smear of the culture on
    the slide.

2.  Air dry

3.  Heat fix by passing slide briefly through flame.

4.  Add gentian violet dye - let stand one minute.  Rinse with
    tap water.  Rinse with Gram's iodine.

5.  Add Gram's iodine - let stand one minute.  Drain slide.

6.  Add decolorizer - let stand 10-15 seconds.  Rinse with
    water.  Rinse with safranin.

7.  Add safranin dye to counterstain - one minute.  Rinse with
    tap water.  Blot dry with paper towel.

    View slide under microscope.

    Gram-positive organisms appear blue-violet; gram-negative
    bacteria retain only the counterstain and hence appear red
    when safranin is used.
                                 17

-------
                           REFERENCES


Control of Communicable Diseases in Man, 10th ed., APHA.  J.  E.
Gordon, Ed.  Published by American Public Health Association,
1970 Broadway.  New York, N.Y.  1965.

Standard Methods for the Examination of Water and Wastewater,
12th ed., APHA, AWWA, WPCF.  Published by American Public Health
Association, 1790 Broadway, New York, N.Y.  1965.

Buck, J. D.  Occurrence of False-Positive Most Probable Number
Tests for Fecal Streptococci in Marine Waters.  Appl.  Microbiology.
18:562.  1969.

Cockburn, T. A. and Cassanos, J. G.  Epidemiology of Endemic
Cholera.  Public Health Reports.  75:791.  1960.

Diesch, S. L. And McCulloch, W. F.  Isolation of Pathogenic
Leptospires from Waters used for Recreation.  Public Health Reports
81:299.  1966.

Geldreich, E. E.  Sanitary Significance of Fecal Coliforms in the
Environment.  U. S. Department of the Interior.  FWPCA Publ.
WP-20-3.  1966.

Greenberg, A. E. and Kupka, E.  Tuberculosis Transmission by Waste
Waters - A Review.  Sewage and Industrial Wastes 29:524.  1957.

Greenberg, A. E. and Ongerth, H. J.  Salmonellosis in Riverside,
California.  Journal AWWA.  58:1145.  1966.

Janssen, W. A. and Meyers, C. D.  Fish: Serologic Evidence of
Infection with Human Pathogens.  Science.  159:547.  1968.

Jeter, H. L.  Bacteriological Indicators of Water Pollution, in
Water Quality Studies, U. S. Department of the  Interior, FWPCA
Training Program.  October 1969.

Kabler, P. W. et al.  Public Health Hazards of  Microbial Pollution
of Water.  Proceedings of the Rudolfs Research  Conference.  1961.

Kenner, B. A. et al.  Fecal Streptococci II.  Quantification of
Streptococci in Feces.  Am. J. Public Health.   50:1553.  1960.

Mailman, W. L. and Seligman, E. B., Jr.  A Comparative Study of
Media for Detection of Streptococci in Water  and Sewage.  Am.  J.
Public Health.  40:286.  1950.

-------
Metcalf, T. G. and Stiles, W. C.  Enteroviruses Within an Estuarine
Environment.  Am. J. Epidemiology.  88:379.  1968.

Prescott, S. C., et al.  Water Bacteriology.  John Wiley and Sons,
Inc.  1946.

Seligmann, R. and Reitler, R.  Enteropathogens in Water with Low
Esch. coli Titer.  Journal AWWA. 57:1572.  1 65.

Sutter, V. L., Hurst, V., and Lane, C. W.  Quantification of
Pseudomonas aeruginosa in Feces of Healthy Human Adults.  Health
Laboratory Science.  3":245.  1967.

Taylor, F, B. et al.  The Case for Water-borne Infectious Hepatitis.
Am. J. Public Health.  56:2093.  1966.
This outline was prepared by K. G. Shimmin, Section Chief
Microbiology, Environmental Protection Agency, Region IX,
620 Central Avenue, Alameda, California  94501.

-------
                 ANALYTICAL METHODS


                        FOR


            METAL AND PESTICIDE ANALYSIS
                        By

                    Ho Young
                    EPA, Region IX
                    San Francisco CA
Presented at the Workshop on Sampling, Monitoring  and Analysis
  of Water and Wastewater, March  6-12, 1974, Honolulu HI.

-------
                ANALYTICAL METHODS FOR METALS


     Metals that are analyzed in wastes consist of large numbers
of cations of the alkali, alkaline earth, noble, heavy metal
series etc.

I.  Methods for metal analyses:  The metal analyses depends on
    the structures of the metal ions, the energy levels of
    their electrons and the excitation energy.

    A.  Emission flame photometry:  It measures the amount of
        light emitted by the excited atom which is aspirated
        into a flame and atomized.  It is commonly used for
        analyzing Na, K, Ca etc.

    B.  Atomic absorption spectrophotmetry:   This technique is
        different from the above in that this method measures
        the light absorbed.  When a sample is aspirated into a
        flame and atomized, it absorbs at a certain wavelength
        of the light source.

        1.  Instrument:   Atomic absorption spectrophotometer.

            a.  A light beam is directed through the flame into
                a monochromator, and onto a detector that measures
                the amount of light absorbed.

            b.  Light absorption is more sensitive than light
                emission because it depends upon the presence
                of free unexcited atoms.

            c.  In the usual flames, the ratio of the unexcited
                to excited atoms at a given moment is very high.

            d.  Because each metallic element has its own charac-
                teristic absorption wavelength,  a source lamp
                composed of that element is employed, making the
                method relatively free of spectral or radiation
                interferences.

            e.  The amount absorbed in the flame is proportional
                to the concentration of the element in the sample.

            f.  Interference

                1.   The most troublesome interference results
                    from the lack of absorption of atoms bound
                    in molecular combination in the flame which
                    is not sufficiently hot to dissociate the
                    new molecule.

-------
                ANALYTICAL METHODS FOR METALS


     Metals that are analyzed in wastes consist of large numbers
of cations of the alkali, alkaline earth, noble, heavy metal
series etc.

I.  Methods for metal analyses:  The metal analyses depends on
    the structures of the metal ions, the energy levels of
    their electrons and the excitation energy -

    A.  Emission flame photometry:  It measures the amount of
        light emitted by the excited atom which is aspirated
        into a flame and atomized.  It is commonly used for
        analyzing Na, K, Ca etc.

    B.  Atomic absorption spectrophotmetry:   This technique is
        different from the above in that this method measures
        the light absorbed.  When a sample is aspirated into a
        flame and atomized, it absorbs at a certain wavelength
        of the light source.

        1.  Instrument:  Atomic absorption spectrophotometer.

            a.  A light beam is directed through the flame into
                a monochromator, and onto a detector that measures
                the amount of light absorbed.

            b.  Light absorption is more sensitive than light
                emission because it depends upon the presence
                of free unexcited atoms.

            c.  In the usual flames, the ratio of the unexcited
                to excited atoms at a given moment is very high.

            d.  Because each metallic element has its own charac-
                teristic absorption wavelength,  a source lamp
                composed of that element is employed, making the
                method relatively free of spectral or radiation
                interferences.

            e.  The amount absorbed in the flame is proportional
                to the concentration of the element in the sample.

            f.  Interference

                1.  The most troublesome interference results
                    from the lack of absorption of atoms bound
                    in molecular combination in the flame which
                    is not sufficiently hot to dissociate the
                    new molecule.

-------
                 2.   The presence of other atoms which absorb
                     at the same wavelength.

                 3.   Interference caused by ionization:   Barium
                     may undergo ionization in the flame and
                     the ground state population is thereby reduced.
                     This interference can be overcome by the
                     addition of an excess of a cation having a
                     similar or lower ionization potential.

         2.   Sample  Preparation

             a.   Special Extraction Procedure:  When the concen-
                 tration of the metal is not sufficiently high
                 to  determine directly certain metals may be
                 chelated and extracted with organic solvents.

                 1.   Chelating agent:  Ammonium pyrrolidine
                     dithiocarbahate  (APDC) for cadmium, iron,
                     manganese, copper, silver, lead and hexavalent
                     chromium.

                 2.   Organic solvent:  methyl isobutyl ketone
                     (MIBK).

             b.   Digestion of sediment

                 1.   Place 2 g of sediment in 250 ml beaker.

                 2.   Add 10 ml of cone. HN03
                         0.5 ml of H202 (30%)

                 3.   Cover beaker with a watch glass and allow
                     mixture to gently reflux for 2 hrs. on a
                     hot plate.1

                 4.   Remove watch glass and evaporate to dryness.
                     If the dry residue is a dark color, add a
                     couple of drops of cone.  HNC>3 and continue
                     to heat.   If the residue is still a dark
                     color after repeating this process, proceed
                     with the  ashing.1

                 5.   Ash the sample at 400-425°C for 1 hr.  in a
                     muffle furnace.

                 6.   Cool to room temperature.
1  Modification by Alameda Laboratory, EPA, Region IX.

                             -  2  -

-------
                7.  Add 10 ml of acid mixture2.
                        8 ml of 10% NH4C1
                        0.4 ml of Ca (NOs) 23

                8.  Heat gently for 15 minutes and cool for
                    5 minutes or longer.

                9.  Transfer sample to a centrifuge tube - rinse
                    the digestion beaker with 10 ml redistilled
                    water until a final volume of 30 ml.

               10.  Centrifuge for 10 minutes at 20,000 RPM.

               11.  Transfer the supernatant into a 100 ml
                    volumetric flask.

               12.  Rinse the digestion beaker with   30 ml of
                    redistilled water and transfer into the
                    centrifuge tube.  Centrifuge and add the
                    supernatant to the previous mixture.

               13.  Rinse and centrifuge a third and final time.

               14.  Adjust the final volume to 100 ml with
redistilled water.
AA analysis.
                                        The mixture is ready for
            Fuel and oxidant combinations and wavelength settings
            needed for metal determinations, listed in the table below.
       Fuel and Oxidant
           Wave Length  Sensitivity  Interferences
                                                         Fe, HC1, V
                                                         H2S04 Ti
                                                         acetic acid

                                                         Al, Si, Mg

                                                         Fe, Ni

                                                         HNC-3 & Ni

                                                         Si, Al, Na
                                                         K, Ca


                                                         Si
2  200 ml cone. HNO3, 50 ml cone. HC1, 750 ml redistilled H20
3  Ca(N03)2 ' 4 H20  11.8 g/100 ml
                               - 3 -
Metal
Aluminum
Barium
Bei yllium
Caomium
Chromium
Copper
Iron
Lead
Magnesium
Manganese
Silver
Zinc
*ug/l of
Combination
Nitrous oxide-acetylene
11 ii ii
ii ii ii
Air- acetylene
M M
n M
M "
n "
II H
II "
II '<
II "
metal for 1% absorption in
nm
309.3
553.6
234.8
228.8
357.9
324.7
248.3
283.3
285.2
279.4
328.1
213.8
an aqueous
ug/1*
1,000
200
100
40
150
200
300
500
15
150
100
40
solution.

-------
         Analysis of Mercury:  Flameless AA procedures

         a.  Procedure

             1.  The mercury is reduced to the elemental
                 state and aerated from solution in a closed
                 system.

             2.  The mercury vapor passes through a cell
                 positioned in the light path of atomic
                 absorption spectrophotometer

             3.  Absorbance, at 253 nm is measured as a
                 function of mercury concentration.

         b.  Interference

             1.  Possible interference from sulfide is
                 eliminated by the addition of potassium
                 permanganate.

             2.  Copper: > 10 mg/1

             3.  Chloride:  During the oxidation step chlorides
                 are converted to free chlorine which will
                 also absorb radiation at 253 nm.
Prepared by
Ho Lee Young, Ph.D.
Chief, Chemistry Section
Laboratory Support Branch
EPA, Region IX
                          _ 4 _

-------
                                                 (Metals)
       TABLE 1

Concentration Ranges
                                   Optimum
                                 Concentration
Detection Limit
Metal mg/1
Aluminum
Arsenic
Cadmium
Calcium
Chromium
t
u. Copper
1 Iron
Lead
Magnesium
Manganese
Potassium
Silver
Sodium
Zinc
0.1
0.05
0.001
0.003
0.01
0.005
0.004
0.01
0.0005
0.005
0.005
0.01
0.001
0.005
Sensitivity
mg/1
0.4
1.0
0.004
0.07
0.02
0.04
0.006
0.06
0.005
0.04
0.01
0.05
0.003
0.02
Range
mg/1
10
10
0.1
1
1
0.1
0.1
1
0.01
0.1
0.01
0.1
1
0.1

1000
100
2
200
200
10
20
10
2
20
2
20
200
2

-------
                     PESTICIDE ANALYSIS


     Most of the pesticides used are synthetic, organic compounds.
They can be grouped into organohalogens, organophosphates,
organosulfates,  organonitrogens and carbamates pesticides.

I.  Analyses of Water and Wastewater Sample.

    A.  Extraction

        1.  One liter sample is extracted with 60 ml of 15%
            ethyl ether in hexane by shaking vigorously for
            2 minutes.

        2.  Repeat extraction

        3.  The sample container is rinsed with each aliquot of
            extracting solvent prior to extraction of the sample.

        4.  Combine the organic solvents from both extractions.

    B.  Florisil clean up

        1.  Prepare a column by placing cotton plug in bottom,
            pouring 4" Florisil in column followed by 3/4"
            anhydrous sodium sulfate.

        2.  Pre-rinse column with 100 ml hexane and discard
            hexane.

        3.  Add extract to column (25-50 ml) and rinse beaker
            three times each with 5 ml hexane.  Use caution in
            not allowing column to run dry.

        4.  When extract meniscus coincides with top of column
            packing, rinse column with 5 ml hexane; when meniscus
            again coincides with top of column packing, add 200 ml
            10% ethyl ether in hexane to elute the sample.

    C.  Concentration of extracts

        1.  Collect the eluant in a 500 ml Kuderna Danish flask,
            fitted with an ampoule.   Connect Snyder column to
            the flask and place in steam bath and evaporate
            until there is action in only two balls.

        2.  Remove Kuderna Danish flask from steam bath, tilt
            slightly and rotate to insure rinsing of all inner
            surface.
                            — 6 —

-------
         3.  Concentrate the sample to 0.5 ml then remove the
             ampoule from the Kuderna Danish flask.

         4.  Analyze the sample using a gas chromatograph,
             without further dilution or concentration, unless
             the chromatogram indicates otherwise.

     D.  Gas chromatographic analysis

         1.  Instrument

             a.  Column:  either spiral or U shape

                 1.  Solid phase:  Gas-chrom Q, 80-100 mesh, or
                     60-80 mesh.

                 2.  Liquid phase:  OV-17, OV-101, OV-210, QF-1

             b.  Carrier gas:  Nitrogen, helium or argon.

             c.  Detector:  Flame ionization and flame photo-
                 metric detector, electron capture, coulometric
                 detector, microcoulson electroconductivity
                 detector.

             d.  recorder.

         2.  Temperature

             a.  Injection - port temperature: 220-230° C.

             b.  Column temperature:  175-200°C.

             c.  Detector temperature:  Should be about 5-10°
                 above column temperature.

         3.  Identification:  Comparing with the retention time
             of the standards.

         4.  Quantitation:  Computed from the area under the peak
             in the chromatogram.

II.   Extraction of Pesticides from Sediments and Tissues

     A.  Extract

         1.  Mix the sediment thoroughly, measure up to 100 g into
             blender cup.

         2.  Add 100 ml acetonitrile and 60 g prehexane-rinsed
             anhydrous sodium sulfate and blend at moderate to
             high speed for 2 minutes.

-------
          3.   Pour into (hexane rinsed)  Buchner funnel and extract
              solvent.

          4.   Carefully transfer residue from Buchner funnel and
              filter  paper into blender  cup.   Add another 100 ml
              mixed solvent,  blend for 1 minute.

          5.   Pour into same  Buchner funnel and filter paper.
              Extract solvent,  combining extract with that
              obtained in step  No. 3.

      B.   Back Partition

          1.   Transfer extracts to 1000  ml separatory funnel con-
              taining 400 ml  distilled water, 100 ml sodium
              sulfate satuarated water,  and 150 ml 5% ethyl-ether
              in hexane.

          2.   Shake vigorously  2 minutes,  then let stand for
              10 minutes,  allowing the two phases to separate.

          3.   Save organic layer, washing twice with 50 ml portions
              of water.

          4.   Transfer to 250 ml beaker  and evaporate over 70°  C
              water bath to 25  ml.

      C.   Florisil clean up:  Same as  above

      D.   Concentration of extracts:   Same as above

      E.   GC  Analysis:   Same  as above

III.   Thin layer chromatography for pesticides analysis

      A.   Extraction^ clean up and concentration as above

      B.   The final solution  is spotted  on a glass plate covered
          with 0.25mm silica-gel G or  magnesium oxide and
          developed in a solvent saturated chamber (hexane:
          acetone = 2:1)

      C.   If  necessary, the developed  spots can be made visible
          by  spraying with an appropriate reagent, e.g. 1-naphthol.

      D.   Another identification technique is to  remove the spot,
          extract the material, and run  an IR scan.


 Prepared by
 Ho Lee Young,  Ph.D.
 Chief, Chemistry Section
 Laboratory  Support  Branch
 EPA, Region IX
                                  — 8 —

-------
                                              Table 1
                       Summary of Gas Chromatographic Analyses of Pesticide Residues
                Extraction
                 Solvent
                           Clean Up
narbamates  Methylene Chloride  Acetonitrile Partition
                                Florisil Column
Organo-
 halogens

Organo-
 nitrogen

Organo-
phosphate
15% Methylene       Acetonitrile Partition
Chloride in Hexane  Florisil Column

Methylene Chloride  Florisil Column
15% Methylene
Chloride in Hexane  Florisil Column
Chlorinated
phenoxy     ethyl ether
acid
                                            Liquid Phase
                                            6%   QF-1
                                            4%   SE-30
                                           Carrier   Column
                                             Gas      Temp.  Detector
                                          Argon
                                            flethane
                                            1% Carbowax  20M   He
Acetonitrite Partition   1.5%
                                                   Ov-17
                                            1.95% QF-1
                                            1.5% OV-17
                                                +
                                            2.95% QF-1
                                               or
                                            5% OV-210
                   Electron
           200° C  Capture

           155° C  Electrolytic
                   Conductivity

N2         215° C  Flame
                   Photometric
                   Detector

Argojj^--            Microcoulo-
—Methane          metric or
                   Electrolytic
                   Conductivity
                      or
                   Electron
                   Capture
                                               - 9 -

-------
•

TABLE
2


RETENTION TIMES OF ORGANOCHLORINE PESTICIDES RELATIVE TO ALDRIN
Liquid Phase
Column Temp.
Pesticide
«-BHC
Lindane
Heptachlor
Aldrin
Kelthane
Heptachlor Epoxide
Y-Chlordane
Endosulfan I
p,p'-DDE
Dieldrin
Endrin
o,p'-DDT
Endosulfan II
p,p'-DDD
p,p'-DDT
Methoxychlor •
Aldrin (Minutes
Absolute)
3% DC-200
+
5% QF-1
200 C
RRt3
0.40
0.51
0.80
1.00
1.19
1.38
1.53
1.77
1.93
2.10
2.43
2.62
2.62
2.68
3.41
5.26
3.76
#
OV-17
200 C
RRt3
0.45
0.61
0.79
1.00
1.52
1.58
1.82
2.00
2.67
2.54
3.21
3.97
3.97
4.13
5.19
11.17
3.84
3%
OV-101
175 C
RRt3
0.33
0.42
0.76
1.00
1.12
1.30
1.55
1.70
2.18
2.08
2.33
3.02
2.45
2.'94
3.97
6.88
2.64
3%
OV-210
160 C
RRt3-
0.54
0.75
0.82
1.00
2.46
2.16
2.12
2.89
2.91
3.65
4.46
4.04
5.96
5.61
6.28
13.52
2.28
Relative
Sensitivity
to EC Detccto


1.0
1.0
1.0
1.0
0.1
0.5
0,5
0.4
0.5 ;
0.5
0.3
0.1
0.3
0.1
0.2
0.1

 All columns glass, 6 ft. long x 4 mm ID, solid support Gas-Chrom Q (80/100  mesh),
 nitrogen carrier flow 80 ml/min.
2
 Sensitivity factors relative to aldrin.
2
 Retention times relative to aldrin.       -  10  -

-------
           304  (g)  WATER QUALITY  GUIDELINES
                         By

                     Ho  Young
                     EPA,  Region IX
                     San Francisco CA
Presented at the Workshop on Sampling, Monitoring and Analysis
  of Water and Wastewater, March 6-12, 1974, Honolulu HI.

-------
                 304(g) WATER QUALITY GUIDELINES
  I.  Section 304(g) of the Water Act requires that the Adminis-
      trator shall promulgate guidelines establishing test pro-
      cedures for the analysis of pollutants.

 II.  Guidelines were issued in the Federal Register, Vol. 38,
      No. 199, Part II, on October 16, 1973.

III.  Objectives of guidelines establishing test procedures:

      A.  To establish reliable procedure(s) for analyses of
          various pollutants.

      B.  To assure the effluent discharge of pollutants from a
          point source or group of point sources meets the
          effluent discharge limitations set forth by Section
          302.

      C.  To achieve the water quality in a specific portion  of
          the navigable water which shall assure protection of
          public water supplies, agricultural, and industrial
          uses, protection and propagation of a balanced popula-
          tion of shellfish, fish and wildlife, and allow recre-
          ational activities in and on the water.

 IV.  Test procedure to be used by:

      A.  Any applicant for a Federal license or permit to con-
          duct any activity including, but not limited to, the
          construction or operation of facilities which may
          result in any discharge into the navigable waters.

      B.  Permit applicants to demonstrate that effluent dis-
          charges meet applicable pollutant discharge limita-
          tions:  National Pollutants Discharge Elimination
          System (NPDES).

      C.  The State and other enforcement activities in routine
          or random monitoring of effluents to verify effective-
          ness of pollution control measures.

-------
                                -2-
   V.  Approved test procedures for pollutants and parameters:
         Parameters
                             Units
            Approved Test Procedures
          Standard             EPA
          Methods!   ASTM2   Methods3
 General Analytical Methods

  1. Alkalinity as CaC03
  2. BOD5
  3. Chemical oxygen demand
  4. Total solids
  5. Total dissolved solids
  6. Total suspended solids
  7. Total volatile solids
  8. Ammonia (as N)
  9. Kjeldahl (as N)
 10. Nitrate (as N)

 11. Total phosphorus
     Acidity as CaC03
     Total organic carbon
     Hardness (as  CaCOS)
12,
13,
14.
15.  Nitrite (as N)
mg/liter
mg/liter
mg/liter
mg/liter
mg/liter
mg/liter
mg/liter
mg/liter
mg/liter
mg/liter

mg/liter

mg/liter
mg/liter
mg/liter


P'
P-
P.
P.

P.
P.

P-
P-
P.
P.
P-

P.
P-


370
489
495
535

537
536

469
458
461
526
532

257
179


P.

P-






P-

P«

P-
P-
P.


143

219






124

42

148
702
170


p. 6,8

p. 17
p. 280
p. 275
p. 278
p. 282
p. 134,141
p. 149,157
p. 170
p. 175
p. 235
p. 246,25

p. 221
p. 76,78
p. 185
p. 195
 Analytical Methods  for Trace Metals
 16-  Aluminum
 17.  Antimony
 18.  Arsenic
 19.  Barium
 20.  Beryllium

 21.  Boron
 22.  Cadmium

 23.  Calcium
 24.  Chromium (+6)
                                          210
mg/liter
mg/liter
mg/liter  p. 65,62
           p. 210
           p. 67
           p. 210
mg/liter
mg/liter
                           mg/liter
                           mg/liter
                                       p.  69
                                       p.  210
                                       p.  422
                           mg/liter    p.  84
                           mg/liter    p.  429
p. 692

p. 692
p. 98

p. 13




p. 101

p. 102
p. 94
    •^Standard Methods for
water, 13th Edition, 137T.

    21972 Annual Book for
Part 23; American Society

    ^Methods for Chemical
mental Protection Agency, 1971.
                          the Examination of_ Water and Waste-


                          ASTM Standards; Water, Atmosphere,
                          for Testing and Metals.

                          Analysis of Water  and Wastes, Environ-

-------
-3-
         Approved Test Procedures
Standard

25.

26.
27.

28.

29.

30.

31.
32.
33.
34.
35.

36.
37.
38.
39.
40.
41.
42.
43.

Parameters
Chromium (total)

Cobalt
Copper

Iron

Lead

Magnesium

Manganese
Mercury
Molybdenum
Nickel
Potassium

Selenium
Silver
Sodium
Thallium
Tin
Titanium
Vanadium
Zinc

Analytical Methods for
44.
45.

46.

47.
48.
49.
50.
51.
52.

53.
Organic nitrogen
Ortho-phosphate (as

Sulfate (as 804)

Sulfide (as S)
Sulfite (as SO3)
Bromide
Chloride
Cyanide
Fluoride

Chlorine
Units
mg/liter

mg/liter
mg/liter

mg/liter

mg/liter

mg/liter

mg/liter
mg/liter
mg/liter
mg/liter
mg/liter

mg/liter
mg/liter
mg/liter
mg/liter

mg/liter
mg/liter
mg/liter

Methods
P-
P.

P.
P-
P-
P-
P-
P-
P-
P-
P-


P-
P-
P-

P-
P-



P-
P-
P-
Nutrients , Anions ,
mg/liter
P) mg/liter

mg/liter

mg/liter
mg/liter
mg/liter
mg/liter
mg/liter
mg/liter

mg/liter
P-
P-

P-
P-
P-
P-

P-
P-
P-
P-
P-
210
426

210
430
210
433
210
436
210
416
210


443
283
285

210
317



157
210
444
and
468
532

331
334
551
337

96
397
171
174
382
ASTM
P-
P-
P-
P-
P-
P-
P-
P-

P-

P-


P-
P-



P-




P-

692
403
692
692
410
692
152
692

692

692


692
326



326




692

EPA
Methods
P-


P-

P-

P-

P.

P-



P.



P-




P.

104


106

108

110

112

114



115



118




120

Organics

P-

P-
P-

P.
P-
P-
P-
P-

P-

42

51
52

261
216
23
556
191

223
P.
P.
246
P.
P.
P-


P-
P.
P-


149
235,
, 259
286
288
294


29
41
64



-------
                                -4-
                                        Approved Test Procedures
         Parameters
 Units
Standard
Methods
ASTM
                                                           EPA
                                                         Methods
54. Oil and Grease
55. Phenols
56. Surfactants
57. Algicides*
58. Benzidine

59. Chlorinated organic
    comp.*
60. Pesticides*
                            mg/liter   p.  254
mg/liter
mg/liter
mg/liter
mg/liter
p. 502
p. 339
J. Asso
                                                 p.
                                                 p.
                        445
                        619
                    P.
                    P.
            232
            131
                            mg/liter

                            mg/liter
                                      Chem. 54:1383-1387, 1971
Analytical Methods for Physical and Biological Properties
 61.  Color  platinum-cobalt
     units  or  dominant wave-
     length, hue,  luminance,
     purity
 62.  Specific  conductance
 63.  Turbidity

 64.  Fecal  streptococci
 65. Coliform  (fecal)
mho/cm.
jackson
unit
number/
100 ml.

number/
100 ml.
Radiological Parameters

67. Alpha  (total)
68. Alpha-counting error
69. Beta  (total)
70. Beta-counting error
71. Radium  (total)
pCi/liter
pCi/liter
pCi/liter
pCi/liter
pCi/liter
           p. 160
           p. 392
 p. 323
 p. 350

 p. 689
 p. 690
 p. 691
 p. 669
 p. 684
 p.  598
 p.  598
 p.  598
 p.  598
 p.  611
 p.  617
p. 163
p. 467
                    p. 38
                                                          p.  284
                                                          p.  308
p. 509
p. 512
p. 478
p. 478
p. 674
 VI.  Application for alternate test procedures:

      A.  Send application for approval of an alternative test
          procedure to the Regional Administrator.

          1.  Name and address of the responsible person or firm
              making the discharge (if not the applicant).

    *Interim procedures prepared by the Methods Development and
Quality Assurance Research Laboratory, National Environmental
Research Center, Cincinnati, Ohio.

-------
                           -5-
      2.   The applicable ID number of the existing or pend-
          ing permit, issuing agency, and type of permit for
          which the alternative test procedure is requested.

      3.   The pollutant or parameter for which approval of
          an alternate testing procedure is being requested.

      4.   Justification for using the alternative testing
          procedures.

      5.   The detailed description of the proposed alternate
          test procedure with supporting data.

  B.   Within 90 days of receipt by the Regional Administra-
      tor of an application for an alternate test procedure,
      the Regional Administrator shall notify the applicant
      and the appropriate State agency of approval or rejec-
      tion, or shall specify the additional information
      which is required to determine whether to approve the
      proposed test procedure.
spared on March 1,  1974

-------
                      SAMPLE PRESERVATION
 I.   Purposes

     A.   To inhibit bacterial growth:  nutrients such as carbon
         sources, nitrogenous compounds and phosphorus compounds.

     B.   To prevent precipitation and adsorption to container:
         metals.

     C.   To prevent salt formation:  acids and alkaline.

II.   Preservatives use

     A.   Bacterial growth:  HgCl2, acid, refrigeration or
         freezing.

     B.   Precipitation:  acid

     C.   Salt formation:  acid for organic base, and alkali for
         cyanides and organic acid.

-------
Ul
           TUESDAY, OCTOBER 16, 1973
           WASHINGTON, D.C.
           Volume 38 • Number 199
           PART II
           ENVIRONMENTAL
              PROTECTION
                AGENCY
             WATER PROGRAMS


           Guidelines Establishing Test Procedures
              for Analysis of Pollutants

-------
 28758
     RULES  AND  REGULATIONS
     Title 4O—Protection of Environment
      CHAPTER  I—ENVIRONMENTAL
          PROTECTION AbENCY
     SUBCHAPTER D—WATER PROGRAMS
 PART 136—GUIDELINES ESTABLISHING
   TEST PROCEDURES FOR THE ANALY-
   SIS OF POLLUTANTS
   Notice was  pubished in the FEDERAL
 REGISTER issue  of June 29, 1973 (38  PR
 17318) at 40 CFR 130, that the Environ-
 mental  Protection  Agency  (EPA)  was
 giving consideration to the testing pro-
 cedures  required  pursuant  to  section
 304(g) of the  Federal  Water Pollution
 Control Act  Amendments of 1972  (86
 Stat. 816, et seq., Pub. L. 92-500  (1972))
 hereinafter referred to as the Act. These
 considerations were given in the form of
 proposed  guidelines   establishing  test
 procedures.
   Section 304(g) of the Act requires that
 the  Administrator   shall  promulgate
 guidelines establishing test  procedures
 for  the analysis of pollutants that shall
 include factors which must be provided
 in:  1, any certification pursuant to sec-
 tion 401 of the  Act, or 2, any permit ap-
 plication pursuant to section 402 of  the
 Act. Such test procedures are to be used
 by permit applicants to demonstrate that
 effluent discharges meet applicable pol-
 lutant discharge limitations,  and by  the
 States and other enforcement activities
 in routine or random monitoring of ef-
 fluents to verify effectiveness of pollu-
 tion control measures.
   These guidelines require that.discharge
 measurements,  including but  not limited
 to the pollutants and parameters listed
 in Table I,  be performed by the test
 procedures indicated; or under certain
 circumstances by other test  procedures
 for  analysis that may be more advan-
 tageous to use, when  such  other test
 procedures have the approval of the Re-
 gional  Administrator  of  the  Region
 where such  discharge  will  occur,  and
 when  the Director of  an approved State
 National  Pollutant  Discharge Elimina-
 tion System (NPDES)  Program (here-
 inafter referred to as the Director)  for
 the  State in which such discharge will
 occur has no objection to such approval.
   The list of test procedures in  Table I
 is published  herein as final rulemaking
 and represents major departures  from
 the list of proposed test procedures which
 was published  in  38 FR 17318, dated
 June 29, 1973. These revisions were made
 after  carefully  considering  all  written
 comments which were received pertain-
 ing  to the proposed test procedures.  All
 written comments are on file and avail-
 able for public  review with the  Quality
 Assurance Division, Office  of Research
 and Development, EPA, Washington, D.C.
  The principal revisions to the proposed
 test procedures are as follows:
  1.  Where several  reliable  test proce-
 dures  for  analysis  are available  from
 the given references for a given pollutant
 or parameter, each such test procedure
has  been  approved for  use for making
 the measurements  required by sections
401 and 402 and related sections of the
Act.  Approved test procedures have been
selected to assure an acceptable level of
intercomparability  of  pollutants  dis-
charge data. For several pollutants and
parameters it has still been necessary to
approve only a single test procedure to
assure this level of acceptability. This is
a  major departure  from the proposed
test procedures  which  would have  re-
quired the use of  a single  reference
method for each pollutant or parameter.
  2. Under certain circumstances a test
procedure  not shown on the approved
list may  be considered  by an applicant
to be more advantageous to use. Under
guidelines in §§ 136.4 and 136.5 it may be
approved by the Regional Administrator
of the Region where the discharge will
occur, providing the  Director has no ob-
jections. Inasmuch as there is no longer
a  single   approved  reference  method
against which a comparison can be made,
the  procedures for  establishing  such
comparisons that were  required by  the
proposed  test procedures  in  § 130.4 (b)
have been  deleted from this final guide-
line for test procedures for the  analysis
of pollutants.
  3. A mechanism is  also provided  to
assure national  uniformity of such ap-
provals of alternate  test procedures for
the   analysis of  pollutants.  This  is
achieved through a centralized,  internal
review within the EPA of all applications
for the use of alternate  testing proce-
dures. These will  be reviewed and ap-
proved or disapproved  on the  basis  of
submitted  information and other avail-
able  information  and  laboratory tests
which may be required by the Regional
Administrator.
  As 'deemed necessary, the Administra-
tor will expand or  revise these guide-
lines to provide the most responsive and
appropriate  list  of  test  procedures  to
meet the requirements of sections 304(g),
401 and 402 of the Act,  as amended.
  These final guidelines establishing test
procedures for the analysis of pollutants
supersede the interim list of test proce-
dures published in the FEDERAL REGISTER
on April 19, 1973 (38  FR 9740)  at 40 CFR
Part 126 and subsequent procedures pub-
lished on July 24,  1973 (38 FR 19894)
at 40 CFR Part  124. Those regulations
established interim  test procedures for
the submittal of applications under sec-
tion 402 of the Act.  Because of the im-
portance  of these  guidelines  for test
procedures for the analysis of pollutants
to the National Pollution Discharge Elim-
ination System (NPDES), the Adminis-
trator finds good cause  to declare that
these guidelines shall be effective Octo-
ber 16,1973.
                    JOHN QUARLES,
               Acting Administrator.

  OCTOBER 3, 1973.
PART 136—TEST PROCEDURES FOR THE
      ANALYSIS OF  POLLUTANTS
Sec.
136.1  Applicability.
136.2  Definitions.
136.3  Identification of test procedures.
136.4  Application for  alternate test proce-
       dures.
130:6  Approval of alternate test procedures.
  AUTHORITY:  Sec. 304(g) of Federal Water
Pollution Control Act Amendments of 1972
86 Stat. 816, et seq., Pub. L. 92^500).
§ 136.1  Applicability.

   The  procedures  prescribed   herein
shall, except as noted in § 136.5, be used
to perform the measurements indicated
whenever the waste constituent specified
is required to be measured for:
   (a)  An  application  submitted to  the
Administrator, or to a State having an
approved NPDES program, for a permit
under section 402 of the  Federal Water
Pollution  Control  Act  as  amended
 (FWPCA), and,
   (b)  Reports required to be submitted
by  dischargers   under  the   NPDES
established by Parts 124 and 125 of this
chapter, and,
   (c) Certifications issued by States pur-
suant to section 401 of the FWPCA, as
amended.
§  136.2   Definitions.
   As used in this part, the term:
   (a)  "Act" means the Federal Water
Pollution  Control Act, as amended, 33
U.S.C. 1314,  et seq.
   (b)  "Administrator" means the Ad-
ministrator  of the U.S. Environmental
Protection Agency.
   (c)  "Regional Administrator" means
one of the EPA Regional Administrators.
   (d)  "Director" means the  Director of
the State Agency authorized  to carry
out an approved National Pollutant Dis-
charge  Elimination   System  Program
under section 402 of the Act.
   (e)  "National   Pollutant   Discnargy
Elimination  System  (NPDES)" means
the national system for the  issuance ol
 permits under section 402 of the Act and
includes any State or interstate program
 which has been approved by the Admin-
istrator, in whole or in part, pursuant to
 section 402 of the Act.
   (f) "Standard Methods" means Stand-
 ard Methods for the Examination of
 Water and  Waste Water, 13th  Edition,
 1971. This publication is  available from
 the American Public Health Association,
 1015 18th St. NW.,  Washington, D.C.
 20036.
   (g)  "ASTM"  means Annual  Book of
 Standards, Part  23, Water, Atmospheric
Analysis, 1972. This publication  is avail-
 able from  the  American  Society  for
 Testing  and Materials, 1916 Race  St.,
Philadelphia, Pennsylvania 19103.
   (h) "EPA Methods" means Methods
for Chemical  Analysis  of  Water and
 Wastes,  1971, Environmental Protection
Agency, Analytical Quality Control Lab-
oratory,  Cincinnati, Ohio. This publica-
tion is   available from the   Super-
intendent  of Documents, U.S. Govern-
ment Printing Office, Washington, D.C.
20402  (Stock Number 5501-0067).
§ 136.3  Identification • of  test  proce-
     dures.
  Every  parameter   or  pollutant  for
which an effluent limitation is now spec-
ified 'pursuant to sections 401  and 402
of the Act is named together with test
descriptions and  references in Table I
The discharge  parameter  values for
which reports are required must be de-
                               FEDERAL REGISTER,  VOL. 38, NO. 199—TUESDAY, OCTOBER

-------
termlned by one of the standard ana- gional Administrator or the Director in
lytical methods cited and described the R^on or State where the discharge
in Table I, or under certain circum- will occur may determine for a par-
stances by other methods that may be ticular discharge that additional param-
more advantageous to use when such eters or pollutants must be reported.
other methods have been previously ap- Under such circumstances, additional
proved by the Regional Administrator of test procedures for analysis of pollutants
the Region in which the discharge will may be specifled by the Regional Ad-
occur, ana p g ministrator or Director upon the recom-
wfll occur does not object to the use of rnendation of the Director of the
such alternate test procedures. Methods Development and Quality As-
Under certain circumstances the Re- surance Research Laboratory.
TABLE I— LIST or APPBOVED TEST PaocEDUBEa
Parameter and units
General analytical methods:
1. Alkalinity as CaCO img
CaCO'Alter.
2. B.O.D. five day mgAiter.
3. Chemical oiyeen de-
mand (C.O.D.) mg/
liter.
4. Total solids mg/btor 	
6. Total dissolved (filter-
able) solids mg/liter.
6. Total suspended (non-
filterable) solids mg/
liter.
7 Total volatile solids mg/
liter.
8. Ammonia (as N) mg/
liter.
9. KleMalil nitrogen (as N)
mg/llter.
10. Nitrate (as N) rag/Uter.
H. Total phosphorus (as P)
mg/uter.
12. Acidity me CaCOi/Uter
13. Total organic carbon
(TOO mgAiter.
14. Hardness— total mg
CaCOi/Uter.
16. Nitrite (as N) mgAittr.
Analytical methods for trace
metals:
16, ilomlnum— total ' mg/
liter.
17. Antimony— total ' mg/
liter.
19. Barium— total * ing/liter
-*0. Beryllium— total * mg/
ItUr.
22. Cadmium- -total ' mg/
liter.
23. Cticlum— total ' mg/liter
24. Chromium VI mgAiter
Method
Titration- electronvtrtc, manual or auto-
mated method— methyl orange.
Modified wlnkler or probe method 	
Gravimetric 10»-106° C 	
Glass fiber filtration 1RO° C 	
Glass fiber filtration 103-105° C 	
Gravimetric 550° C. 	 	 	
Distillation— nesslerlzatlon or titration au-
tomated phenolate.
Digestion + distillation— nesslerizatton or
titration utomated digestion phenolate.
Cadmium reduction; brucine sul/ate. au-
tomated cadmium or hydrazine. reduc-
tion.
Penulfate digestion and single reagent
(ascorbic acid), or manual digestion,
and automated single reagent or Stan-
nous chloride.
Electrometric end point or phenolphthal-
eln end point.
EDTA titration; automated colorimetric
atomic absorption.
Manual or automated colorimetric dlaioti-
zation.

Digestion plus silver dlethyldithiocarba-
mate; atomic absorption.'


Atomic absorption; colorimetilc 	 	
EUTA titration; atomic absorption. 	
Extraction and atomic absorption; colori-
metric.

Standard
methods
p. 370
p. 489 	
p. 535. .

p. 537. .
p. 536 	

p. 469 	
p. 158 	
p. 161 	
p 528
p. 632

p. 267
p. 179 	

p. 210

p 95
p 62
p. 210 	

p 210
p. 69
p. 210 	
p. 422 	
P.S4 	
p. 429

Beferences
ASTM EPA
methods
D. 143 n 6.
p. 8.
	 p. 280.
	 p. 275.
	 p. 278.
	 p. 282.
	 p. 134.
p. 141.
	 p. 149.
p. 157.
. p. 124 	 p. 170.
	 p. 175.
p. 185.
p. 42 p. 286.
:. 	 p. 248.
p. 269.
p. 148 	
p. 702. . . p. 221.
.. p. 170 	 p. 78.
p. 78.
	 p. 185.
p. 196.
	 p. 98.

p. 13.





.. p. 692 	 p. 101.
.. p. 69! 	 p. 102.
	 	 . . p. W.

Parameter and units
25, Chromium— total5 mg/
liter.
28. Cobalt— total ' mg/liter,
27. Copper— total ! mg/litar.
28. Iron— total ' mg/Utar
29 Lead total ' mg/liter
30. Magnesium— total 'mg/
liter.
31. Manganese— total ' mg/
liter.
33. Molybdenum— total '
mg/llter.
34. Niokel— total ' mg/liter .
36 Potassium — total ' mg/
liter.
36. Selenium-total m«/litor.
37. Silver-total1 	 	
38. Sodium— total 'mg/liter .
3'J. Thallium-total 'mg/liter.
40. Tin— total ' mg/liter 	
41. Titanium — total mg/
liter.
42. Vanadium— total1 mg/
liter.
43. Zinc— total ' mg/liter...
Analytical methods for nu-
trients, anions, and organics:
44. Organic nitrogen (as N)
mg/liter.
•15. Ortho-phosphate (as P)
mg/liter.
46. Sulfate (as SO,) mg/
liter.
47. Sulflde (as S) mg/liter. _
48. Sulflte (as 30i) mg/
liter.
50. Chloride mg/llter 	
51. Cyanide— total mgAiter,
52 Fluoride mg/liter
53. Chlorine— total residual
rag/liter.
54. Oilandgre:isemg/Utcr..
58. Surfactants rag/liter 	
58. Benzldiae m^/liter 	
69. Chlorinated organic
compounds (except
pesticides) mg/liter.
SO. Pesticides ing/liter 	
Analytical methods (or
physical and biological
parameters-
61. Color platinum-cobalt
units or doui Inant
wsve-lentrth, hue,
luminance, purity.
62. Specific conductance
| mho/cm it 25" C.
'• 63. Turbidity Jackson
1 'inlte.
] See Note at end oi Table I
Method
Atomic absorption; colorimetric- 	

do 	 	 	
do

Atomic absorption 	 . 	
Atomic absorption ' 	

Atomic absorption; colorimetric; flame
photometric.
Atomic absorption ' 	
Flame photometric; atomic absorption . .
	 do 	 	 	 - 	 	 	
	 do 	 	 	 - 	
Atomic Absorption; Colorimetric 	
KJeldahl nitrogen minus ammonia
nitrogen.
Direct single reagent; automated single
reagent or jtannous chloride.
Gravimetric; lurbidimetric; automated
colorirnetric— barium chluninilate.
Titrimstric— iodine 	
do
Silver nitrate; mart-uric oitrite; automated
colorimetric- ferric yanide.
Distillation— silver nitrate titration or
pyridine pyrazolone colorimetric.
Distillation— fiPADNS 	
Colorimetric; amperomeuic titration 	
Liquid-Liquid extraction with trichloro-
trifluoroethane.
Colorimetric, 4 AAP 	





Wheatstone bridge 	 	 	

Refareaoas
Standard
methods
- P.
P-
p.
P-
p
P.
p
P-
p
P-
P.
. P.

p
P.
p.
- p.
-'V

p
- p.
p.
p.
p.
p.
p.
p.

p.
p.
p.
p.
p.
- p.
p.
- p-
p.




- p
p
- p

210 _
426..
?,lf)
no
?1 n
m
"in
4
-------
 28760
                          RULES AND  REGULATIONS
     Parameter and units
                                      Method
                                                                      References
                                                            Standard
                                                            methods
                                                                       ASTM
                                                        EPA
                                                       raetho as
MFN; membrane filter, plate count ....... p. 689
                                 p. 690
                                 P- 891
MPN: Membrane niter.. .................. p. 669
                                 p. 684
    64. Focal streptococci
        bacteria numberAOO
        ml.
    68. Conform bacteria
        (fecal) number/100
        ml.
    66. Coliform bacteria     ..... do ......... , ........................... p. 664 .................................
        (total) number/100                                     p. 679 .................................
        ml.
 Radiological parameters:
    67. Alpha— total pCi/llter.. Proportional counter; scintillation counter p. 898 ....... p. 809 ...................
    68. Alpha— counting error ..... do ..................................... p. 898 ....... p. 612 ...................
        pCl/llter.
    69. Beta— total pCl/llter... Proportional countert ..................... p. 898 ------- p. 478 ...................
    70. Beta— counting error ........ do ..................................... p. 698 ....... p. 478 ...................
        pClfliter.
    71. Radium— total pCl/    Proportional counter; scintillation counter., p. 611 ....... p. 074 ...................
        liter.                                               p. 617.. ............ ... ................

   1 A number of such systems manufactured by various companies are considered to be comparable in their per-
 formance. In addition, another technique, based on Combustion-Methane Detection, is also acceptable.
   ' For the determination of total metals the sample Is not filtered before processing. Choose a volume of sample
 appropriate for the expected level of metals. If much suspended material is present, as little as 60-100 ml of well-mined
 sample wi II most probably be sufficient. (The sample volume required may also vary proportionally with the number
 of metals to be determined.)
   Transfer a representative aliquot of the well-mixed sample to a Griffin beaker and add 3 ml of concentrated distilled
 HNOi. Place the beaker on a hotplate and evaporate to dryness making certain that the sample does not boil. Cool
 the beaker and add another 3 ml portion of distilled concentrated HNOj. Cover the beaker with a watch glass and
 return to the hotplate. Increase the temperature of the hotplate so that a gentle reflux action occurs. Conti nue heating,
 adding additional acid as necessary until the digestion is complete, generally indicated by a light colored residue.
 Add (1:1 with distilled water) distilled concentrated HC1 In an amount sufficient to dissolve the residue upon warm-
 ing. Wash down the beaker walls and the watch glass with distilled water and filter the sample to remove silicates
 and other insoluble material that could clog the atomizer. Adjust the volume to some predetermined value based
 on the expected metal concentrations. The sample Is now ready for analysis. Concentrations so determined shall be
 reported as "total".                                                     ,
   > See D. C. Manning,  "Technical Notes", Atomic Absorption Newsletter, Vol. 10, Nol 6 p. 123, 1971. Available
 from Pwkin-Elmer Corporation, Main Avenue, Norwalfe, Connecticut 06862.
   < Atomic absorption method available from Methods Development and Quality Assurance Research Laboratory,
 National Environmental Research Center, USEPA, Cincinnati, Ohio 46268.
   1 For updated method, see: Journal of the American Water Works Association 64, No. 1, pp. 20-26 (Jan. 1972) ar
 ASTM Method D 3223-73, American Society for Testing and Materials Headquarters, 1916 Race St., Philadelphia,
 Pa. 19103.
   • Interim procedures for algicldes, chlorinated organic compounds, and pesticides can be obtained from the Methods
 Development and Quality Assurance Research Laboratory, National Environmental Research Center, USEPA,
 Cincinnati, Ohio 48268.
   * Benzldlne may be estimated by the method of M.A. El-Dib, "Colorimetric Determination of Aniline Derivative!
 in Natural Waters", El-Dib, M.A., Journal of the Association of Official Analytical Chemists, Vol. 84, No. 6, Nov.
 1971, pp. 1383-1387.
   fAs a prescreening measurement.

 § 136.4   Application  for  alternate   teat
      procedures.

   (a) Any person may  apply  to  the Re-
 gional  Administrator  In   the  Region
 where the discharge occurs for approval
 of an alternative test procedure.
   (b) When the discharge for which an
 alternative test procedure  is proposed
 occurs within a State having a permit
 program  approved pursuant  to section
 402 of the Act, the applicant shall sub-
 mit his application to the Regional Ad-
 ministrator through the Director of the
 State agency having  responsibility for
 issuance  of NPDES permits within such
State.
   (c) Unless and until  printed applica-
tion forms are made available, an appli-
                    cation for  an  alternate test procedure
                    may be made by letter in triplicate. Any
                    application for an alternate test proce-
                    dure under this subchapter shall:
                      (1) Provide the name and address of
                    the responsible person or firm  making
                    the discharge (if not the applicant) and
                    the applicable ID number of the  existing
                    or pending permit,  issuing agency, and
                    type of permit for  which  the alternate
                    test procedure is requested, and  the dis-
                    charge serial number.
                      (2) Identify the pollutant or parame-
                    ter  for  which  approval of an alternate
                    testing procedure is being  requested.
                      (3)  Provide   justification  for  using
                    testing  procedures  other  than  those
                    specified in Table I.
   (4)  Provide  a detailed description of
 the proposed alternate  test procedure,
 together  with  references to  published
 studies of the applicability of the alter-
 nate  test  procedure to  the effluents in
 question.

 § 136.5   Approval of alternate lest pro-
      cedures.
   (a)  The  Regional Administrator  of
 the region in which  the discharge will
 occur  has final  responsibility  for ap-
 proval of  any  alternate test procedure.
   (b)  Within thirty  days of  receipt of
 an application, the Director will forward
 such application,  together with his rec-
 ommendations, to the Regional Admin-
 istrator. Where the Director recommends
 rejection of  the  application for  scien-
 tific and technical reasons which he pro-
 vides,  the Regional Administrator shall
 deny the  application, and shall forward
 a copy of the  rejected application and
 his  decision to  the Director of the State
 Permit Program and to  the Director of
 the Methods Development  and Quality
 Assurance Research Laboratory.
   
-------
           1-      METHOD FOR ORGANOCHLORINE PESTICIDES IN INDUSTRIAL EFFLUENTS

           1.   Scope and Application

               1.1   This method covers the determination of various organochlorine

                    pesticides, including some pesticidal degradation products and related

                    compounds in industrial effluents.   Such compounds are composed of

                    carbon,  hydrogen,  and chlorine,  but may also contain oxygen,  sulfur,

                    phosphorus, nitrogen or other halogens.

            £J  1.2   The following compounds may be determined individually by this method

    _      ^       with a sensitivity of 1 yg/liter:   BHC,  lindane,  heptachlor,  aldrin,
I-  O  —  x-   -       heptachlor epoxide, dieldrin, endrin, Captan,  DDE, ODD, DDT,  methoxy-
.__    f>
_j  ~  z;          chlor, endosulfan, dichloran, mirex, pentachloronitrobenzene  and  tri-
—'  _  ^  o
O    i  ri   ££
d.  uj  Q-          fluralin.  Under favorable circumstances, Strobane,  toxaphene,
    CD  ^~ ^ |      chlordane (tech.)  and others may also be determined.   The  usefulness
    £c  "-1    -
    rc  £5    •       °f tne meth°d for  other specific pesticides must  be demonstrated  by
    CJ  >_  W)
    CO  CO  "*
    Q     W       the analyst  before any attempt is made to apply it  to sample analysis.
             •
           "Jj  1.3  When organochlorine pesticides exist as complex mixtures,  the
           to
                    individual  compounds may be difficult to distinguish.  High, low,  or

                    otherwise unreliable results may be obtained through  misidentifica-

                    tion and/or  one compound obscuring another of lesser  concentration.

                    Provisions  incorporated in this method are intended to minimize the

                    occurrence  of such interferences.

           2.   Summary

               2.1  The method  offers  several analytical alternatives,  dependent on the

                    analyst's assessment of the nature and extent of interferences  and/or

                    the complexity of  the pesticide mixtures found.  Specifically,  the

                    procedure describes the use of an effective co-solvent for efficient

                    sample extraction; provides,  through use of column chromatography

-------
                                    1-2




         and liquid-liquid partition,  methods  for  elimination  of non-pesticide




         interferences and the pre-separation  of pesticide mixtures.   Identifi-




         cation is made by selective gas  chromatographic  separations  and may




         be corroborated through the use  of two or more unlike columns.




         Detection and measurement  is  accomplished by electron capture,  micro-




         coulometric or electrolytic conductivity  gas chromatography.   Results




         are reported in micrograms  per liter.




    2.2  This method is recommended  for use only by experienced pesticide




         analysts or under the close supervision of such  qualified  persons.




3.   Interferences




    3.1  Solvents, reagents,  glassware, and other  sample  processing hardware




         may yield discrete artifacts  and/or elevated baselines causing




         misinterpretation of gas chromatograms.   All of  these materials must




         be demonstrated to be free  from  interferences under the conditions




         of the analysis.   Specific  selection  of reagents and  purification of




         solvents by distillation in all-glass  systems may be  required.




         Refer to Part I,  Sections  1.4 and  1.5, (1).




    3.2  The interferences in industrial  effluents are high and varied and




         often pose great  difficulty in obtaining  accurate and precise




         measurement of organochlorine pesticides.   Sample clean-up procedures




         are generally required and  may result  in  the loss of  certain  organo-




         chlorine pesticides.   Therefore, great care should be exercised in




         the selection and use of methods for  eliminating or minimizing




         interferences.   It is not possible to  describe procedures  for over-




         coming  all  of the interferences  that may  be  encountered in industrial




         effluents.

-------
                                   1-3






    3.3  Polychlorinated Biphenyls (PCB's) - Special attention is called




         to industrial plasticizers and hydraulic fluids such as the PCB's




         which are a potential source of interference in pesticide analysis.




         The presence of PCB's  is indicated by a large number of partially




         resolved or unresolved peaks which may occur throughout the entire




         chromatogram.  Particularly severe PCB interference will require




         special separation procedures (2,3).




    3.4  Phthalate Esters - These compounds, widely used as plasticizers,




         respond to the electron capture detector and are a source of inter-




         ference in the determination of organochlorine pesticides using




         this detector.  Water leaches these materials from plastics, such




         as polyethylene bottles and tygon tubing.  The presence of phthalate




         esters is implicated in samples that respond to electron capture but




         not to the microcoulometric or electrolytic conductivity halogen




         detectors or to the flame photometric detector.




    3.5  Organophosphorus Pesticides - A number of organophosphorus pesticides,




         such as those containing a nitro group, eg, parathion, also respond




         to the electron capture detector and may interfere with the determina-




         tion of the organochlorine pesticides.  Such compounds can be




         identified by their response to the flame photometric detector (4).




4.   Apparatus and Materials




    4.1  Gas Chromatograph   Equipped with glass lined injection port.




    4.2  Detector Options:




         4.2.1  Electron Capture   Radioactive (tritium or nickel 63)




         4.2.2  Microcoulometric Titration




         4.2.3  Electrolytic Conductivity

-------
                                 1- 4






 4.3  Recorder - Potentiometric strip chart   (10  in.) compatible with




      the detector.




 4.4  Gas Chromatographic Column Materials:




      4.4.1  Tubing - Pyrex  (180 cm long x 4 mm ID)




      4.4.2  Glass Wool - Silanized




      4.4.3  Solid Support - Gas-Chrom Q (100-120 mesh)




      4.4.4  Liquid Phases - Expressed as weight percent coated on




             solid support.




             4.4.4.1  OV-1,  3%




             4.4.4.2  OV-210, 5%




             4.4.4.3  OV-17, 1.5% plus QF-1, 1.95%




             4.4.4.4  QF-1,  6% plus SE-30, 4%




 4.5  Kuderna-Danish  (K-D) Glassware  (Kontes)




      4.5.1  Snyder Column - three ball (macro) and two ball  (micro)




      4.5.2  Evaporative Flasks - 500 ml




      4.5.3  Receiver Ampuls - 10 ml,graduated




      4.5.4  Ampul Stoppers




 4.6  Chromatographic Column   Chromaflex (400 mm long x 19 mm ID) with




      coarse fritted plate on bottom and Teflon stopcock;  250 ml reservoir




      bulb at top of column with flared out funnel shape at top of bulb  - a




      special order (Kontes K-420540-9011).




 4.7  Chromatographic Column - pyrex  (approximately 400 mm long x 20 mm  ID)




      with coarse fritted plate on bottom.




 4.8  Micro Syringes - 10, 25, 50 and 100 pi




 4.9  Separatory Funnels - 125 ml, 1000 ml and 2000 ml with Teflon stopcock.




4.10  Blender - High speed, glass or stainless steel cup.

-------
                                     1-5



    4.11  Graduated cylinders - 100 and 250 ml




    4.12  Florisil - PR Grade (60-100 mesh); purchase activated at 1250 F




          and store in the dark in glass containers with glass stoppers or




          foil-lined screw caps.  Before use, activate each batch overnight




          at 130 C in foil-covered glass container.  Determine lauric-acid




          value (See Appendix I).




5.  Reagents, Solvents, and Standards




    5.1   Ferrous Sulfate -  (ACS) 30% solution in distilled water.




    5.2   Potassium Iodide - (ACS) 10% solution in distilled water.




    5.3   Sodium Chloride -  (ACS) Saturated solution in distilled water




          (pre-rinse NaCl with hexane).




    5.4   Sodium Hydroxide   (ACS) 10 N in distilled water.




    5.5   Sodium Sulfate - (ACS) Granular, anhydrous.




    5.6   Sulfuric Acid - (ACS) Mix equal volumes of cone. H-SO. with




          distilled water.




    5.7   Diethyl Ether - Nanograde, redistilled in glass, if necessary.




          5.7.1  Must contain 2% alcohol and be free of peroxides by




                 following test:  To 10 ml of ether in glass-stoppered




                 cylinder previously rinsed with ether; add one ml of




                 freshly prepared 10% KI solution.   Shake and let stand




                 one minute.   No yellow color should be observed in either layer.




          5.7.2  Decompose ether peroxides by adding 40 g of 30% ferrous  sulfate




                 solution to each  liter of solvent.   CAUTION:   Reaction  maybe




                 vigorous if the solvent contains a high concentration of




                 peroxides.




          5.7.3  Distill deperoxidized ether in glass and add 2% ethanol.

-------
                                    1-6




    5.8  Acetonitrile,  Hexane,  Methanol, Methylene Chloride,  Petroleum



         Ether (boiling range 30-60 C)  - nanograde, redistill in glass



         if necessary



    5.9  Pesticide Standards -  Reference grade.



6.  Calibration



    6.1  Gas chromatographic operating  conditions  are considered acceptable



         if the response to dicapthon is at least  50% of full scale when



         < 0.06 ng is injected for electron capture detection and < 100 ng is



         injected for microcoulometric  or electrolytic conductivity detection.



         For all quantitative measurements, the  detector must be operated



         within its linear response range and the  detector noise level should



         be less than 2% of full scale.



    6.2  Standards are injected frequently as a  check on the  stability of



         operating conditions.   Gas chromatograms  of several  standard



         pesticides are shown in Figures 1, 2, 3 and 4 and provide reference



         operating conditions for the four recommended columns.



    6.3  The elution order and retention ratios  of various organochlorine



         pesticides are provided in Table 1,  as  a  guide.



7.  Quality Control



    7.1  Duplicate and spiked sample analyses are  recommended as quality control



         checks.  When the routine occurrence of a pesticide  is  being observed,



         the use of quality control charts is recommended (5).



    7.2  Each time a set of samples is  extracted,  a method blank is determined



         on a volume of distilled water equivalent to that used  to dilute the



         sample.

-------
                                     1-7






8-   Sample Preparation




    8.1  Blend the sample if suspended matter is present and adjust pH to




         near neutral (pH 6.5-7.5) with 50% sulfuric acid or 10 N sodium




         hydroxide.




    8.2  For a sensitivity requirement of 1 ug/1, when using microcoulometric




         or electrolytic conductivity methods for detection take 100 ml of




         sample for analysis.  If interferences pose no problem, the sensitivity




         of the electron capture detector should permit as little as 50 ml of




         sample to be used.  Background information on the extent and nature




         of interferences will assist the analyst in choosing the required




         sample size and preferred detector.




    8.3  Quantitatively transfer the proper aliquot into a two-liter separatory




         funnel and dilute to one liter.




9.   Extraction




    9.1  Add 60 ml of 15% methylene chloride in hexane (v:v) to the sample in




         the separatory funnel and shake vigorously for two minutes.




    9.2  Allow the mixed solvent to separate from the sample, then draw the




         water into a one-liter Erlenmeyer flask.  Pass the organic layer




         through a column containing 3-4 inches of anhydrous sodium sulfate,




         and collect it in a 500 ml K-D flask equipped with a 10 ml ampul.




         Return the water phase to the separatory funnel.   Rinse the Erlen-




         meyer flask with a second 60 ml volume of solvent; add the solvent




         to the separatory funnel and complete the extraction procedure a




         second time.  Perform a third extraction in the same manner.




    9.3  Concentrate the extract in the K-D evaporator on  a hot water bath.

-------
                                      1-8




     9.4  Analyze by gas chromatography unless a need for cleanup is  indicated.




          (See Section 10).




10.  Clean-up and Separation Procedures




     10.1  Interferences in the form of distinct peaks and/or high background




           in the initial gas chromatographic analysis, as well as the physical




           characteristics  of the extract (color;  cloudiness, viscosity)  and




           background knowledge of the sample will indicate whether clean-up




           is required.   When these interfere with measurement of the pesticides,




           or affect column life or detector sensitivity, proceed as  directed




           below.




     10.2  Acetonitrile Partition - This procedure is used to isolate fats  and




           oils from the sample extracts.  It should be noted that not all




           pesticides are quantitatively recovered by this procedure.  The




           analyst must be  aware of this and demonstrate the efficiency of




           the partitioning for specific pesticides.   Of the pesticides listed




           in Scope (1.2) only mirex is not efficiently recovered.




           10.2.1  Quantitatively transfer the previously concentrated extract




                   to a 125 ml separatory funnel with enough hexane to bring




                   the final volume to 15 ml.   Extract the sample four times




                   by shaking vigorously for one minute with 30 ml portions




                   of hexane-saturated acetonitrile.




           10.2.2  Combine  and transfer the acetonitrile phases to a  one-liter




                   separatory funnel and add 650 ml of distilled water and




                   40 ml of saturated sodium chloride solution.   Mix  thoroughly




                   for 30-45 seconds.   Extract with two 100 ml  portions of

-------
                                   1-9







              hexane by vigorously shaking about  15  seconds.




      10.2.3  Combine the hexane extracts  in a one-liter  separatory  funnel




              and wash with two 100 ml portions of distilled  water.   Dis-




              card the water layer and pour the hexane layer  through a




              3-4 inch anhydrous sodium sulfate column into a 500 ml K-D




              flask equipped with a 10 ml  ampul.   Rinse the separatory




              funnel and column with three 10 ml  portions of  hexane.




      10.2.4  Concentrate the extracts to  6-10 ml in the  K-D  evaporator




              in a hot water bath.




      10.2.5  Analyze by gas chromatography unless a need for further




              cleanup is indicated.




10.3  Florisil Column Adsorption Chromatography




      10.3.1  Adjust the sample extract volume to 10 ml.




      10.3.2  Place a charge of activated  Florisil (weight determined by




              lauric-acid value, see Appendix I)  in  a Chromaflex column.




              After settling the Florisil  by tapping the  column, add about




              one-half inch layer of anhydrous granular sodium sulfate  to




              the top.




      10.3.3  Pre-elute the column, after  cooling, with 50-60 ml of




              petroleum ether.  Discard the eluate and just prior to




              exposure of the sulfate layer to air,  quantitatively transfer




              the sample extract into the  column by  decantation and  subse-




              quent petroleum ether washings.  Adjust the elution rate  to




              about 5 ml per minute and, separately,  collect  up to three




              eluates in 500 ml K-D flasks equipped  with  10 ml  ampuls.




              (See Eluate Composition 10.4).

-------
                                  1-10


              Perform  the  first  elution with  200 ml  of 6% ethyl  ether in

              petroleum ether, and the second elution with 200 ml  of 15%

              ethyl  ether  in  petroleum ether.   Perform the third elution

              with 200 ml  of  50% ethyl ether  - petroleum ether and the

              fourth elution  with 200 ml  of 100% ethyl ether.

      10.3.4  Concentrate  the eluates to  6-10 ml in  the K-D evaporator

              in a hot water  bath.

      10.3.5  Analyze by gas  chromatography.

10.4  Eluate Composition - By using an equivalent quantity of  any  batch of

      Florisil as determined  by  its lauric acid value, the pesticides will

      be separated into the eluates indicated below:

                                6% Eluate

             Aldrin               DDT                       Pentachloro-
             BHC                Heptachlor                  nitrobenzene
             Chlordane          Heptachlor Epoxide          Strobane
             ODD                Lindane                      Toxaphene
             DDE                Methoxychlor                 Trifluralin
                                  Mirex                      PCB's

                    15% Eluate                50% Eluate

                  Endosulfan  I             Endosulfan II
                    Endrin                 Captan
                  Dieldrin
                  Dichloran
                  Phthalate esters

      Certain thiophosphate pesticides will occur in each of the above

      fractions as well as the 100% fraction.   For additional  information

      regarding eluate composition, refer to  the FDA Pesticide Analytical

      Manual (6).

-------
                                    1-11
11.   Calculation of Results

    11.1  Determine the pesticide concentration by using the absolute calibra-

          tion procedure described below or the relative calibration procedure

          described in Part  I, Section 3.4.2.  (1).

          (1)    Micrograms/liter =  (A)   (B)   (Vt)

                                       (VA)   (vs)

                 A =  ng standard
                      Standard  area

                 B =  Sample  aliquot  area

                 V. = Volume of  extract injected

                 V  = Volume of  total  extract

                 V  = Volume of  water  extracted  (ml)

 12.   Reporting  Results

     12.1  Report results  in  micrograms per liter  without correction  for

          recovery data.   When duplicate  and spiked  samples  are analyzed,all

          data  obtained  should be reported.

-------
                                         1-12

                                        REFERENCES

 1.   "Method for Organic Pesticides in Water and Wastewater,"  Environmental
     Protection Agency, National Environmental Research Center, Cincinnati, Ohio
     45268, 1971.

 2.   Monsanto Methodology for Aroclors - Analysis of Environmental Materials for
     Biphenyls, Analytical Chemistry Method 71-35, Monsanto Company, St. Louis,
     Missouri 63166, 1970.

 3.   "Method for Polychlorinated Biphenyls in Industrial Effluents," Environmental
     Protection Agency, National Environmental Research Center, Cincinnati, Ohio
     45268, 1973.

 4.   "Method for Organophosphorus Pesticides in Industrial Effluents,"  Environ-
     mental Protection Agency, National Environmental Research Center, Cincinnati
    -Ohio 45268, 1973.

 5.   "Handbook for Analytical Quality Control in Water and Wastewater Laboratories,"
     Chapter 6, Section 6.4,  U.S. Environmental Protection Agency, National Environ-
     mental Research Center,  Analytical Quality Control Laboratory,  Cincinnati,
     Ohio 45268, 1973.

 6.   "Pesticide Analytical Manual," U.S. Dept. of Health,  Education and Welfare,
     Food and Drug Administration, Washington, D.C.

 7.   "Analysis of Pesticide Residues in Human and Environmental Samples," U.S.
     Environmental Protection Agency, Perrine Primate Research Laboratories,
     Perrine, Florida  33157, 1971.

 8.   Mills, P.A., "Variation of Florisil Activity:  Simple Method for Measuring
     Adsorbent Capacity and its Use in Standardizing Florisil Columns," Journal
     .of the Association of Official Analytical Chemists, 51,  29 (1968).

 9.   Goerlitz, D.F.  and Brown, E., "Methods for Analysis of Organic Substances
     in Water," Techniques of Water Resources Investigations  of the United States
     Geological Survey, Book  5, Chapter A3, U.S.  Department of the Interior,
     Geological Survey, Washington, D.C. 20402, 1972, pp.  24-40.

10.   Steere, N.V., editor, "Handbook of Laboratory Safety," Chemical Rubber
     Company, 18901  Cranwood  Parkway, Cleveland,  Ohio 44128,  1971, pp. 250-254.

-------
                                       1-13





                                       Table  1




    RETENTION RATIOS OF VARIOUS ORGANOCHLORINE  PESTICIDES  RELATIVE  TO  ALDRIN
Liquid Phase

Column Temp.
Argon/Methane
Carrier Flow
Pesticide
Trifluralin
"-EMC
PCNB
Lindane
Dichloran
Heptachlor
Aldrin
Heptachlor Epoxide
Endosulfan I
p,p'-DDE
Dieldrin
Captan
Fncirin
j,I-.f-DDT
p.p'-DDD
lirulosulfan II
P.i !-UDT
Mi rex
* -r 'ioxvchlor
^ 'rin
I1*1 in absolute)
1.5% OV-17
2.95% QF-1
200 C
60 ml/min
RR
0.39
0.54
0.68
0.69
0.77
0.82
1.00
1.54
1.95
2.23
2.40
2.59
2.93
3.16
3.48
3.59
4.18
6.1
7.6
3.5
5%
OV-210
180 C
70 ml/min
RR
1.11
0.64
0.85
0.81
1.29
0.87
1.00
1.93
2.48
'2.10
3.00
4.09
3.56
2.70
3.75
4.59
4.07
3.78
6.5
2.6
3%
OV-1
180 C
70 ml/min
RR
0.33
0.35
0.49
0.44
0.49
0.78
1.00
1.28
1.62
2.00
1.93
1.22
2.18
2.69
2.61
2.25
3.50
6.6
5.7
4.0
6% QF-1
4% SE-30
200 C
60 ml/min
RR
0.57
0.49
0.63
0.60
0.70
0.83
1.00
1.43
1.79
1.82
2.12
1.94
2.42
2.39
2.55
2.72
3.12
4.79
4.60
5.6
'  ,i columns glass,  180 cm X 4 mm ID, solid support Gas-Chrom Q (100/120 mesh)

-------
                                      1-1





                                   APPENDIX I





13.   Standardization of Florisil Column by Weight Adjustment Based on Adsorption




     of Laurie Acid.



     13.1  A rapid method for determining adsorptive capacity of Florisil is




           based on adsorption of lauric acid from hexane solution (6) (8).




           An excess of lauric acid is used and amount not adsorbed is measured




           by alkali titration.  Weight of lauric acid adsorbed is used to




           calculate, by simple proportion, equivalent quantities of Florisil




           for batches having different adsorptive capacities.




     13.2  Apparatus




           13.2.1  Buret. -- 25 ml with 1/10 ml graduations.




           13.2.2  Erlenmeyer flasks.  -- 125 ml narrow mouth and 25 ml, glass




                   stoppered.




           13.2.3  Pipet. -- 10 and 20 ml transfer.




           13.2.4  Volumetric flasks.  -- 500 ml.




     13.3  Reagents and Solvents




           13.3.1  Alcohol,  ethyl. --  USP or absolute, neutralized to




                   phenolphthalein.




           13.3.2  Hexane.  -- Distilled from all  glass apparatus.




           13.3.3  Lauric acid. --Purified, CP.




           13.3.4  Lauric acid solution. -- Transfer 10.000 g lauric acid to




                   500 ml volumetric flask, dissolve in hexane, and dilute to




                   500 ml (1 ml = 20 mg).




           13.3.5  Phenolphthalein Indicator.  --  Dissolve 1 g in alcohol  and




                   dilute to 100 ml.

-------
                               1-2
      13.3.6  Sodium hydroxide. -- Dissolve 20 g NaOH (pellets, reagent




              grade) in water and dilute to 500 ml (1NJ .   Dilute 25 ml




              IN NaOH to 500 ml with water (0.05N).  Standardize as follows:




              Weigh 100-200 mg lauric acid into 125 ml Erlenmeyer flask.




              Add 50 ml neutralized ethyl alcohol and 3  drops phenol-




              phthalein indicator; titrate to permanent  end point.   Calculate




              mg lauric acid/ml 0.05 N_ NaOH (about 10 mg/ml).




13.4  Procedure




      13.4.1  Transfer 2.000 g Florisil to 25 ml glass stoppered Orlenmeyer




              flasks.  Cover loosely with aluminum foil  and heat overnight




              at 130°C.  Stopper, cool to room temperature, add 20.0 ml




              lauric acid solution (400 mg) , stopper, and shake occasionally




              for 15 min.  Let adsorbent settle and pipet 10.0 ml of




              supernatant into 125 ml Erlenmeyer flask.   Avoid inclusion




              of any Florisil.




      13.4.2  Add 50 ml neutral alcohol and 3 drops indicator solution;




              titrate with 0.05N to a permanent end point.




13.5  Calculation of Lauric Acid Value and Adjustment of Column Weight




      13.5.1  Calculate amount of lauric acid adsorbed on Florisil  as




              follows :




              Lauric Acid value = mg lauric acid/g Florisil = 200   (ml




              required for titration X mg lauric acid/ml  0.05N_NaOH).




      13.5.2  To obtain an equivalent quantity of any batch of Florisil,




              divide 110 by lauric acid value for that batch and multiply




              by 20 g.   Verify proper elution of pebticides by 15.6.

-------
                               1-3
13.6  Test for Proper Elution Pattern and Recovery of Pesticides:




      Prepare a test mixture containing aldrin,  heptachlor epoxide,




      p.p'-DDE, dieldrin,  Parathion and malathion.   Dieldrin and




      Parathion should elute in the 15% eluate;  all  but  a trace of




      malathion in the 50% eluate and the others in  the  6% eluate.

-------
                          20
 15                10
RETENTION TIME IN MINUTES
Figure  1. Column Packing: 1.5% OV-17 + 1.95% QF-1,  Carrier  Gas:  Argon/Methane  at 60 ml/min,
                     Column Temperature: 200 C,  Detector: Electron  Capture.

-------
                                                  .
        15                10                 5                 0
                      RETENTION TIME IN MINUTES
Figure 2. Column  Packing:  5%  OV-210, Carrier Gas: Argon/Methane
          at 70 ml/min, Column Temperature: 180 C, Detector:
          Electron  Capture.

-------
25
             20                15               10
                            RETENTION TIME IN MINUTES
Figure  4. Column Packing:  3%  OV-1, Carrier  Gas: Argon/Methane at 70  ml/min,
         Column Temperature:  180 C, Detector:  Electron Capture.

-------
   25
20
 15                10
RETENTION TIME IN MINUTES
Figure 3. Column  Packing: 6% QF-1 + 4% SE-30, Carrier Gas: Argon/Methane at 60 ml/min,
                   Column  Temperature: 200  C,  Detector:  Electron Capture.

-------
       2.  METHOD FOR ORGANOPIIOSPHORUS PESTICIDES IN INDUSTRIAL EFFLUENTS
o  ^
I—

2:  5
    Q_
    Q_
    «a:
     .. CO
   co
   Scope and Application


   1.1  This method covers the determination of various organophosphorus


        pesticides and may be extended to pesticidal degradation products


        and related compounds.  Such compounds are composed of carbon,


        hydrogen, and phosphorus, but may also contain sulfur, oxygen,


        halogen or nitrogen.


   1.2  The following compounds may be determined individually by this method


        with a sensitivity of 1 ug/1 :  Disyston, Diazinon, malathion, Methyl


        Parathion, Parathion, demeton, and Guthion.  Under favorable circum-


        stances other organophosphorus pesticides may also be determined.


        However, the usefulness of the method for other specific compounds


        must be demonstrated by the analyst before applying it to sample
 •
bQ      analysis.


. .  1.3  When organophosphorus pesticides exist as complex mixtures, the


fzi       individual compounds may be difficult to distinguish.  High, low, or


        otherwise unreliable results may be obtained through misidentifica-


        tion and/or one compound obscuring another of lesser concentration.


        Provisions incorporated in this method are intended to minimize the


        occurrence of such interferences.


  Summary


  2.1  The method offers  several analytical alternatives, dependent on the


       analyst's assessment of the nature and extent of interferences and


       the complexity of  the pesticide mixtures found.  Specifically, the


       procedure describes the use of an effective co-solvent for efficient


       sample extraction; provides, through use of column chromatography

-------
                                 2-2






         and  liquid-liquid partition, methods  for  the  elimination  of non-




         pesticide  interferences  and the  preseparation of pesticide mixtures.




         Identification  is made by  selective gas chromatographic  separation




         and  may  be corroborated  through  the use of two or more unlike




         columns.   Detection  and  measurement are best  accomplished by  flame




         photometric gas  chromatography using  a phosphorus specific filter.




         The  electron capture detector, though non-specific,  may  also  be




         used for those  compounds to which  it  responds.   Results  are reported




         in micrograms per liter.



    2.2  This method is  recommended for use only by experienced pesticide




         analysts or under the close supervision of such qualified persons.




3.   Interferences




    3.1  Solvents,  reagents,  glassware, and other  sample processing hardware




         may  yield discrete artifacts and/or elevated  baselines causing




         misinterpretation of gas chromatograms.   All  of these materials




         must be  demonstrated to  be free  from  interference under  the




         conditions of the analysis.  Specific selection of reagents and puri-




         fication of solvents by  distillation  in all-glass systems may be




         required.   Refer to  Part 1, Sections  1.4,  1.5 (1).




    3.2  The  interferences in industrial  effluents  are high and varied and




         often pose great difficulty in obtaining  accurate and precise




         measurement of  organophosphorus  pesticides.   Sample  clean-up




         procedures are  generally required  and may result in  the  loss  of




         certain  organophosphorus pesticides.  Therefore, great care should be




         exercised  in the selection and use of methods for eliminating or




         minimizing interferences.  It is not  possible to describe procedures

-------
                                    2-3






         for overcoming all of the interferences  that  may be encountered  in




         industrial effluents.




    3.3  Compounds such as organochlorine pesticides,  polychlorinated




         biphenyls and phthalate esters interfere with the analysis  of organo




         phosphorus pesticides by electron capture gas chromatography.  When




         encountered these interferences are overcome  by the use of  the




         phosphorus specific flame photometric detector.  If such a  detector




         is not available, these interferences may be  removed from the sample




         by using the clean-up procedures described in the EPA Methods for




         those compounds  (2)  (3).




    3.4  Elemental sulfur will interfere with the determination of organo-




         phosphorus pesticides by flame photometric and electron capture  gas




         chromatography.  The elimination of elemental sulfur as an  inter-




         ference is described in Section 10.5, Clean-up and Separation




         Procedures.




4.   Apparatus and Materials




    4.1  Gas Chromatograph   Equipped with glass lined injection port.




    4.2  Detector Options:




         4.2.1  Flame Photometric - 526 mg phosphorus  filter.




         4.2.2  Electron Capture - Radioactive (tritium or nickel-63)




    4.3  Recorder   Potentiometric strip chart (10 in.) compatible with the




         detector.

-------
                                 2-4







 4.4  Gas Chromatographic Column Materials:




      4.4.1  Tubing   Pyrex (180 cm long x 4 mm ID)




      4.4.2  Glass Wool - Silanized




      4.4.3  Solid Support - Gas-Chrom Q (100-120 mesh)




      4.4.4  Liquid Phases   Expressed as weight percent coated on




             solid support.




             4.4.4.1  OV-1, 3%




             4.4.4.2  OV-210, 5?0




             4.4.4.3  OV-17, 1.5% plus QF-1, 1.95%




             4.4.4.4  QF-1, 6% plus SE-30, 4%




 4.5  Kuderna-Danish  (K-D) Glassware (Kontes)




      4.5.1  Snyder Column - three ball  (macro) and two ball  (micro)




      4.5.2  Evaporative Flasks - 500 ml




      4.5.3  Receiver Ampuls - 10 ml, graduated




      4.5.4  Ampul Stoppers




 4.6  Chromatographic Column - Chromaflex  (40.0 mm x 19 mm  ID) with coarse




      fritted  plate and Teflon stopcock on bottom; 250 ml reservoir bulb




      at top of column with flared out funnel shape at top of bulb - a




      special order (Kontes K-420S40-9011).




 4.7  Chromatographic Column   Pyrex (approximately 400 mm long x 20 mm  ID)




      with coarse fritted plate on bottom.




 4.8  Micro Syringes - 10, 25, 50 and 100 ul




 4.9  Separatory Funnels - 125 ml, 1000 ml and 2000 ml with Teflon stopcock.




4.10  Micro-pipets - disposable (140 mm long x 5 mm ID)




4.11  Blender - High speed, glass or stainless steel cup.




4.12  Graduated cylinders - 100 and 250 ml

-------
                                      2- 5




    4.13  Florisil   PR Grade (60-100 mesh);  purchase activated at  1250 F  and




          store in the dark in glass containers with glass  stoppers  or




          foil-lined screw caps.  Before use, activate each batch overnight




          at 130 C in foil-covered glass container.   Determine lauric acid




          value (See Appendix I).




    4.14  Alumina - Woelm, neutral; deactivate by pipeting 1 ml of distilled




          water into 125 ml ground glass-stoppered Erlenmeyer flask.  Rotate




          flask to distribute water over surface of glass.   Immediately add




          19.0 g fresh alumina through small powder funnel.  Shake flask




          containing mixture for two hours on a mechanical shaker (4).




5.  Reagents, Solvents, and Standards




    5.1   Ferrous Sulfate - (ACS) 30% solution in distilled water.




    5.2   Potassium Iodide - (ACS) 10% solution in distilled water.




    5.3   Sodium Chloride - (ACS) Saturated solution  (pre-rinse Nad with




          hexane) in distilled water.




    5.4   Sodium Hydroxide - (ACS) 10 N in distilled water.




    5.5   Sodium Sulfate - (ACS) Granular, anhydrous.




    5.6   Sulfuric Acid - (ACS) Mix equal volumes of cone. H2S04 with




          distilled water.




    5.7   Uiethyl Ether - Nanograde, redistilled in glass, if necessary.




          5.7.1  Must contain 2% alcohol and be free of peroxides by following




                 test:  To 10 ml of ether in glass-stoppered cylinder previously




                 rinsed with ether, add one ml of freshly prepared 10% KI




                 solution.  Shake and let stand one minute.  No yellow color




                 should be observed in either layer.

-------
                                     2-6




          5.7.2   Decompose  ether peroxides  by adding 40 g of 30% ferrous




                 sulfate  solution  to  each liter of solvent.   CAUTION:




                 Reaction may be vigorous if the solvent contains a high




                 concentration of  peroxides.




          5.7.3   Distill  deperoxidized ether in glass and add 2% ethanol.




    5.8  Acetonitrile,  Hexane, Methanol,  Methylene Chloride, Petroleum Ether




         (boiling range 30-60 C) - Nanograde, redistill in glass if necessary.




    5.9  Pesticide Standards  - Reference  Grade.




6.  Calibration



    6.1  Gas chromatographic  operating conditions are considered acceptable




         if the  response  to dicapthon is  at least 50% of full scale when




         < 1.5 ng is injected for  flame photometric detection and < 0.06 ng is




         injected for electron capture detection.  For all quantitative




         measurements the detector must be  operated within its linear  response




         range and the detector noise level should be less than 2% of  full  scale.




    6.2  Standards are injected frequently  as a check on the stability of




         operating conditions. Gas chromatograms of several standard  pesticides




         are shown in Figures 1, 2, 3 and 4 and provide reference operating




         conditions for the four recommended columns.




    6.3  The elution order  and retention  ratios  of various organophosphorus




         pesticides are provided in Table 1,  as  a guide.



7.  Quality Control




    7.1  Duplicate and spiked sample  analyses are recommended as quality control




         checks.   When the  routine occurrence of a pesticide is being  observed,




         the use  of quality control charts  is recommended (5).

-------
                                  2 - 7




    7.2  Each time a set of samples is extracted,  a method  blank  is  determined




         on a volume of distilled water equivalent to that  used to dilute  the




         sample.




8.   Sample Preparation




    8.1  Blend the sample, if suspended matter is  present,  and adjust pH to




         near neutral (pH 6.5-7.5) with 50% sulfuric acid or 10 N sodium




         hydroxide.




    8.2  For a sensitivity requirement of 1 ug/1,  when using flame photometric




         or electron capture for detection, take 100 ml of sample for analysis.




    8.3  Quantitatively transfer a 100 ml aliquot  of sample into  a two-liter




         separator/ funnel and dilute to one liter.




9.   Extraction




    9.1  Add 60 ml of 15% methylene chloride in hexane (v:v) to the  sample in




         the separatory funnel and shake vigorously for two minutes.




    9.2  Allow the mixed solvent to separate from the sample, then  draw the




         water into a one-liter Erlenmeyer flask.   Pass the organic  layer




         through a column containing 3-4 inches of anhydrous sodium  sulfate,




         and collect it in a 500 ml K-D flask equipped with a 10  ml  ampul.




         Return the water phase to the separatory funnel.  Rinse  the Erlenmeyer




         flask with a second 60 ml volume of solvent, add the solvent to the




         separatory funnel, and complete the extraction procedure a  second time




         Perform a third extraction in the same manner.




    9.3  Concentrate the extract in the K-D evaporator on a hot water bath.




    j.-\  Analyze by gas chromatography unless a need for cleanup  is  indicated.




         (Sec Section 10).

-------
                                     2-8




10.   Clean-up and Separation Procedures




     10.1  Interferences in the form of distinct peaks and/or high background




           in the initial gas chroraatographic analysis, as well as the




           physical characteristics of the extract (.color, cloudiness, viscosity)




           and background knowledge of the sample will indicate whether clean-up




           is required.   When these interfere with measurement of the pesti-




           cides, or affect column life or detector sensitivity, proceed as




           directed below.




     10.2  Acetonitrile  Partition - This procedure is used to isolate fats and




           oils from the sample extracts.   It should be noted that not all




           pesticides are quantitatively recovered by this procedure.  The




           analyst must  be aware of this and demonstrate the efficiency of




           the partitioning for specific pesticides.




           10.2.1  Quantitatively transfer the previously concentrated extract




                   to a  125 ml separatory funnel with enough hexane to bring




                   the final volume to 15 ml.   Extract the sample four times




                   by shaking vigorously for one minute with 30 ml portions




                   of hexane-saturated acetonitrile.




           10.2.2  Combine and transfer the acetonitrile phases to a one-liter




                   separatory funnel and add 650 ml of distilled water and




                   40 ml of saturated sodium chloride solution.  Mix thoroughly




                   for 30-45 seconds.   Extract with two 100 ml portions  of




                   hexane by vigorously shaking about 15 seconds.




           10.2.3  Combine the hexane extracts in a one-liter separatory




                   funnel and wash with two 100 ml portions of distilled




                   water.   Discard the water layer and pour the hexane layer

-------
                             2 - 9




              through a 3-4 inch anhydrous  sodium sulfate  column  into a




              500 ml K-D flask  equipped with a  10 ml  ampul.   Rinse  the




              separator/ funnel and column  with three 10 ml  portions of




              hexane.




      10.2.4  Concentrate the extracts to 6-10  ml in  the K-D evaporator




              in a hot water bath.




      10.2.5  Analyze by gas chromatography unless a  need  for further




              clean-up is indicated.




10.3  Florisil Column Adsorption Chromatography




      10.3.1  Adjust the sample extract volume  to 10  ml.




      10.3.2  Place a charge of activated Florisil (weight determined by




              lauric-acid value, see Appendix I) in a Chromaflex  column.




              After settling the Florisil by tapping  the  column,  add




              about one-half inch layer of anhydrous  granular sodium




              sulfate to the top.




      10.3.3  Pre-elute the column, after cooling, with 50-60 ml  of




              petroleum ether.   Discard the eluate and just prior to




              exposure of the sulfate layer to  air, quantitatively




              transfer the sample extract into  the column  by decantation




              and subsequent petroleum ether washings.  Adjust the




              elution rate to about 5 ml per minute and,  separately,




              collect up to four eluates in 500 ml K-D flasks equipped




              with 10 ml ampuls.  (See Eluate Composition, 10.4.)




              Perform the first elution with 200 ml of 6%  ethyl ether  in




              petroleum ether,  and the second elution with 200 ml of  15%




              ethyl ether in petroleum ether.  Perform the third  elution

-------
                             2 -10

              with  200  ml  of 50%  ethyl  ether -  petroleum ether and the

              fourth  elution with 200 ml  of 100% ethyl  ether.

      10.3.4  Concentrate  the eluates to  6-10 ml in the K-D evaporator

              in a  hot  water bath.

      10.3.5  Analyze by gas chromatography.

10.4  Eluate Composition - By using  an  equivalent quantity of any batch

      of Florisil as  determined by its  lauric-acid value, the pesticides

      will be separated into the  eluates  indicated below:

         6% Eluate                            15% Eluate

         Demeton                             Diazinon
         Disyston                            Malathion (trace)
                                             Methyl Parathion

         50% Eluate                          100% Eluate

         Malathion                           Guthion (80%)
         Guthion (20%)

      For additional  information  regarding eluate composition, refer to the

      FDA Pesticide Analytical  Manual (6).

10.5  Removal of Sulfur -  If elemental  sulfur interferes with the gas

      chromatographic analysis, it can  be removed by the use of an

      alumina microcolumn.

      10.5.1  Adjust  the sample extract volume  to 0.5 ml in a K-D

              apparatus, using a  two-ball Snyder microcolumn.

      10.5.2  Plug  a  disposable pipet with a small quantity of glass wool.

              Add enough alumina  to  produce a 3 cm column after settling.

              Top the alumina with a 0.5  cm layer of anhydrous sodium

              sulfate.

      10.5.3  Quantitatively transfer the concentrated  extract to the

              alumina microcolumn using a 100 ul syringe.  Rinse the

-------
                                     2-11

                   ampul with 200 yl of hexane and add to  the  microcolumn.

           10.5.4  Elute the microcolumn with 3 ml of hexane and  discard the

                   first eluate which contains the elemental sulfur.

           10.5.5  Next elute the column with 5 ml of 10%  hexane  in methylene

                   chloride.  Collect the  eluate in a 10 ml graduated  ampul.

           10.5.6  Analyze by gas chromatography.

           NOTE:   If the electron capture  detector is to be used  methylene

                  chloride must be removed.   To do this, attach the ampul to a

                  K-D apparatus (500 ml flask and 3-ball Snyder column)  and

                  concentrate to about 0.5 ml.  Adjust volume  as  required prior

                  to analysis.

11.   Calculation of Results

    11.1  Determine the pesticide concentration by using the  absolute  calibra-

          tion procedure described below or the relative calibration procedure

          described in Part I, Section 3.4.2.(1).

          (1)     Micrograms/liter =  (A)  (B)   (Vt)

                                      CV  (Vs}

                 A = ng standard
                     Standard area

                 B - Sample aliquot area

                 V.= Volume of extract injected (yl)

                 V = Volume of total extract  (yl)

                 V = Volume of water extracted (ml)

12 .   Reporting Results

    12.1  Report results in micrograms per liter without correction for

          recovery data.  When duplicate and spiked samples  are analyzed all

          data obtained should be reported.

-------
2-12
TABLE 1
RETENTION TIMES OF SOME ORGANOPHOSPMOROUS PESTICIDES
RELATIVE TO PARATHION
Liquid Phase

Column Temp.
Nitrogen
Carrier Flow
Pesticide
Naled

DDVP
Phosdrin
Demeton

Thimet
Diazinon
Disulfoton
Dimethoate
Ronnel
Merphos
Malathion
Methyl Parathion
Parathion
Phosphamidon
DEF
Ethion
Trithion
F,PN
Guthion
Parathion
(min absolute)
1.5% OV-17
+
1.95% QF-1
215 C
70 ml/min
RR
with solvent

0.16
0.26
0.46

0.35
0.40
0.46
0.65
0.65
0.69
0.86
0.82
1.00
0.98
1.25
2.04
2.21
4.23
6.65
4.5

6% QF-1
+
4% SE-30
215 C
70 ml/min
RR
0.11
0.15
0.16
0.24
0.26
0.43
0.35
0.38
0.45
0.57
0.60
0.67
0.78
0.80
1.00
1.06
1.12
1.58
1.66
3.32
4.15
6.6

5%
OV-210
200 C
60 ml/min
RR
with solvent

0.13
0.23
0.20
0.38
0.23
0.25
0.31
0.58
0.43
0.34
0.73
0.81
1.00
1.30
0.78
2.27
1.18
3.. 37
4.44
5.7

7%
OV-1
200 C
60 ml/min
RR
with solvent

0.29
0.36
0.74

0.47
0.59
0.62
0.72
0.83
1.23
0.92
0.79
1.00
0.87
1.78
2.26
2.57
3.84
4.68
3.1

 All  columns  glass,  180  xm  x  4  mm  ID,  solid  support  Gas-Chrom  Q,  100/120  mesh.
I
"Anomalous, multipeak  response  often  encountered.

-------
                                      2-13
                                    REFERENCES

 (1)   "Methods for Organic Pesticides in Water  and Wastewater,"  U.S.  Environ-
      mental  Protection Agency,  National Environmental  Research  Center,
      Analytical Quality Control Laboratory,  Cincinnati,  Ohio  45268,  1971.

 (2)   "Method for Organochlorine Pesticides in  Industrial Effluents," U.S.
      Environmental Protection Agency, National Environmental  Research Center-
      Analytical Quality Control Laboratory,  Cincinnati,  Ohio  45268,  1973.

 (3)   "Method for Polychlorinated Biphenyls (PCB's) in  Industrial  Effluents,"
      U.S.  Environmental Protection Agency, National Environmental  Research
      Center, Analytical Quality Control Laboratory, Cincinnati, Ohio 45268,
      1973.

 (4)   Law,  L. M. and Goerlitz, D. F., "Microcolumn Chromatographic  Clean-up
      for the Analysis of Pesticides in Water," Journal of the Association
      of Official Analytical Chemists, 53,  1276 (1970).

 (5)   "Handbook for Analytical Quality Control  in Water and Wastewater
      Laboratories", Chapter 6,  Section 6.4,  U.S.  Environmental  Protection
      Agency, National Environmental Research Center, Analytical Quality
      Control Laboratory, Cincinnati, Ohio  45268,  1972.

 (6)   "Pesticide Analytical Manual," U.S. Department of Health,  Education,
      and Welfare, Food and Drug Administration, Washington, D.C.

 (7)   "Analysis of Pesticide Residues in Human  and Environmental Samples;"
      U.S.  Environmental Protection Agency, Perrine Primate Research
      Laboratories, Perrine, Florida 33157, 1971.

 (8)   Mills,  P. A., "Variation of Florisil  Activity:  Simple Method for
      Measuring Adsorbent Capacity and Its  Use  in Standardizing Florisil
      Columns," Journal of the Association  of Official  Analytical  Chemists,
      51_, 29  (196TT

 (9)   Goerlitz, D. F.  and Brown, E., "Method for Analysis of Organic  Substances
      in Water'" Techniques of Water Resources  Investigations  of the  United
      States  Geological Survey,  Book 5, Chapter A3, U.S.  Department of the
      Interior, Geological Survey, Washington,  D.C. 20402, 1972, pp.  24-40.

(10)   Steere, N. V., editor, "Handbook of Laboratory Safety,"  Chemical Rubber
      Company, 18901 Cranwood Parkway, Cleveland,  Ohio  44128,  1971, pp.  250-254

-------
                                      2-1
                                   APPENDIX I





13.   Standardization of Florisil  Column by Weight Adjustment Based on Adsorption




     of Lauxic Acid.



     13.1  A rapid method for determining adsorptive capacity of Florisil is




           based on adsorption of lauric acid from hexane solution (6) (8).




           An excess of lauric acid is used and amount not adsorbed is measured




           by alkali titration.   Weight of lauric acid adsorbed is used to




           calculate, by simple proportion, equivalent quantities of Florisil




           for batches having different adsorptive capacities.




     13.2  Apparatus




           13.2.1  Buret. -- 25 ml with 1/10 ml graduations.




           13.2.2  Erlenmeyer flasks.  -- 125 ml narrow mouth and 25 ml, glass




                   stoppered.




           13.2.3  Pipet. -- 10 and 20 ml transfer.




           13.2.4  Volumetric flasks.  -- 500 ml.




     13.3  Reagents and Solvents




           13.3.1  Alcohol,  ethyl. --  USP or absolute, neutralized to




                   phenolphthalein.




           13.3.2  Hexane.  -- Distilled from all  glass apparatus.




           13.3.3  Lauric acid.  --Purified, CP.




           13.3.4  Lauric acid solution. -- Transfer 10.000 g lauric acid to




                   500 ml volumetric flask, dissolve in hexane, and dilute to




                   500 ml (1 ml  = 20 mg).




           13.3.5  Phenolphthalein Indicator.  --  Dissolve 1 g in alcohol  and




                   dilute to 100  ml.

-------
                              2  - 2
      13.3.6  Sodium hydroxide. -- Dissolve 20 g NaOH (pellets, reagent




              grade) in water and dilute to 500 ml (IN).   Dilute 25 ml




              IN NaOH to 500 ml with water (0.05NJ.   Standardize as follows:




              Weigh 100-200 mg lauric acid into 125  ml Erlenmeyer flask.




              Add 50 ml neutralized ethyl alcohol and 3 drops phenol -




              phthalein indicator; titrate to permanent end point.   Calculate




              mg lauric acid/ml 0.05 N_ NaOH (about 10 mg/ml).




13.4  Procedure




      13.4.1  Transfer 2.000 g Florisil to 25 ml glass stoppered Erlenmeyer




              flasks.  Cover loosely with aluminum foil and heat overnight




              at 130°C.  Stopper, cool to room temperature, add 20.0 ml




              lauric acid solution (400 mg) , stopper, and shake occasionally




              for 15 min.  Let adsorbent settle and  pipet 10.0 ml of




              supernatant into 125 ml Erlenmeyer flask.  Avoid inclusion




              of any Florisil.




      13.4.2  Add 50 ml neutral alcohol and 3 drops  indicator solution;




              titrate with 0.05N_ to a permanent end  point.




13.5  Calculation of Lauric Acid Value and Adjustment of Column Weight




      13.5.1  Calculate amount of lauric acid adsorbed on Florisil as




              follows :




              Lauric Acid value = mg lauric acid/g Florisil,= 200 - (ml




              required for titration X mg lauric acid/ml  0.05N_NaOH).




      13.5.2  To obtain an equivalent quantity of any batch of Florisil,




              divide 110 by lauric acid value for that batch and multiply




              by 20 g.  Verify proper elution of pesticides by 13.6.

-------
                             2 - 3
13.6  Test for Proper  Elution  Pattern  and Recovery  of  Pesticides:




      Prepare a test mixture containing aldrin,  heptachlor  epoxide,




      p,p'-DDE, dieldrin,  Parathion  and malathion.   Dieldrin  and




      Parathion should elute in  the  15% eluate;  all but  a trace  of




      malathion in  the 50% eluate  and  the others in the  6%  eluate.

-------
0
2
10
12
                   4         6         8
                RETENTION TIME  IN MINUTES
Figure 1. Column Packing: 1.5%  OV-17  + 1.95 %  QF-1,
Carrier Gas:  Nitrogen at  70 ml/min,  Column Temperature:  215  C,
Detector: Flame Photometric (Phosphorus).

-------
0
10
           2468
            RETENTION TIME IN MINUTES
Figure 2. Column Packing: 5% OV-210,  Carrier Gas: Nitrogen
at 60 ml/min,  Column Temperature: 200 C, Detector:
Flame Photometric (Phosphorus).

-------
0
2
10
12
                     4         6        8
                 RETENTION TIME IN MINUTES
Figure 3. Column Packing: 6% QF-1 +4% SE-30, Carrier Gas: Nitrogen
at 70 ml/min,  Column Temperature:  125 C,  Detector:  Flame
Photometric (Phosphorus).

-------
            2         4          6          8         10
                RETENTION TIME IN  MINUTES
Figure 4. Column Packing: 3% OV-1, Carrier Gas: Nitrogen at
60 ml/min,  Column Temperature:  200 C,  Detector: Flame
Photometric (Phosphorus).
                                     U.S. GOVERNMEKT P«IKTII« OfTICt 1973- 759-555/1146

-------
       1.
       2.
_r ^
=>  ^
><
o
   «=  UJ

   Ij  °-
   UJ  C"1-
3-   METHOD FOR POLYCHLORINATED BIPHENYLS [PCB'S] IN INDUSTRIAL



 Scope and Application


 1.1  This method covers the determination of certain polychlorinated


      biphenyl (PCB) mixtures including:  Aroclors 1221, 1232, 1242, 1248,


      1254, 1260 and 1016.


 1.2  The method is an extension of the method for organochlorine pesticides


      in industrial effluents (1).   It is designed so that determination of


      both the PCB's and the organochlorine pesticides may be made on the


      same sample.


 1.3  The limit of detection is approximately 1 yg/1 for each Aroclor mixture.


 Summary


 2.1  The PCB's and the organochlorine pesticides are co-extracted by

 jLjjJ
 M   liquid-liquid extraction and, insofar as possible, the two classes of


 •M   compounds separated from one another prior to gas chromatographic


   e   determination.  A combination of the standard Florisil column cleanup


   ,   procedure and a silica gel microcolumn separation procedure (2)(3) are


      employed.  Identification is made from gas chromatographic patterns
    o
    23
-J  UJ

2—  O—
    on
    to
      obtained through the use of two or more unlike columns.  Detection
       LU
O  «C  _
r:  =c  oo
5  to  r^
    bO
    1)
           T»
           ID
      and measurement is accomplished using an electron capture, microcoulo-


      metric, or electrolytic conductivity detector.  Techniques for confirm-


      ing qualitative identification are suggested.
 HH

 Interferences


 3.1   Solvents, reagents, glassware, and other sample processing hardware


      may yield discrete artifacts and/or elevated baselines causing mis-


      interpretation of gas chromatograms.   All of these materials must be


      demonstrated to be free from interferences under the conditions of


      the analysis.   Specific selection of reagents and purification of


      solvents by distillation in all-glass systems may be required.  Refer

-------
                                      3-2




         to  (4),  Part  I,  Sections  1.4  and  1.5.




    3.2   The  interferences  in  industrial effluents  are  high  and varied and




         pose great  difficulty in  obtaining  accurate  and precise measurement




         of  PCB's and  organochlorine pesticides.  Separation and cleanup  pro-




         cedures  are generally required to eliminate  these interferences; however,




         such techniques  may result in the loss  of  certain organochlorine com-




         pounds.   For  this  reason  great care should be  exercised in the selection




         and use  of  methods for eliminating  or minimizing interferences.   It




         is  not possible  to describe procedures  for overcoming all  of the inter-




         ferences that may  be  encountered  in industrial wastes.




    3.3  Phthalate esters,  certain organophosphorus pesticides, and elemental




         sulfur will interfere when using  electron  capture for detection.  These




         materials do  not interfere when the microcoulometric or electrolytic




         conductivity  detectors are used in  the  halogen mode.




    3.4  Organochlorine pesticides and other halogenated compounds  constitute




         interferences in the  determination  of PCB's.   Most  of these are




         separated by  the method described below.   However,  certain compounds,




         if  present  in the  sample, will occur with  the  PCB's.  Included are:




         Sulfur^  Heptachlor, aldrin, DDE,  technical chlordane, mirex,  and to




         some extent o,p'-DDT  and  p,p'-DDT.




4.  Apparatus and Materials




    4.1  Gas Chromatograph  - Equipped  with glass lined  injection part.




    4.2  Detector Options:




         4.2.1 Electron  Capture - Radioactive  (tritium or nickel-63)




         4.2.2 Microcoulometric Titration




         4.2.3 Electrolytic Conductivity




    4.3  Recorder -  Potentiometric strip chart  (10  in.) compatible  with




         detector system.

-------
                                    3-3




 4.4  Gas Chromatographic Column Materials;




      4.4.1  Tubing - Pyrex  (180 cm long X 4 mm ID)




      4.4.2  Glass Wool - Silanized




      4.4.3  Solid Support - Gas-Chrom Q  (100-120 mesh)




      4.4.4  Liquid Phases - Expressed as weight percent coated on solid




             support:




             4.4.4.1  SE-30 or OV-1, 3%




             4.4.4.2  OV-17. 1.5% + QF-1, 1.95%




 4.5  Kuderna-fDanish  (K-D~, Glassware (Kontes)




      4.5.1  Snyder Columns - three ball  Oacro)




      4.5.2  Evaporate Flasks   500 ml




      4.5.3  Receiver Ampuls - 10 ml, graduated




      4.5.4  Ampul stoppers




 4.6  Chromatographic Column - Chromaflex C400 mm long X 19 mm ID) with




      coarse fritted plate on bottom and Teflon stopcock; 250 ml reservoir




      bulb at  top of column with flared out funnel shape at top of bulb -




      a special order (Kontes K-420540-9011).




 4.7  Chromatographic Column - Pyrex (approximately 400 mm long X 20 mm ID)




      with a coarse fritted plate on bottom.




 4.8  Micro Column Pyrex - constructed according to Figure 1.




 4.9  Capillary pipets disposable (5-3/4 in.) with rubber bulb.  (Scientific




      Products P5205-1).




4.10  Low pressure regulator - 0 to 5 PSIG - with low-flow needle valve




      (See Figure 1, Matheson Model 70).




4.11  Beaker - 100 mi




4.12  Micro syringes - 10, 25, 50 and 100 pi.




4.13  Separatory Funnels   125 ml, 1000 ml, and 2000 ml with Teflon stopcocks

-------
                                     3-4




    4.14   Graduated Cylinders  -  100 ml,  250 ml.




    4.15   Blender - High  speed,  glass or stainless  cup.




    4.16   Florisil - PR Grade  (60-100 mesh);  purchase  activated at 1250 F




          and store in the dark  in glass containers  with  glass  stoppers or




          foil-lined screw caps.   Before use, activate each  batch overnight




          at 130 in foil-covered glass container.   Determine lauric-acid




          value (See Appendix  I).




    4.17   Silica gel - Davison code 950-08-08-226  (60/80  mesh).




    4.18   Glass Wool - Hexane  extracted.




    4.19   Centrifuge Tubes - Pyrex calibrated (15 ml).




5.  Reagents, Solvents and Standards




    5.1   Ferrous Sulfate - (ACS)  30% solution in distilled  water.




    5.2   Potassium Iodide -  (ACS) 10% solution in  distilled water.




    5.3   Sodium Chloride - (ACS)  Saturated solution (pre-rinse NaCl  with




          hexane) in distilled water.




    5.4   Sodium Hydroxide -  (ACS) 10 N in distilled water.




    5.5   Sodium Sulfate  - (ACS)  Granular, anhydrous,  conditioned for




          4 hours @ 400 C.




    5.6   Sulfuric Acid - (ACS)  Mix equal volumes of cone. H_SO. with




          distilled water.




    5.7   Diethyl Ether - Nanograde, redistilled in glass, if necessary.




          5.7.1  Must contain  2% alcohol and be free of peroxides by




                 following test:   to 10 ml of ether in glass-stoppered




                 cylinder previously rinsed with ether, add  one ml of




                 freshly  prepared 10% KI solution.   Shake and let stand




                 one minute.   No yellow color should be observed in



                 either layer.

-------
                                      3-5

         5.7.2  Decompose ether peroxides by adding 40 g of 30% ferrous

                sulfate solution to each liter of solvent.   CAUTION:

                Reaction may be vigorous if the solvent contains a high

                concentration of peroxides.

         5.7.3  Distill deperoxidized ether in glass and add 2% ethanol.
                                                             ^
    5.8  n-Hexane - Pesticide quality (NOT MIXED HEXANES).

    5.9  Acetonitrile, Hexane, Methanol, Methylene Chloride, Petroleum

         Ether (Boiling range 30-60 C) - pesticide quality, redistill in

         glass if necessary.

   5.10  Standards - Aroclors 1221, 1232, 1242, 1248, 1254, 1260,  and 1016.

   5.11  Anti-static Solution - STATNUL, Daystrom, Inc., Weston Instrument

         Division, Newark, N.J.  95212.

6.  Calibration

    6.1  Gas chromatographic operating conditions are considered acceptable

         when the response to dicapthon is at least 50% of  full scale when

         < . 06 ng is injected for electron capture detection and < 100 ng

         is injected for microcoulometric or electrolytic conductivity

         detection.  For all quantitative measurements, the detector  must be

         operated within its linear response range and the  detector noise level

         should be less than 2% of full  scale.

    6.2  Standards are injected frequently as a check on the stability of

         operating conditions, detector and column.  Example chromatograms

         are shown in Figures 3 through 8 and provide reference operating

         conditions.

    Quality Control

    7.1  Duplicate and spiked sample analyses are recommended as a quality

         control  check.   When the routine occurrence of a pollution parameter

         is observed,  quality control  charts are also recommended  (5).

-------
                                        3-6





    7.2  Each time a set  of samples is extracted,  a method blank is determined




         on a volume of distilled water equal  to that  used to dilute the sample.




8.   Sample Preparation




    8.1  Blend the sample if suspended matter  is present and adjust pH to




         near neutral (pH 6.5-7.5) with 50% sulfuric acid or 10 N sodium




         hydroxide.




    8.2  For a sensitivity requirement of 1 jjg/1,  when using microcoulometric




         or electrolytic  conductivity methods  for detection take 100 ml of




         sample for analysis.   If interferences  pose no problem, the sensitivity




         of the electron  capture detector should permit as little as 50 ml of




         sample to be used.  Background information on the extent and nature




         of interferences will assist the analyst  in choosing the required




         sample size and  preferred detector.




    8.3  Quantitatively transfer the proper aliquot into a two-liter separatory




         funnel and dilute to one liter.




9.   Extraction



    9.1  Add 60 ml of 15% methylene chloride in  hexane (v:v)  to the sample




         in the separatory funnel and shake vigorously for two minutes.




    9.2  Allow the mixed  solvent to separate from the  sample, then draw the




         water into a one-liter Erlenmeyer flask.   Pass the organic layer




         through a column containing 3-4  inches  of anhydrous  sodium sulfate,




         and collect it in a 500 ml K-D flask  equipped with a 10 ml ampul.




         Return the water phase to the separatory  funnel.   Rinse the Erlen-




         meyer flask with a second 60 ml  volume  of solvent; add the solvent




         to the separatory funnel and complete the extraction procedure a




         second time.  Perform a third extraction  in the same manner.




    9.3  Concentrate the  extract to 6-10  ml in the K-D evaporator on a hot




         water bath.

-------
                                   3-7





9.4  Qualitatively  analyze  the  sample by  gas  chromatography with an




     electron  capture  detector.   From the response obtained decide:




     a.   If  there are  any organochlorine  pesticides present,




     b.   If  there are  any PCB's  present,




     c.   If  there is a combination of a and b,




     d.   If  elemental  sulfur  is  present,




     e.   If  the  response is too  complex to determine a, b, or c.




     f.   If  no response, concentrate to 1.0 ml or less, as required,




          according  to  EPA Method (4), pg.  28  and repeat the analysis




          looking for a, b,  c, d,  and e.   Samples containing Aroclors




          with  a  low percentage of chlorine, eg. 1221 and 1232, may




          require this  concentration in order  to achieve the detection




          limit of 1 yg/1.   Trace  quantities of PCB's are often masked




          by  background which  usually occur in the samples.




9.5  If condition a_ exists, quantitatively determine the organochlorine




     pesticides  according to  (1).




9.6  If condition b exists, PCB's  only are present, no further separation




     or cleanup  is  necessary.  Quantitatively determine the PCB's according




     to 11. below.




9.7  If condition £ exists, compare peaks obtained from the sample to




     those of standard Aroclors  and make  a judgment as to which Aroclors




     may  be present.   To separate  the PCB's from the organochlorine




     pesticides,  continue as outlined in  10.4.




9.8  If condition id exists separate the sulfur from the sample using the




     method outlined in (10.3) followed by the method in (10.5).




9.9  If condition e exists then the following macro cleanup and separation




     procedures  (10.2  and 10.3)  should be employed and, if necessary,




     followed by the micro separation procedures (10.4 and 10.5).

-------
                                      3-8





10.   Cleanup and Separation Procedures



     10.1  Interferences in the form of distinct peaks and/or high background



           in the initial gas chromatographic analysis, as well  as, the



           physical characteristics of the extract (color, cloudiness,



           viscosity)  and background knowledge of the sample will indicate



           whether cleanup is required.   When these interfere with measure-



           ment of the pesticides,  or affect column life or detector sen-



           sitivity, proceed as directed below.



     10.2  Acetonitrile Partition - This procedure is used to remove fats and



           oils from the sample extracts.   It should be noted that not  all



           pesticides are quantitatively recovered by this procedure.   The




           analyst must be aware of this and demonstrate the efficiency of



           the partitioning for the compounds of interest.



           10.2.1  Quantitatively transfer the previously concentrated  extract



                   to a 125 ml separatory funnel with enough hexane to  bring



                   the final volume to 15  ml.  Extract the sample four  times



                   by shaking vigorously for one minute with 30 ml  portions



                   of hexane-saturated acetonitrile.



           10.2.2  Combine and transfer  the acetonitrile phases to  a one-liter



                   separatory funnel  and add 650 ml  of distilled  water  and



                   40  ml of saturated sodium chloride solution.   Mix thor-



                   oughly for 30-35 seconds.   Extract with  two 100  ml portions



                   of  hexane by vigorously shaking about 15 seconds.



           10.2.3  Combine the hexane extracts in a one-liter separatory funnel



                   and wash with two  100 ml portions  of distilled water.   Dis-



                   card the water layer  and pour the  hexane layer through a



                   3-4 inch anhydrous sodium sulfate  column into  a  500  ml K-D

-------
                                 3-9





              flask equipped with a 10 ml ampul.   Rinse the separator/




              funnel and column with three 10 ml  portions of hexane.




      10.2.4  Concentrate the extracts to 6-10 ml in the K-D evaporator




              in a hot water bath.




      10.2.5  Analyze by gas chromatography unless a need for further




              cleanup is indicated.




10.3  Florisil Column Adsorption Chromatography




      10.3.1  Adjust the sample extract volume to 10 ml.




      10.3.2  Place a charge of activated Florisil (weight determined




              by lauric-acid value, see Appendix  I)  in a Chromaflex




              column.  After settling the Florisil by tapping the column,




              add about one-half inch layer of anhydrous granular sodium




              sulfate to the top.




      10.3.3  Pre-elute the column, after cooling, with 50-60 ml  of




              petroleum ether.   Discard the eluate and just prior to




              exposure of the sulfate layer to air,  quantitatively




              transfer the sample extract into the column by decantation




              and subsequent petroleum ether washings.   Adjust the




              elution rate to about 5 ml per minute  and, separately,




              collect up to three eluates in 500  ml  K-D flasks equipped




              with 10 ml ampuls.   (See Eluate Composition below).




              Perform the first elution with 200  ml  of 6% ethyl ether




              in petroleum ether, and the second  elution with 200 ml  of




              15°0 ethyl ether in petroleum ether.   Perform the third




              elution with 200  ml of 50°o ethyl ethf-r   petroleum  ether




              and the fourth elution with 200 ml  of IOC9, ethyl ether.

-------
                                   3-10

            Eluate Composition  - By using an equivalent quantity of  any

            batch of Florisil as determined by its  lauric acid value,  the

            pesticides will be  separated into the eluates indicated  below:

                                   6% Eluate  ,,,,.,

            Aldrin              DDT                 Pentachloro-
            BHC                 Heptachlor           nitrobenzene
            Chlordane           Heptachlor Epoxide   Strobane
            ODD                 Lindane              Tojcaphene
            DDE                 Methoxychlor         Trifluralin
                                Mirex     •           PCB's

                     15%  Eluate               50% Eluate

                     Endosulfan  I             Endosulfan II
                     Endrin                   Captan
                     Dieldrin
                     Dichloran
                     Phthalate esters

            Certain  thiophosphate  pesticides will occur in each of the

            above  fractions as  well as  the 100% fraction.  For additional

            information  regarding  eluate composition,  refer to the FDA

            Pesticide  Analytical Manual (6).

     10.3.4 Concentrate  the eluates to  6-10 ml in the  K-D evaporator

            in a hot water bath.

     10.3.5 Analyze  by gas chromatography.

10.4 Silica Gel Micro-Column Separation  Procedure  C?)

     10.4.1 Activation for Silica  Gel

            10.4.1.1  Place about  20 gm of silica gel  in a 100 ml  beaker.

                       Activate  at  180 C for approximately 16 hours.   Transfer

                       the silica gel to a 100 ml glass stoppered bottle.

                       When cool, cover  with about 35 ml of 0.50% diethyl

                       ether in  benzene  (volume:volume).  Keep bottle

                       well sealed. If  silica gel collects on the  ground

                       glass surfaces, wash off with the above solvent

-------
                              3-11




                  before resealing.  Always maintain an excess




                  of the mixed solvent in bottle (approximately 1/2 in




                  above silica gel).  Silica gel can be effectively




                  stored in this manner for several days.




10.4.2  Preparation of the Chromatographic Column




       10.4.2.1   Pack the lower 2 mm ID Section of the microcolumn




                  with glass wool.  Permanently mark the column




                  120 mm above the glass wool.  Using a clean rubber




                  bulb from a disposable pipet seal the lower end




                  of the microcolumn.  Fill the microcolumn with




                  0.50% ether in benzene (v:v) to the bottom of




                  the 10/30 joint  (Figure 1).  Using a disposable




                  capillary pipet, transfer several aliquots of the




                  silica gel slurry into the microcolumn.  After




                  approximately 1 cm of silica gel collects in




                  the bottom of the microcolumn, remove the rubber




                  bulb seal, tap the column to insure that the




                  silica gel settles uniformly.  Carefully pack




                  column until the silica gel reaches the 120 ± 2




                  mm mark.   Be sure that there are no air bubbles




                  in the column.  Add about 10 mm of sodium sulfate




                  to the top of the silica gel.  Under low humidity




                  conditions, the silica gel may coat the sides of




                  the column and not settle properly.  This can be




                  minimized by wiping the outside of the column




                  with an anti-static solution.

-------
                 3-12




10.4.2.2  Deactivation of the Silica Gel




          a.  Fill the microcolumn to the base of




              the 10/30 joint with the 0.50% ether-




              benzene mixture, assemble reservoir




              (using spring clamps) and fill with




              approximately 15 ml of the 0.50% ether-




              benzene mixture.  Attach the air




              pressure device (using spring clamps)




              and adjust the elution rate to approxi-




              mately 1 ml/min. with the air pressure




              control.  Release the air pressure and




              detach reservoir just as the last of




              the solvent enters the sodium sulfate.




              Fill the column with n-hexane (not mixed




              hexanes) to the base of the 10/30 fitting.




              Evaporate all residual benzene from the




              reservoir, assemble the reservoir section




              and fill with 5 ml of n-hexane.   Apply




              air pressure and adjust the flow to 1




              ml/min.  (The n-hexane flows slightly




              faster than the benzene).   Release the air




              pressure and remove the reservoir just as




              the n-hexane enters the sodium sulfate.




              The column is now ready for use.




          b.  Pipet a 1.0 ml aliquot of the concentrated




              sample extract (previously reduced to a




              total volume of 2.0 ml) on to the column.

-------
                            3-13





                      As the last of the sample passes into




                      the sodium sulfate layer, rinse down




                      the internal wall of the column twice




                      with  0.25 ml of n-hexane.  Then assemble




                      the upper section of the column.  As the




                      last  of the n-hexane rinse reaches the




                      surface of the sodium sulfate^ add enough




                      n-hexane (volume predetermined, see




                      10.4.3 below) to just elute all of the




                      PCB's present in the sample.   Apply air




                      pressure and adjust until the flow is




                      1 ml/min.  Collect the desired volume of




                      eluate (predetermined, see 10.4.3 below)




                      in an accurately calibrated ampul.   As the




                      last  of the n-hexane reaches  the surface




                      of the sodium sulfate, release the air




                      pressure and change the collection ampul.




                  c.  Fill  the column with 0.50% diethyl  ether




                      in benzene, again apply air pressure and




                      adjust flow to 1 ml/min.   Collect the




                      eluate until all of the organochlorine




                      pesticides  of interest have been eluted




                      (volume predetermined, see 10.4.3 below).




                  d.  Analyze the eluates by gas chromatography.




10.4.3  Determination of Elution  Volumes




        10.4.3.1  The elution volumes  for the PCB's  and the




                  pesticides depend upon a number of factors which

-------
                     3-14



         are  difficult  to  control.  These  include



         variation  in:



         a.   Mesh size  of  the  silica  gel



         b.   Adsorption properties  of the  silica gel



         c.   Polar  contaminants present in the  eluting




              solvent



         d.   Polar  materials present  in the sample  and



              sample solvent



         e.   The dimensions of the  microcolumns



         Therefore, the optimum elution volume  must



         be  experimentally determined each time a factor



         is  changed.  To determine  the elution  volumes,



         add standard mixtures of Aroclors and  pesticides



         to  the column  and serially collect 1 ml elution



         volumes.   Analyze the individual  eluates by  gas



         chromatography and determine the  cut-off volume



         for n-hexane and  for  ether-benzene.  Figure  2



         shows the  retention order  of the  various PCB



         components and of the pesticides.  Using this



         information, prepare  the mixtures required for



         calibration of the microcolumn.



10.4.3.2 In  determining the volume  of hexane required to



         elute the  PCB's the sample volume Cl n>l) and the



         volume of  n-hexane used  to rinse  the column  wall



         must be considered.   Thus, if it  is determined



         that a 10.0 ml elution volume is  required  to



         elute the  PCB's,  the  volume  of hexane  to be  added

-------
                                 3-15





                         in  addition  to the  sample  volume  but  including




                         the rinse  volume  should be 9.5  ml.




               10.4.3.3   Figure  2 shows that as the average  chlorine




                         content of a PCB  mixture decreases  the  solvent




                         volume  for complete elution increases.  Quali-




                         tative  determination  (9.4)  indicates  which




                         Aroclors are present  and provides the basis




                         for selection  of  the  ideal  elution  volume.  This




                         helps to minimize the quantity  of organochlorine




                         pesticides which  will elute along with  the low




                         percent chlorine  PCB's and  insures  the  most




                         efficient  separations possible  for  accurate




                         analysis.









               10.4.3.4   For critical analysis where  the PCB's and




                         pesticides are  not  separated completely, the




                         column should  be  accurately  calibrated  according




                         to  (10.4.3.1)  to  determine  the percent  of




                         material of  interest that elutes  in each fraction.




                         Then flush the  column with  an additional 15 ml of




                         0.50% ether  in  benzene followed by 5 ml of n-




                         hexane and use  this reconditioned column for




                         the sample separation.  Using this technique one




                         can accurately  predict the  amount (%) of materials




                         in each  micro column fraction.




10.5  Micro Column Separation of Sulfur, PCB's,  and Pesticides




      10.5.1  See procedure for  preparation  and  packing micro column in




              PCB analysis section  (10.4.1 and 10.4.2).

-------
                       3-16




10.5.2  Microcolumn Calibration




        10.5.2.1  Calibrate the microcolumn for sulfur and




                  PCB separation by collecting 1.0 ml fractions




                  and analyzing them by gas chromatography to




                  determine the following:




                  1)  The fraction with the first eluting PCB's




                      (those present in 1260),




                  2)  The fraction with the last eluting PCB's




                      (those present in 1221),




                  3)  The elution volume for sulfur,




                  4)  The elution volume for the pesticides of




                      interest in the 0.50% ether-benzene fraction.




                  From these data determine the following:




                  1)  The eluting volume containing only sulfur




                      (Fraction I),




                  2)  The eluting volume containing the last of




                      the sulfur and the early eluting PCB's




                      (Fraction II),




                  3)  The eluting volume containing the remaining




                      PCB's (Fraction III),




                  4)  The ether-benzene eluting volume containing




                      the pesticides of interest (Fraction IV).



10.5.3  Separation Procedure




        10.5.3.1  Carefully concentrate the 6% eluate from the




                  florisil column to 2.0 ml in the graduated




                  ampul  on a warm water bath.




        10.5.3.2  Place  1.0 ml (50%) of the concentrate into




                  the microcolumn with a 1 ml  pipet.  Be careful

-------
                 3-17






          not to get any sulfur crystals into the pipet.




10.5.3.3  Collect Fractions I and II in calibrated centri




          fuge tubes.




          Collect Fractions III and IV in calibrated ground




          glass stoppered ampules.




10.5.3.4  Sulfur Removal (9)  - Add 1 to 2 drops of mercury




          to Fraction II stopper and place on a wrist-action




          shaker.  A black precipitate indicates the presence




          of sulfur.  After approxiately 20 minutes the




          mercury may become  entirely reacted or deactivated




          by the precipitate.  The sample should be quantita-




          tively transferred  to a clean centrifuge tube and




          additional mercury  added.  When crystals are present




          in the sample, three treatments may be necessary to




          remove all the sulfur.  After all the sulfur has




          been removed from Fraction II (check using gas




          chromatography) combine Fractions II and III.




          Adjust the volume to 10 ml and analyze gas chroma-




          tography.   Be sure  no mercury is transferred to




          the combined Fractions II and III, since it can




          react with certain  pesticides.




          By combining Fractions II and III, if PCB's are




          present,  it is possible to identify the Aroclor(s)




          present and a quantitative analysis can be per-




          formed accordingly.  Fraction I can be discarded




          since it  only contains the bulk of the sulfur.




          Analyze Fractions III and IV for the PCB's and

-------
                                     3-18


                            pesticides.  If  DDT  and  its homologs, aldrin,

                            heptachlor,  or technical chlordane  are present

                            along  with  the PCB's,  an additional micro -

                            column separation  can  be performed  which may help

                            to  further  separate  the  PCB's  from  the pesticides

                             (See 10.4).

11 .   Quantitative  Determination

     11.1  Measure the  volume  of n-hexane eluate, containing  the PCB's  and

           inject  1 to  5 ul  into the gas  chromatograph.  If necessary,  adjust

           the volume of the eluate to give linear  response to the electron

           capture detector.  The microcoulometric  or the electrolytic  detector

           may be  employed to  improve specificity for samples having higher

           concentrations of PCB's.

     11.2  Calculations

           11.2.1   When a single Aroclor  is present,  compare  quantitative

                   Aroclor reference standards  (e.g., 1242, 1260) to the un-

                   known.  Measure  and sum the  areas  of  the unknown and the

                   reference Aroclor and  calculate  the result as follows:

                                          [A]  [B]  [V J
                         Microgram/liter  =               x  [N]
                         .   _   ng  of Standard  Injected
                               I   of Standard  Peak  Areas
                                                           mm
                         B  =   Z   of Sample  Peak  Areas  =  (mm  )

                         V.  =   Volume of sample injected  (yl)

                         V  =   Volume of Extract  (pi)  from which  sample
                               is  injected into gas  chromatograph

                         V  =   Volume of water sample  extracted  (ml)

                         N  =   2 when micro  column used
                               1 when micro  column not  used

-------
                       3-19



             Peak Area •= Peak height (mm x Peak Width at

                         1/2 height



11.2.2  For complex situations, use the calibration method



        described below.  Small variations in components between



        different Aroclor batches make it necessary to obtain



        samples of several specific Aroclors.  These reference



        Aroclors can be obtained from Dr. Ronald Webb, Southest



        Environmental Research Laboratory, EPA, Athens, Georgia



        30601.  The procedure is as follows:



        11.2.2.1  Using the OV-1 column, chromatograph a known



                  quantity of each Aroclor reference standard.



                  Also chromatograph a sample of p,p'-DDE.



                  Suggested concentration of each standard is



                  0.1 ng/yl for the Aroclors  and 0.02 ng/pl for



                  the p,p'-DDE.



        11.2.2.2  Determine the relative retention time (RRT) of



                  each PCB peak in the resulting chromatograms



                  using p,p'-DDE as 100.  See Figures 3 through 5



                        RT  x  100
                  RRT =
                          RTDDE
                  RRT = Relative Retention Time



                  RT  = Retention time of peak of interest



                  RT
                    DDE = Retention time of p,p'-DDE



                  Retention time is measured as that distance in



                  mm between the first appearance of the solvent



                  peak and the maximum for the compound.



        11.2.2.3  To calibrate the instrument for each  PCB



                  measure the area of each peak.

-------
               3-20




          Area = Peak height On)  x Peak width at 1/2



          height.   Using Tables 1  through 6 obtain the



          proper mean weight f actor ;  then determine

                                   2
          the response factor ng/iran .



                   (ng- ) (mean weight  percent]

            ,  2                100
          ng/mm  =
          n§i = n§ °f Aroclor Standard Injected



          Mean weight percent = obtained from Tables 1

                                through 6 .



11.2.2.4  Calculate the RRT value and the area for each



          PCB peak in the sample chroma tograra.  Compare



          the sample chromatogram to those  obtained for



          each reference Aroclor standard.   If it is



          apparent that the PCB peaks present are due to



          only one Aroclor then calculate the concentration



          of each PCB using the following formula:



          ng PCB = ng/mm  x Area



          Where Area = Area (mm )  of sample peak



          ng/mm  = Response factor for that peak measured.



          Then add the nanograms of PCB's present in the



          injection to get the  total number of nanograms



          of PCB's present.   Use the following formula to



          calculate the concentration of PCB's in the sample:



                             [ngj   [VtJ

          Micrograms/ Liter = -^^ - t—^- x [N]

                             i vsJ   1 i J



          Vg  = volume of water  extracted (ml)



          Vt = volume of extract (ul)

-------
                                     3-21




                                V.  = volume of sample injected (pi)




                                Eng = sum of all the PCB's in nanograms for




                                      that Aroclor identified




                                N = 2 when microcolumn used




                                N = 1 when microcolumn not used




                                The value can then be reported as Micrograms/




                                Liter PCB's reported as the Aroclor.   For




                                samples containing more than one Aroclor, use




                                Figure 9 chromatogram divisional flow chart




                                to assign a proper response factor to each




                                peak and also identify the "most likely"




                                Aroclors present.  Calculate the ng of each




                                PCB isomer present and sum them according




                                to the divisional flow chart.  Using the




                                formula above, calculate the concentration of




                                the various Aroclors present in the sample.




12.   Reporting Results




     12.1  Report results in micrograms per liter without correction for




           recovery data.  When duplicate and spiked samples are analyzed,




           all data obtained should be reported.

-------
                          3-22
                           Table 1

                 Composition of Aroclor 1221 (8)


RRTa
11
14
16
19
21
28
32
p7
140
Total
Mean
Weight
Percent
31.8
19.3
10.1
2.8
20.8
5.4
1.4
1.7
93.3

Relative
Std. Dev.b
15.8
9.1
9.7
9.7
9.3
13.9
30.1
48.8


Number of
Chlorines0
1
1
2
2
2
2") 85%
3J 15%
2] 10%
3J 90%
3
3

     aRetention time relative to p,p'-DDE=100.   Measured from
first appearance of solvent.  Overlapping peaks that are
quantitated as one peak are bracketed.
     bstandard deviation of seventeen results as a percentage
of the mean of the results.
     CFrom GC-MS data.  Peaks containing mixtures of isomers
of different chlorine numbers are bracketed.

-------
                          3-23
                            Table 2

                 Composition of Aroclor 1232 C8)


RRTa
11
14
16
[20
121
28
32
37
40
47
54

58
70
78
Total
Mean
Weight
Percent
16.2
9.9
7.1
17.8
9.6
3.9
6.8
6.4
4.2
3.4

2.6
4.6
•
1.7
9<.2

Relative
Std. Dev.b
3.4
2.5
6.8
2.4
3.4
4.7
2.5
2.7
4.1
3.4

3.7
3.1
7.5


Number of
Chlorines0
1
1
2
2
2
21 40%
3j 60%
3
3
3
4
3] 33%
4j 67%
4
4] 90%
5J 10%
4

     aRetention time relative to p,p'-DDE=100.  Measured from
first appearance of solvent.  Overlapping peaks that are
quantitated as one peak are bracketed.
     ^Standard deviation of four results as a mean of the
results.
     cFrom GC-MS data.  Peaks containing mixtures of isomers
of different chlorine numbers are bracketed.

-------
                          .3-24
                           Table 3

                 Composition of Aroclor 1242 (.8)

RRTa
11
16
21
28

32
37
40
47
54

58
70

78
84
98
104
125
146


Total
Mean
Weight
Percent
1.1
2.9
11.3
11.0

6.1
11.5
11.1
8.8
6.8

5,6
10.. 3

3.6
2.7
1.5
2.3
1.6
1.0


98.5

Relative
Std. Dev.b
35.7
4.2
3.0
5.0

4.7
5.7
6.2
4.3
2.9

3.3
2.B

4.2
9.7
9.4
16.4
20.4
19.9




Number of
Chlorines0
1
2
2
21 25%
3J 75%
3
3
3
4
3] 33%
4j 67%
4
4] 90%
5J 10%
4
5
5
5
5] 85%
6J 15%
51 75%
1
6J 25%

     aRetention time relative to p,p'-DDE=100.  Measured from
first appearance of solvent.
     ^Standard deviation of six results as a percentage of
the mean of the results.
     °From GC-MS data.  Peaks containing mixtures of isomers
of different chlorine numbers are bracketed.

-------
                          3-25
                            Table 4

                  Composition of Aroclor 1248


RRTa
21
28
32
47
40
47
54

58
70
78
84
98
104

112
125
146
Total
Mean
Weight
Percent
1.2
5.2
3.2
8.3
8.3
15.6
9.7

9.3
19.0
6.6
4.9
3.2
3.3

1.2 '
2.6
1.5
103.1

Relative
Std. Dev.b
23.9
3.3
3.8
3.6
3.9
1.1
6.0

5.8
1.4
2.7
2.6
3.2
3.6

6.6
5.9
10.0


Number of
Chlorines0
2
3
3
3
31 85%
4j 15%
4
3] 10%
4j 90%
4
4] 80%
5J 20%
4
5
5
4] 10%
5j 90%
5
51 90%
6j 10%
5] 85%
6j 15%

     aRetention time relative to p,p'-DDE=100.   Measured from
first appearance of solvent.
     ^Standard deviation of six results as a percentage of
the mean of the results.
     cFrom GC-MS data.  Peaks containing mixtures of isomers
of different chlorine numbers are bracketed.

-------
                           3-26
                            Table  5

                  Composition of Aroclor 1254 C®/


RRTa
47
54
58
70

84
98
104
125
-146
160
174
203
232
Total
Mean
Weight
Percent
6.2
2.9
1.4
13.2

17.3
7.5
13.6
15.0
10.4
1.3
8.4
1.8
1.0
100.0

Relative
Std. Dev.b
3.7
2.6
2.8
2.7

1.9
5.3
3.8
2.4
2.7
8.4
5.5
18.6
26.1


Number of
Chlorines0
4 .
4
4
4] 25%
5J 75%
5
5
5
5] 70%
6J 30%
51- 30%
6j 70%
6
6
6
7

     Detention time relative to p/p'-DDE=100.  Measured from
firs't appearance.of solvent.
     ^Standard deviation of six results as a percentage of the
mean of the results.
     cFrom GC-MS data.  Peaks containing mixtures of isomers
are bracketed.

-------
                           3-27
                            Table 6

                 Composition of Aroclor 1260

RRTa
70
84
T 98
UL04

117
125
146
160
174
203

[232
L244

280
332
372
448
528
Total
Mean
Weight
Percent
2.7
4.7
3.8

3.3
12.3
14.1
4.9
12.4
9.3


9.8

11.0
4.2
4.0
.6
1.5
98.6

Relative
Std. Dev.b
6.3
1.6
3.5

6.7
3.3
3.6
2.2
2.7
4.0


3.4

2.4
5.0
8.6
25.3
10.2



Number of
Chlorines0
5
5
5
6
6
.5'
6.
6
6'
6


d
60%
40%

1 15%
1 85%

1 50%
J 50%

6"! 10%
7j 90%

6
7
7
7
8
8
8
e
10%
90%






     aRetention time relative to p,p"-DDE=100.   Measured from
first appearance of solvent.  Overlapping peaks that are
quantitated as one peak are bracketed.
     ^Standard deviation of six results as a mean of the
results.
     cFrom GC-MS data.  Peaks containing mixtures of isomers
of different chlorine numbers are bracketed.
     ^Composition determined at the center of peak 104.
     Composition determined at the center of peak 232.

-------
                            3-28
COMPRESSED,
AIR	
SUPPLY
$9-
               SHUT-OFF
                VALVE
0-5
PSIG }
                                            PRESSURE
              REGULATOR
                                            NEEDLE
                                             VALVE
                               cm
                       FLEXIBLE
                        TUBING
               SILICA GEL
                  5 cm  '

                    I cm*
                                GLASS
                                WOOL
                                     10/30
                                   15ml
                                RESERVOIR
                                  §  10/30
                                  23cm  x 4.2mm I.D.
                                    cm   x  2 mm I.D.
          FIGURE  I.   MICROCOLUMN  SYSTEM

-------
     TSULFUR
                                        3-2y
   50i-
O
h-
a:
u
a.
    0
                  HEPTACHLOR
                              DOE
              M IREX
                                  ALDRIN
                      OP'  8  PP*   DDT
         1    TECHNICAL   CHLORODANE
                                                                    I r-CHLOROANE
'/    \\  \ N
                     4        6
                              VOLUME n-HEXANE   ml

                  Figure 2.   Aroclor  Elution   Patterns
                                    14
                                       La^.
16

-------
                                     3-30


                                   REFERENCES

 (1)  "Method for Organochlorine Pesticides in  Industrial  Effluents,"  U.S.
     Environmental Protection Agency, National  Environmental  Research Center,
     Analytical Quality Control Laboratory,  Cincinnati, Ohio  45268,  1973.

 (2)  Leoni, V., "The Separation of Fifty Pesticides  and Related Compounds
     and Polychlorinated Biphenyls into Four Groups  by Silica Gel  Micro-
     column Chromatography," Journal of Chromatography, 62, 63 (1971).

 (3)  McClure, V. E., "Precisely Deactivated  Adsorbents Applied to  the
     Separation of Chlorinated Hydrocarbons," Journal of  Chromatography,  70,
     168  (1972).

 (4)  "Methods for Organic  Pesticides in Water  and Wastewater," U.S.  Environ-
     mental Protection Agency, National Environmental Research Center,
     Analytical Quality Control Laboratory,  Cincinnati, Ohio  45268,  1971.

 (5)  "Handbook for Analytical Quality Control  in Water and Wastewater
     Laboratories," Chapter 6, Section 6.4,  U.S. Environmental Protection
     Agency, National Environmental Research Center, Analytical Quality
     Control Laboratory, Cincinnati, Ohio 45268, 1972. '

 (6)  "Pesticide Analytical Manual," U.S. Dept.  of Health, Education,  and
     Welfare, Food and Drug Administration,  Washington, D.C.

 (7)  Bellar, T. A. and Lichtenberg, J. J., "Method for the Determination of
     Polychlorinated Biphenyls in Water and  Sediment," U.S. Environmental
     Protection Agency, National Environmental  Research Center, Analytical
     Quality Control Laboratory, Cincinnati, Ohio 45268,  1973.

 (8)  Webb, R. G. and McCall, A. C., "Quantitative PCB Standards for  Electron
     Capture Gas Chromatography."  Presented at the  164th National ACS
     Meeting, New York, August 29. 1972.   (Submitted to the Journal  of
     Chromatographic Science for publication).

 (9)  Goerlitz, D. F. and Law, L. M., "Note on  Removal of  Sulfur Interferences
     from  Sediment Extracts for Pesticide Analysis," Bulletin of Environmental
     Contamination and Toxicology, 6_, 9  (1971).

(10)  Mills, P. A., "Variation of Florisil Activity:  Sample Method for Measuring
     Adsorbent Capacity and its Use in Standardizing Florisil Columns,"
     Journal of the Association of Official  Analytical Chemists, 51,  29 (1968).

(11)  Steere, N. V., editor, "Handbook of Laboratory  Safety,"  Chemical Rubber
     Company, 18901 Cranwood Parkway: Cleveland, Ohio 44128,  1971, pp.  250-254.

-------
                                     3-1






                                   APPENDIX I





13.   Standardization of Florisil Column by Weight Adjustment Based on Adsorption




     of Laurie Acid.




     13.1  A ic .Id method for determining adsorptive capacity of Florisil is




           based on adsorption of lauric acid from hexane solution (6) (8).




           An excess of lauric acid is used and amount not adsorbed is measured




           by alkali titration.  Weight of lauric acid adsorbed is used to




           calculate, by simple proportion, equivalent quantities of Florisil




           for batches having different adsorptive capacities.




     13.2  Apparatus




           13.2.1  Buret.  -- 25 ml with 1/10 ml graduations.




           13.2.2  Erlenmeyer flasks. -- 125 ml narrow mouth and 25 ml, glass




                   stoppered.




           13.2.3  Pipet.  -- 10 and 20 ml transfer.




           13.2.4  Volumetric flasks. -- 500 ml.




     13.3  Reagents and Solvents




           13.3.1  Alcohol, ethyl. -- USP or absolute, neutralized to




                   phenolphthalein.




           13.3.2  Hexane. --  Distilled from all glass apparatus.




           13.3.3  Lauric acid. --Purified, CP.




           13.3.4  Lauric acid solution. -- Transfer 10.000 g lauric acid to




                   500 ml  volumetric flask, dissolve in hexane, and dilute to




                   500 ml  (1 ml = 20 mg).




           13.3.5  Phenolphthalein Indicator.  -- Dissolve 1 g in alcohol  and




                   dilute  to 100 ml.

-------
                               3-2
      13.3.6  Sodium hydroxide.  -- Dissolve 20 g NaOH (pellets, reagent




              grade) in water and dilute to 500 ml (IN).   Dilute 25 ml




              IN NaOH to 500 ml  with water (0.05NJ).   Standardize as follows:




              Weigh 100-200 mg lauric acid into 125  ml Erlenmeyer flask.




              Add 50 ml neutralized ethyl alcohol and 3  drops phenol -




              phthalein indicator; titrate to permanent  end point.   Calculate




              mg lauric acid/ml  0.05 N_NaOH (about 10 mg/ml).




13.4  Procedure




      13.4.1  Transfer 2.000 g Florisil to 25 ml glass stoppered Erlenmeyer




              flasks.   Cover loosely with aluminum foil  and heat overnight




              at 130°C.  Stopper- cool  to room temperature, add 20.0 ml




              lauric acid solution (400 mg),  stopper, and  shake occasionally




              for 15 min.   Let adsorbent settle and  pipet  10.0 ml  of




              supernatant into 125 ml Erlenmeyer flask.  Avoid inclusion




              of any Florisil.




      13.4.2  Add 50 ml neutral  alcohol and  3 drops  indicator solution;




              titrate with 0.05N_ to a permanent end  point.




13.5  Calculation of Lauric Acid Value  and Adjustment of Column Weight




      13.5.1  Calculate amount of lauric acid adsorbed on  Florisil  as




              follows:




              Lauric Acid value  = mg lauric  acid/g Florisil.= 200  - (ml




              required for titration X  mg lauric acid/ml 0.05N NaOH).




      13.5.2  To obtain an equivalent quantity of any batch of Florisil,




              divide 110 by lauric acid value for that batch  and multiply




              by 20 g.   Verify proper elutiori of pesticides by 13.6.

-------
                                3-3
13.6  Test for Proper Elution Pattern and Recovery of Pesticides:




      Prepare a test mixture containing aldrin, heptachlor epoxide,
                                                *.



      p,p'-DDE, dieldrin, Parathion and malathion.  Dieldrin and




      Parathion should elute in the 15% eluate; all but a trace of




      malathion in the 50% eluate and the others in the 6% eluate.

-------
   \
               37
                  AROCLOR 1242
Figure 3. Column: 3%  OV-1, Carrier Gas: Nitrogen  at 60 ml/mm,
         Column Temperature: 170 C,  Detector: Electron Capture

-------
I
                 70
                              AROCLOR 1254
                         104
                             125
                                  146
                                        174
                                                       232
Figure  4. Column:  3%  OV-1, Carrier Gas: Nitrogen at 60 ml/min,
         Column Temperature: 170 C, Detector: Electron Capture.

-------
                               AROCLOR 1260
                               280
                                                        528
Figure  5. Column: 3% OV-1, Carrier Gas: Nitrogen  at  60  ml/min,
         Column  Temperature: 170 C,  Detector:  Electron Capture

-------
                               AROCLOR 1242
               I
              JL
               I
I
I
                                                           I
0
3
6
       21
       24
                       9       12      15      18
                       RETENTION TIME IN MINUTES
Figure  6. Column: 1.5%  OV-17  + 1.95%  QF-1, Carrier Gas: Nitrogen
at 60  ml/min,  Column  Temperature: 200 C, Detector: Electron Capture

-------
                                                          AROCLOR 1254
0
12
15
18
21
24
27
30
33
36
39
42
45
  Figure 7. Column: 1.5%  OV-17 +  1.95%
           Detector: Electron  Capture.
               RETENTION TIME IN  MINUTES
              QF-1, Carrier Gas:  Nitrogen at 60 ml/min, Column Temperature:   200 C,

-------
I
I
 I
 I
I
I
                                                                I
                                                         I
 I
I
I
J
54
3
              12
15
IB
                                  36
                                  39
                                                                                           42
45
48
51
                                                21      24     27     30     33
                                                 RETENTION  TIME IN MINUTES
Figure 8. Column: 1.5% OV-17 + 1.95%  QF-1, Carrier Gas:  Nitrogen at 60 ml/min,  Column Temperature: 200C, Detector:  Electron Capture

-------
               I RRT of first peak < 47?   I
               IMHMHMM^MHM^^^H^M^MMMMMMMMMMMJI
               YES
                          NO
      Is there a  distinct
      peak with  RRT  78?
      YES
/       v
                        RRT 47-58?
YES
   Use 1242  for
 peaks! RRT 84
          Use 1242 for
         peaks- RRT 70
    Use 1254
    for peaks
   1  RRT  104
NO
 RRT- 70?
            Is there  a distinct
           peak with RRT 117?
          YES
               NO
                    Use 1254 for all
                     peaks! RRT 174
      Use  1260 for
     all other  peaks
                                Use 1260 for
                                  all peaks
Figure 9.  Chromatogram Division  Flowchart (8),
                                   US GOVERNMENT PRINTING OFFICE: 1973- 758-4U6/10 Zl

-------
             4,    METHOD FOR TRIAZINE PESTICIDES IN INDUSTRIAL EFFLUENTS



        1.   Scope and Application


           1.1   This method covers the determination of various symmetrical


                triazine pesticides.


           1.2   The following compounds may be determined by this method with a


                sensitivity of 1 yg/1:  ametryne, atratone, atrazine, GS-13529,


                GS-14254, prometone, prometryne, propazine, and simazine.  The


                usefulness of the method for other specific pesticides must be


                demonstrated by the analyst before any attempt is made to apply


                it to sample analysis.


           1.3   Individual triazines may be difficult to identify and quantitate


                in the presence of other triazines or other nitrogen-containing


                compounds.  Provisions incorporated in this method are intended


                to minimize the effect of such  interferences.


        2.   Summary


           2.i   The method describes an efficient sample extraction procedure and
        M
        ...       provides, through use of column chromatography, a method for the


O    <"  2.2   This  method is recommended for use only by experienced pesticide

oi
        ^       analysts or under the close supervision of such qualified persons.


        I?   Interferences


           3.1   Solvents, reagents,  glassware, and other sample processing hardware


                may yield discrete artifacts  and/or elevated baselines causing

-------
                                 4 -2




        misinterpretation  of gas  chromatograms.   All  of these  materials




        must  be  demonstrated to be  free  from interferences  under the




        conditions  of the  analysis.   Specific selection of  reagents  and




        purification of solvents  by distillation in all-glass  systems may




        be  required.   Refer to  (1)  Part  1,  Sections 1.4 and 1.5.




   3.2  The interferences  in industrial  effluents are high  and varied




        and often pose great difficulty  in  obtaining  accurate  and precise




        measurement of triazine pesticides.   The use  of a specific



        detector supported by an  optional column cleanup procedure will




        eliminate many of  these interferences.




   3.3  Nitrogen containing compounds other than the  triazines may interfere.




4.  Apparatus and Materials




   4.1  Gas Chromatograph  - Equipped with glass-lined injection port.



   4.2  Detector -  Electrolytic Conductivity.




   4.3  Recorder -  Potentiometric strip  chart (10 in) compatible with




        the detector.




   4.4  Gas Chromatographic Column  Materials:




        4.4.1  Tubing - Pyrex (ISO  cm long  X 4 mm ID)




        4.4.2  Glass Wool  - Silanized




        4.4.3  Solid Support - Gas-Chrom Q  (100-120 mesh)




        4.4.4  Liquid Phase - Expressed  as  weight percent coated on




               solid support




               4.4.4.1  Carbowax  20M, 1%




   4.5  Kuderna-Danish (K-D) Glassware  (Kontes)




        4.5.1  Snyder Column - three ball  (K-503000)




        4.5.2  Micro-Snyder Column  - two ball (K-569001)




        4.5.3  Evaporative Flasks - 500  ml  (K-570001)

-------
                                  4  - 3




        4.5.4  Receiver Ampuls - 10 ml, graduated (K-570050)




        4.5.5  Ampul Stoppers




   4.6  Chromatographic Column - Chromaflex (400 mm long X 19 mm ID) with




        coarse fritted plate on bottom and Teflon stopcock; 250 ml




        reservoir bulb at top of column with flared out funnel shape at




        top of bulb - a special order (Kontes K-420540-9011).




   4.7  Chromatographic Column - Pyrex (approximately 400 mm long X 20 mm ID)




        with coarse fritted plate on bottom.




   4.8  Micro Syringes - 10, 25, 50, and 100 yl.




   4.9  Separatory Funnels - 2000 ml with Teflon stopcock.




  4.10  Blender - High speed, glass or stainless steel cup.




  4.11  Graduated Cylinders - 1000 ml.




  4.12  Florisil - PR Grade (60-80 mesh); purchase activated at 1250 F




        and store in the dark in glass containers with glass stoppers or




        foil-lined screw caps.  Before use activate each batch overnight




        at 130 C in foil-covered glass container.  Determine lauric-acid




        value (See Appendix I).




5.   Reagents, Solvents and Standards




   5.1  Ferrous Sulfate - (ACS) 30% solution in distilled water.




   5.2  Potassium Iodide - (ACS) 10% solution in distilled water.




   5.3  Sodium Hydroxide - (ACS) 10 N in distilled water.




   5.4  Sodium Sulfate - (ACS) Granular,  anhydrous.




   5.5  Sulfuric Acid   (ACS)  Mix equal volumes of cone. H SO. with




        distilled water.




   5.6  Diethyl Ether - Pesticide Quality^  redistilled in glass,  if necessary




        5.6.1  Must contain 2% alcohol and be free of peroxides by the




               following test:   To 10 ml  of ether in glass-stoppered

-------
                                  4-4




                cylinder previously  rinsed with ether;  add 1  ml  of




                freshly prepared 10% KI solution.   Shake  and  let stand




                one  minute.   No  yellow color should be  observed  in




                either  layer.




         5.6.2  Decompose  ether  peroxides  by adding 40  g  of 30%  ferrous




                sulfate solution to  each  liter  of  solvent.  CAUTION:




                Reaction may be  vigorous  if the solvent contains a high




                concentration of peroxides.




         5.6.3  Distill deperoxidized ether in  glass and  add  2%  ethanol.




    5.7  Hexane, Methanol, Methylene Chloride,  Petroleum  Ether (boiling




         range 30-60 C) -  pesticide  quality,  redistill  in glass  if necessary.




    5.8  Pesticide Standrads - Reference  grade.




6.  Calibration




    6.1  Gas chromatographic operating conditions  are considered optimum




         when an injection of <  20 ng of  each triazine  will yield a peak




         at least 50% of full scale   deflection with the  modified Coulson




         detector (2).  For all  quantitative measurements,  the detector




         must be operated  within its linear response range and the




         detector noise level should be less than  2% of full  scale.




    6.2  Inject standards  frequently as a check on the  stability of




         operating conditions.  A chromatogram  of  a mixture of several




         pesticides  is  shown in  Figure 1  and provides reference  operating




         conditions  for the recommended column.




    6.3  The elution order and retention  ratios of various triazine




         pesticides  are provided in  Table 1,  as a  guide.

-------
                                   4  - 5



7-  Quality Control




    7.1  Duplicate and spiked sample analyses are recommended as quality




         control checks.  When the routine occurrence of a pesticide is




         being observed, the use of quality control charts is  recommended (3)




8.  Sample Preparation




    8.1  Blend the sample if suspended matter is present and adjust pH to




         near neutral (pH 6.5-7.5) with 50% sulfuric acid or 10 N sodium



         hydroxide.




    8.2  Quantitatively transfer a 1000 ml aliquot into a two-liter




         separatory funnel.




9.  Extraction




    9.1  Add 60 ml methylene chloride to the sample in the separatory




         funnel and shake vigorously for two minutes.




    9.2  Allow the solvent to separate from the sample, then draw the water




         into a one-liter Erlenmeyer flask.  Pass the organic layer through




         a chromatographic column containing 3-4 inches anhydrous sodium




         sulfate, and collect it in a 500 ml K-D flask equipped with a




         10 ml ampul.  Return the water phase to the separatory funnel.




         Rinse the Erlenmeyer flask with a second 60 ml volume of solvent,




         add the solvent to  the separatory funnel, and complete the




         extraction procedure a second time.  Perform a third extraction




         in the same manner.




    9.3  Concentrate the extract to 10 ml in a K-D evaporator on a hot




         water bath.  Disconnect the  Snyder column just long enough to add




         10 ml hexane to the  K-D flask and then continue the concentration




         to about 5-6 ml.   If the need for cleanup is  indicated, continue




         to Florisil Column  Cleanup (10 below).

-------
                                  4 -  6

    9.4  If further cleanup is  not required,  replace the Snyder column

         and flask with a micro-Snyder column and continue the concentration

         to 0.5-1.0 ml.  Analyze this  final concentrate by gas chromatography.

10.   Florisil Column Cleanup

   10.1  Adjust the sample extract to  10 ml with hexane.

   10.2  Place a charge of activated Florisil (weight determined by lauric

         acid value, see Appendix I) in a Chromaflex chromatographic column.

         After settling the Florisil by tapping the column,  add about one-

         half inch layer of anhydrous  granular sodium sulfate to the top.

   10.3  Pre-elute the column,  after cooling, with 50-60 ml  of petroleum

         ether.  Discard the eluate and just  prior to exposure of the

         sulfate layer to air,  quantitatively transfer the sample extract

         into the column by decantation and subsequent petroleum ether

         washings.  Adjust the  elution rate to about 5 ml per minute and

         separately collect the eluates in 500 ml K-D flasks equipped with

         10 ml ampuls.  Perform the first elution with 200 ml of 6% ethyl

         ether in petroleum ether and  the second elution with 200 ml of 15%

         ethyl ether in petroleum ether.   Perform the third  elution with

         200 ml of 50% ethyl ether - petroleum ether and the fourth elution

         with 200 ml of 100% ethyl ether.

   10.4  Eluate Composition - By using an equivalent quantity of any batch

         of Florisil as determined by  its lauric acid value, the pesticides

         will be separated into the eluates indicated below:

               15% Eluate             50% Eluate            100% Eluate

             Propazine (90%)        Propazine (10%)          Atratone
             GS-13529 (30%)         GS-13529  (70%)           GS-14254
             Atrazine (20%)         Atrazine  (80%)           Prometone
                                    Ametryne
                                    Prometryne
                                    Simazine

-------
                                 4-7


     10.5  Concentrate the eluates to 6-10 ml in the K-D evaporator in

           a hot water bath.  Change to the micro-Snyder column and

           continue concentration to 0.5-1.0 ml.

     10.6  Analyze by gas chromatography.

11.   Calculation of Results

     11.1  Determine the pesticide concentration by using the absolute

           calibration procedure described below or the relative cali-

           bration procedure described in Part I, Section 3.4.2(4)

                                   (A)  (B)  (V )
                Micrograras/liter - — T-T
                                               y
                                              S
           A = ng standard
               Standard area
           B = Sample aliquot area

           V  = Volume of extract injected (yl)

           V  = Volume of total extract (yl)

           V  = Volume of water extracted (ml)
            s

12.   Reporting Results

     12.1  Report results in micrograms per liter without  correction  for

           recovery data.  When duplicate and spiked samples  are  analyzed

           all data obtained should be reported.

-------
                          4  -  8
                        TABLE 1
       RETENTION RATIOS OF VARIOUS TRIAZINE
          PESTICIDES RELATIVE TO ATRAZINE
Pesticide
Prometone
Atratone
Propazine
GS-13529
GS-14254
Atrazine
Prometryne
Simazine
Ametryne
Retention Ratio
0.52
0.67
0.71
0.78
0.88
1.00
1.10
1.35
1.48
Absolute retention time of atrazine = 10.1 minutes

-------
                                  4-9
REFERENCES:

(1)  "Methods for Organic Pesticides in Water and Wastewater", U.S.
     Environmental Protection Agency, National Environmental Research
     Center, Analytical Quality Control Laboratory, Cincinnati, Ohio
     45268,  1971.

(2)  Patchett, G. G., "Evaluation of the Electrolytic Conductivity
     Detector for Residue Analyses of Nitrogen-Containing Pesticides",
     Journal of Chromatographic Science, 8_, 155  (1970).

(3)  "Handbook for Analytical Quality Control in Water and Wastewater
     Laboratories", Chapter 6, Section 6.4, U. S. Environmental Protection
     Agency, National Environmental Research Center, Analytical Quality
     Control Laboratory, Cincinnati, Ohio  45268, 1972.

C4)  Cochrane, W. P. and Wilson, B. P., "Electrolytic conductivity
     detection of some nitrogen-containing herbicides", Journal of
     Chromatography, 63, 364  (1971).

-------
                                  4  -  1






                                   APPENDIX I





13.   Standardization of Florisil Column by Weight Adjustment Based on Adsorption




     of Lau/ric Acid.




     13.1  A rapid method for determining adsorptive capacity of Florisil is




           based on adsorption of lauric acid from hexane solution (6)  (8).




           An excess of lauric acid is used and amount not adsorbed is measured




           by alkali titration.  Weight of lauric acid adsorbed is used to




           calculate, by simple proportion, equivalent quantities of Florisil




           for batches having different adsorptive capacities.




     13.2  Apparatus




           13.2.1  Buret. -- 25 ml with 1/10 ml graduations.




           13.2.2  Erlenmeyer flasks.  — 125 ml narrow mouth and 25 ml, glass




                   stoppered.




           13.2.3  Pipet. -- 10 and 20 ml transfer.




           13.2.4  Volumetric flasks.  -- 500 ml.




     13.3  Reagents and Solvents




           13.3.1  Alcohol, ethyl. --  USP or absolute, neutralized to




                   phenolphthalein.




           13.3.2  Hexane. -- Distilled from all glass apparatus.




           13.3.3  Lauric acid. --Purified, CP.




           13.3.4  Lauric acid solution. -- Transfer 10.000 g lauric acid to




                   500 ml volumetric flask, dissolve in hexane, and dilute to




                   500 ml (1 ml = 20 mg).




           13.3.5  Phenolphthalein Indicator. -- Dissolve 1 g in alcohol and




                   dilute to 100 ml.

-------
      13.3.6  Sodium hydroxide. -- Dissolve 20 g NaOH (pellets, reagent




              grade) in water and dilute to 500 ml (1N_) .   Dilute 25 ml




              IN NaOH to 500 ml with water (0.05N).  Standardize as follows:




              Weigh 100-200 mg lauric acid into 125 ml Erlenmeyer flask.




              Add 50 ml neutralized ethyl alcohol and 3 drops phenol -




              phthalein indicator; titrate to permanent end point.  Calculate




              mg lauric acid/ml 0.05 N_ NaOH (about 10 mg/ml).




13.4  Procedure




      13.4.1  Transfer 2.000 g Florisil to 25 ml glass stoppered Erlenmeyer




              flasks.  Cover loosely with aluminum foil and heat overnight




              at 130°C.  Stopper, cool to room temperature, add 20.0 ml




              lauric acid solution (400 mg), stopper, and shake occasionally




              for 15 min.  Let adsorbent settle and pipet 10.0 ml of




              supernatant into 125 ml Erlenmeyer flask.   Avoid inclusion




              of any Florisil.




      13.4.2  Add 50 ml neutral alcohol and 3 drops indicator solution;




              titrate with 0.05N_ to a permanent end point.




13.5  Calculation of Lauric Acid Value and Adjustment of Column Weight




      13.5.1  Calculate amount of lauric acid adsorbed on Florisil as




              follows:




              Lauric Acid value = mg lauric acid/g Florisil,= 200 - (ml




              required for titration X mg lauric acid/ml  0.05N NaOH).




      13.5.2  To obtain an equivalent quantity of any batch of Florisil,




              divide 110 by lauric acid value for that batch and multiply




              by 20 g.  Verify proper elution of pesticides by 13.6.

-------
                              4  - 3
13.6  Test for Proper Elution Pattern and  Recovery of Pesticides:




      Prepare a test mixture containing aldrin,  heptachlor epoxide,




      p,p'-DDE, dieldrin,  Parathion  and malathion.   Dieldrin and




      Parathion should elute in  the  15% eluate;  all but  a trace of




      malathion in the 50% eluate  and the  others in the  6% eluate.

-------
                    4          6         8         10

                          RETENTION TIME  IN MINUTES
14
Figure 1. Column Packing: 1% Carbowax 20M on Gas-Chrom Q (100/120 mesh),
         Column Temperature : 155 C, Carrier Gas: Helium at 80 ml/min.
         Detector:  Electrolytic Conductivity.
                                                      U S GOVEBNMEKT PRINTING OfflCE-1973— 759-555/1148

-------
    5.    METHOD FOR 0-ARYL CARBAMATE PESTICIDES IN INDUSTRIAL EFFLUENTS



  1.   Scope and Application


      1.1  This method covers the determination of various 0-aryl carbamate


           pesticides in industrial effluents.  Such compounds are character-


           ized by the carbamate structure with the oxygen atom  attached to
     i .

           an aromatic ring.


      1.2  The following compounds may be determined individually by this method


           with a sensitivity of 1 yg/liter:  Baygon, carbaryl (Sevin), Matacil,


           Mesurol, and Zectran.  The usefulness of the method for other


           specific pesticides must be demonstrated by the analyst before any


           attempt is made to apply it to sample analysis.


      1.3  The method also detects phenols and can be extended to the detection


           of phenolic hydrolysis products of the compounds above.


  2.   Summary


      2.1  A measured volume of water is extracted with methylene chloride.


           The concentrated extract is cleaned up with a Florisil column.


    ^     Appropriate fractions from the column are concentrated and portions

     «*
    -p     are separated by thin-layer chromatography.  The carbamates are


v •   ~     hydrolyzed on the layer and the hydrolysis products are reacted

c~I  £>
;?-   .     with 2,6-dibromoquinone chlorimide to yield specific colored products
u'  "i
c ... . ^
c- '"*      Quantitative measurement is achieved by visually comparing the
c-
  .  co'
           responses of sample extracts to the responses of standards on the


           same thin-layer.  Identifications are confirmed by changing the pH


           of the layer and observing color changes of the reaction products.


           Results are reported in micrograms per liter.


      2.2  This method is recommended for use only by experienced pesticide


           analysts or under the close supervision of such qualified persons.

-------
                                   5-2




3.   Interferences



    3.1  Direct interferences may be encountered from phenols that may be



         present in the sample.  These materials react with the chromogenic



         reagent and yield reaction products similar to those of the car-



         bamates.  In cases where phenols are suspected of interfering with



         a determination, a different solvent system should be used to



         attempt to isolate the carbamates.



    3.2  Indirect interferences may be encountered from naturally colored



         materials whose presence masks the carbamate reaction.



4.  Apparatus and Materials



    4.1  Thin layer plates - Glass plates (200 X 200 mm), coated with 0.25 mm



         layer of Silica Gel G (gypsum binder)



    4.2  Spotting template



    4.3  Developing chamber



    4.4  Sprayer - 20 ml capacity



    4.5  Kudema-Danish  (K-D) Glassware (Kontes)



         4.5.1  Snyder Column - Three ball (K-503000)



         4.5.2  Micro-Snyder Column - Two ball (K-569001)



         4.5.3  Evaporative Flasks - 500 ml (K-570001)



         4.5.4  Receiver Ampuls - 10 ml graduated (K-570050)



         4.5.5  Ampul Stoppers



    4.6  Chromatographic Column - Chromaflex (400 mm long X 19 mm ID) with



         coarse fritted plate on bottom and Teflon stopcock; 250 ml reservoir



         bulb at top of column with flared out funnel shape at top of bulb -



         a special order (Kontes K-420540-9011).



    4.7  Chromatographic Column - Pyrex (approximately 400 mm long X 20 mm ID)



         with coarse fritted plate on bottom.

-------
                              5-3





    4.8   Micro Syringes - 10, 25, 50 and 100 ul.




    4.9   Separator/ Funnel - 2000 ml, with Teflon stopcock.




    4.10  Blender - High speed, glass or stainless steel cup.




    4.11  Florisil   PR Grade (60-80 mesh); purchase activated at 1250 F




          and store in the dark in glass containers with glass stoppers




          or foil-lined screw caps.  Before use activate each batch over-




          night at 130 C in foil-covered glass container.   Determine lauric




          acid value (See Appendix I).




5.   Reagents, Solvents and Standards




    5.1   Ferrous Sulfate - (ACS) 3% solution in distilled water.




    5.2   Potassium Iodide -  (ACS) 10% solution in distilled  water.




    5.3   Sodium Hydroxide -  (ACS) 10 N in distilled water.




    5.4   Sodium Sulfate - (ACS) Granular, anhydrous.




    5.5   Sulfuric Acid   (ACS) Mix equal volumes of cone. H_SO. with




          distilled water.




    5.6   Diethyl Ether   Nanograde, redistilled in glass, if necessary.




          5.6.1  Must contain 2% alcohol and be free of peroxides by




                 following test:  To 10 ml of ether in glass-stoppered




                 cylinder previously rinsed with ether, add one ml of




                 freshly prepared 10% KI solution.   Shake  and  let stand




                 one minute.   No yellow color should be observed in




                 either layer.




          5.6.2  Decompose ether peroxides by adding 40g of 30% ferrous




                 sulfate solution to each liter of solvent.   CAUTION:




                 Reaction may be vigorous if the solvent contains a high




                 concentration of peroxides.




          5.6.3  Distill deperoxidized ether in glass and  add  2%  ethanol.

-------
                                  5-4




    5.7   Hexane,  Methanol,  Methylene  Chloride,  Petroleum Ether -  Nanograde,




         redistill  in  glass if necessary.




    5.8   Pesticide  Standards - Reference grade.




         5.8.1   TLC standards  - 0.100 pg/yl  in  chloroform.




    5.9   Chromogenic agent  - Dissolve 0.2  g  2,6-dibromoquinone chlorimide




         in 20  ml chloroform.




    5.10 Buffer solution -  0.1 N sodium borate  in  water .




6.   Calibration




    6.1   To insure  even solvent travel up  the  layer,  the tank used for layer




         development must be thoroughly saturated  with  developing solvent




         before it  is  used.  This may be achieved  by  lining the inner walls




         of the tank with chromatography paper  and introducing the solvent




         1-2 hours  before use.




    6.2   Samples and standards should be introduced to  the  layer  using a




         syringe, micropipet or other suitable  device that  permits all




         the spots  to  be about the same size and as small as  possible. An




         air stream directed on the  layer  during spotting will speed




         solvent evaporation and help to maintain  small spots.




    6.3   For qualitative and quantitative  work, spot  a  series of  standards




         representing  0.1 - 1.0 pg of a pesticide.  Tables  1  and  2 present




         color  responses and R,. values for several  solvent  systems.




7.   Quality Control




    7.1   Duplicate  and spiked sample  analyses are  recommended as  quality




         control checks. When the routine occurrence of a  pesticide  is




         being  observed, the use of quality  control charts  is recommended.

-------
                              5-5





8•   Sample Preparation




    8.1   Blend the sample if suspended matter is present and adjust pH




          to near neutral  (pH 6.5-7.5) with 50% sulfuric acid or 10 N




          sodium hydroxide.




    8.2   Quantitatively transfer a one-liter aliquot into a two-liter




          separatory funnel.




9.   Extraction




    9.1   Add 60 ml methylene chloride to the sample in the separatory




          funnel and shake vigorously for two minutes.




    9.2   Allow the solvent to separate from the sample, then draw the water




          into a one-liter Erlenmeyer flask.  Pass the organic layer through




          a chromatographic column containing 3-4 inches of anhydrous sodium




          sulfate, and collect it in a 500 ml K-D flask equipped with a 10 ml




          ampul.  Return the water phase to the separatory funnel.   Rinse




          the Erlenmeyer flask with a second 60 ml volume of solvent, add the




          solvent to the separatory funnel, and complete the extraction




          procedure a second time.  Perform a third extraction in the same




          manner.




    9.3   Concentrate the extract to 10 ml in a K-D evaporator on a hot




          water bath.   Disconnect the Snyder column just long enough to add




          10 ml hcxane to the K-D flask and then continue the concentration




          to about 5-6 ml.   If the need for cleanup  is indicated,  continue




          to Florisil  Column Cleanup (10 below).




    9.4   If further cleanup is not required,  replace the Synder column and




          flask with a micro-Snyder column and continue the concentration




          to 0.5-1.0 ml.   Analyze this final concentrate by thin-layer




          chromatography (Section 11).

-------
                                 5  -  6




10.   Florisil Column Cleanup




     10.1  Adjust the sample extract to 10 ml with hexane.




     10.2  Place a charge of activated Florisil (weight determined by




           lauric acid value,  see Appendix I) in a Chromaflex chromato-




           graphic column.   After settling the Florisil by  tapping the




           column, add about one-half inch layer of anhydrous granular




           sodium sulfate to the top.




     10.3  Pre-elute the column, after cooling, with 50-60  ml of petroleum




           ether.  Discard the eluate  and just prior to exposure of the




           sulfate layer to air, quantitatively transfer the  sample extract




           into the column by decantation and subsequent petroleum ether




           washings.  Adjust the elution rate to about  5 ml per minute




           and separately collect the  eluates in 500 ml K-D flasks equipped




           with 10 ml ampuls.   Perform the first elution with 200 ml  of




           6% ethyl ether in petroleum ether, and the second  elution  with




           200 ml of 15% ethyl ether in petroleum ether.  Perform the




           third elution with  200 ml of 50% ethyl ether in  petroleum  ether




           and the fourth elution with 200 ml of 100% ethyl ether.




           Eluate Composition    By using an equivalent  quantity of any




           batch of Florisil as  determined by its lauric acid value,  the




           pesticides will  be  separated into the eluates  indicated below:




                            50%  Eluate          100% Eluate




                            Sevin (70%)          Sevin (30%)




                            Zectran             Baygon




                                                Matacil

-------
                                5  _  7




     10.4  Concentrate the eluates to 6-10 ml in the K-D evaporator in a




           hot water bath.  Change to the micro-Snyder column and  continue




           concentration to 0.5 -  1.0 ml.




     10.5  Analyze according to 11.  below.




11.   Separation and Detection




     11.1  Carefully spot 10% of the extract on a thin-layer.   On  the  same




           plate spot several pesticides or mixtures for screening purposes




           or a series of 1,2,4,6,8  and 10 yl of specific standards for




           quantitative analysis.




     11.2  Develop the layers 10 cm  in a tank saturated with solvent vapors.




           Remove the plate and allow it to dry.




     11.3  Spray the layer rapidly and evenly with about 10-15 ml  chromogenic




           reagent.   Heat the layer  in an oven at 110 C for 15 minutes.   The




           pesticides will appear  with colors as indicated in Table 2.  Make




           quantitative estimates  by visually comparing the intensity  and




           size of the spots with  those of the series of standard.




     11.4  Spray the layer with sodium borate reagent and observe  the




           color shift of the reaction products.  The color shift  must




           be the same for sample  and standard for identification  to be




           confirmed.




12.   Calculation of Results




     12.1  Determine the concentration of pesticide in a sample by comparing




           the response in a sample  to that of a quantity of standard  treated




           on the same layer.   Divide the result,  in micrograms, by the




           fraction of extract spotted to convert  to micrograms per liter.

-------
                                  5-8




13.   Reporting Results




     13.1  Report results in micrograms per liter without correction for




           recovery data.  When duplicate and spiked samples are analyzed




           all data obtained should be reported.

-------
                                      5-9
REFERENCES CITED:

(1)  "Handbook for Analytical Quality Control in Water and Wastewater
     Laboratories", Chapter 6, Section 6.4, U. S. Environmental Protection
     Agency, National Environmental Research Center, Analytical Quality
     Control Laboratory, Cincinnati, Ohio  45268, 1972.

(2)  "Methods for Organic Pesticides in Water and Wastewater", U. S.
     Environmental Protection Agency, National Environmental Research
     Center, Analytical Quality Control Laboratory, Cincinnati, Ohio
     45268, 1971.

(3)  Finocchiaro, J. M. and Benson, W. R. , "Thin- Layer Chromatopr.-mhy
     of Some Carbamate and Phenyl Urea Pesticides", Journal of the
     Association of Official Agricultural Chemist, 50, 888  (19()~) .

(4)  Smith, D. and Lichtenherg, J. J., "Determination of Phenol? in
     Surface Waters by Thin-Layer Chromatography", Microorganic Matter
     in Water, ASTM STP 448, American Society for Testing and Materials,
     1969, pp. 78-95.

(5)  Stahl, E., "Thin-Layer Chromatography", Academic Press, New York, 1969

(6)  Longbottom, J. E. and Lichtenberg, J. J., "Determination of Carbamate
     and Urea Pesticides in Surface Waters by Thin-Layer Chromatography",
     U. S. Environmental Protection Agency, National Environmental Research
     Center, Analytical Quality Control Laboratory, Cincinnati, Ohio
     45268, 1972.

(7)  Steere, N. V., editor, "Handbook of Laboratory  Safety", Chemical
     Rubber Company, 18901 Cranwood Parkway, Cleveland, Ohio 44128, 1971,
     pp. 250-254.

-------
                                   5 - 10
                             Table 1




R_ Values of 0-Aryl Carbamates Pesticides in Several Solvent Systems

Sevin
Matacil
Zectran
Mesurol
Baygon

0.
0.
0.
0.
0.
A
26
26
34
31
27

0.
0.
0.
0.
0.
B
22
02
22
31
10

0
0
0
0
0
C
.48
.46
.54
.55
.53

0.
0.
0.
0.
0.
D
41
52
53
55
59

0.
0.
0.
0.
0.
E
58
54
60
59
60
F
0.24
0.04
0.24
0.28
0.13
Solvent Systems




A. Hexane/acetone (3:1)




B.  Methylene chloride




C.  Benzene/acetone (4:1)




D.  Benzene/cyclohexane/diethylamine (5:2:2)




D. Ethyl acetate




F.  Chloroform

-------
                            .5 - 11










                         TABLE 2




COLOR RESPONSES AND DETECTION LIMIT FOR 0-ARYL CARBAMATES
Colors
Sevin
Matacil
Zectran
Mesural
Baygon
Before
Buffer
Brown
Gray
Gray
Brown
Blue
After
Buffer
Red-Purple
Green
Green
Tan
Blue
Detection
Limit
(Mg)
0.1
0.1
0.1
0.2
0.1

-------
                                  5  -  1






                                   APPENDIX I





13.   Standardization of Florisil Column by Weight Adjustment Based on Adsorption




     of Laurie Acid.




     13.1  A rapid method for determining adsorptive capacity of Florisil is




           based on adsorption of lauric acid from hexane solution (6) (8).




           An excess of lauric acid is used and amount not adsorbed is measured




           by alkali titration.   Weight of lauric acid adsorbed is used to




           calculate, by simple proportion, equivalent quantities of Florisil




           for batches having different adsorptive capacities.




     13.2  Apparatus




           13.2.1  Buret. -- 25 ml with 1/10 ml graduations.




           13.2.2  Erlenmcyer flasks.  -- 125 ml narrow mouth and 25 ml, glass




                   stoppered.




           13.2.3  Pipet. -- 10 and 20 ml transfer.




           13.2.4  Volumetric flasks.  -- 500 ml.




     13.3  Reagents and Solvents




           13.3.1  Alcohol,  ethyl. --  USP or absolute, neutralized to




                   phenolphthalein.




           13.3.2  Hexane.  -- Distilled from all  glass apparatus.




           13.3.3  Lauric acid.  --Purified, CP.




           13.3.4  Lauric acid solution.  -- Transfer 10.000 g lauric acid to




                   500 ml volumetric flask, dissolve in hexane, and dilute to




                   500 ml (1 ml  = 20 mg).




           13.3.5  Phenolphthalein Indicator.  --  Dissolve 1 g in alcohol  and




                   dilute to 100 ml.

-------
                             5 -  2
      13.3.6  Sodium hydroxide. -- Dissolve 20 g NaOH (pellets, reagent




              grade) in water and dilute to 500 ml (IN).  Dilute 25 ml




              1N_ NaOH to 500 ml with water (0.05N).  Standardize as follows:




              Weigh 100-200 mg lauric acid into 125 ml Erlenmeyer flask.




              Add 50 ml neutralized ethyl alcohol and 3 drops phenol -




              phthalein indicator; titrate to permanent end point.   Calculate




              mg lauric acid/ml 0.05 N_ NaOH (about 10 mg/ml).




13.4  Procedure




      13.4.1  Transfer 2.000 g Florisil to 25 ml glass stoppered Erlenmeyer




              flasks.   Cover loosely with aluminum foil and heat overnight




              at 130°C.  Stopper, cool to room temperature, add 20.0 ml




              lauric acid solution (400 mg) ,  stopper, and shake occasionally




              for 15 min.   Let adsorbent settle and pipet 10.0 ml of




              supernatant into 125 ml Erlenmeyer flask.   Avoid inclusion




              of any Florisil.




      13.4.2  Add 50 ml neutral alcohol and 3 drops indicator solution;




              titrate with 0.05N to a permanent end point.




13.5  Calculation of Lauric Acid Value and Adjustment of Column Weight




      13.5.1  Calculate amount of lauric acid adsorbed on Florisil  as




              follows:




              Lauric Acid value = mg lauric acid/g Florisil,= 200 - (ml




              required for titration X mg lauric acid/ml 0.05N NaOH).




      13.5.2  To obtain an equivalent quantity of any batch of Florisil,




              divide 110 by lauric acid value for that batch  and multiply




              by 20 g.   Verify proper elution of pesticides by 13.6.

-------
                              5 -  3
13.6  Test for Proper Elution  Pattern and Recovery of Pesticides:




      Prepare a test mixture containing aldrin, heptachlor  epoxide,




      p,p'-DDE, dieldrin,  Parathion and malathion.  Dieldrin  and




      Parathion should elute in  the 15% eluate; all but a trace  of




      malathion in the 50% eluate  and the others in the 6%  eluate.
 OS. SOVUKKKT mmm OfFICfc 1973- TS'J-S'.S/l 141

-------
6-  METHOD FOR N-ARYL CARBAMATE AND UREA PESTICIDES IN INDUSTRIAL EFFLUENTS
o
-
CO
        0)
        PCJ
Scope and Application

1.1  This method covers the determination of various N-aryl carbamate

     and urea pesticides in industrial effluents.  Such compounds are

     characterized by the carbamate and urea structures with a nitrogen

     atom attached to an aromatic ring.

1.2  The following compounds may be determined individually by this

     method with a sensitivity of 1 yg/liter:  barban, chloropropham,

     diuron, fenuron, linuron, monuron, neburon, propham, biduron, Swep,

     Urab and Urox.  The usefulness of the method for other specific

     pesticides must be demonstrated by the analyst before any attempt

     is made to apply it to sample analysis.

1.3  The method also detects anilines and can be extended to the

     detection of anilinic hydrolysis products of the compounds above.

Summary

2.1  A measured volume of water is extracted with methylene chloride

     and the concentrated extract is cleaned up with a Florisil column.

     Appropriate fractions from the column are concentrated and portions

     are separated by thin-layer chromatography.  The pesticides are

     hydrolyzed to primary amines, which in turn are chemically converted

     to diazonium salts.  The layer is sprayed with 1-naphthol and the

     products appear as colored spots.  Quantitative measurement is

     achieved by visually comparing the responses of sample extracts to

     the responses of standards on the same thin layer.  Results are

     reported in micrograms per liter.

2.2  This method is recommended for use only by experienced pesticide

     analysts or under the close supervision of such qualified persons.

-------
                                     6-2




3.   Interferences




    3.1  Direct interferences may be encountered from aromatic amines that




         may be present in the sample.  These materials react with the




         chromogenic reagent and yield reaction products similar to those




         of the pesticides.  In cases where amines are suspected of inter-




         fering with a determination, a different solvent system should be




         used to attempt to isolate the pesticides on the layer.




    3.2  Indirect interferences may be encountered from naturally colored




         materials whose presence masks the chromogenic reaction.




4.  Apparatus and Materials



    4.1  Thin-layer plates - Glass plates (200 X 200 mm) coated with 0.25 mm




         layer of Silica Gel G (gypsum binder).




    4.2  Spotting Template




    4.3  Developing Chamber




    4.4  Sprayer - 20 ml capacity




    4.5  Kuderna-Danish (K-D) Glassware (Kontes)




         4.5.1  Snyder Column - three ball (K-503000)




         4.5.2  Micro-Snyder Column - two ball (K-569001)




         4.5.3  Evaporative Flasks - 500 ml (K-570001)




         4.5.4  Receiver Amputs - 10 ml graduated (K-570050)




    4.6  Chromatographic Column - Chromaflex (400 mm long X 19 mm ID) with




         coarse fritted plate on bottom and Teflon stopcock; 250 ml




         reservoir bulb at top of column with flared out funnel shape at




         top of bulb - a special, order (Kontes K-420540-9011).




    4.7  Chromatographic Column - Pyrex (approximately 400 mm long X 20 mm  ID)




         with coarse fritted plate on bottom.




    4.8  Micro Syringes - 10, 25, 50 and 100 jil.

-------
                                  o    3





    4.9  Separatory Funnel - 2000 ml, with Teflon stopcock.




   4.10  Blender - High speed, glass or stainless steel cup.




   4.11  Florisil - PR Grade (60-80  mesh); purchase activated at 1250  F




         and store in the dark in glass containers with glass stoppers or




         foil-lined screw caps.  Before use activate each batch overnight




         at 130 C in foil-covered glass container.  Determine lauric acid




         value (See Appendix I).




5.   Reagents, Solvents and Standards




    5.1  Ferrous Sulfate - (ACS)  30% solution in distilled water.




    5.2  Potassium Iodide - (ACS) 10% solution in distilled water.




    5.3  Sodium Hydroxide - (ACS) 10 N in distilled water.




    5.4  Sodium Sulfate - (ACS) Granular, anhydrous.




    5,5  Sulfuric Acid - (ACS) Mix equal volumes of cone. H^SO.  with




         distilled water.




    5.6  Diethyl Ether - Nanograde,  redistilled in glass, if  necessary.




         5.6.1  Must contain 2% alcohol and be free of peroxides by




                following test:  To  10 ml of ether in glass-stoppered




                cylinder previously  rinsed with ether, add one ml of




                freshly prepared  10% KI solution.  Shake and  let stand




                one minute.   No yellow color should be observed in either




                layer.




         5.6.2  Decompose ether peroxides by adding 40g of 30% ferrous




                sulfate solution  to  each liter of solvent. CAUTION:




                Reaction may be vigorous if the solvent contains a high




                concentration of  peroxides.




         5.6.3  Distill deperoxidized ether in glass and add  2%  ethanol.

-------
                                     6 -  4




    5.7  Hexane, Methanol, Methylene  Chloride,  Petroleum Ether - nanograde,




         redistill  in glass  if necessary.




    5.8  Pesticide  Standards - Reference  grade.




         5.8.1   TLC Standards - 0.100 ug/yl in  chloroform.




    5.'9  Nitrous acid - prepare just  before use  by mixing 1  g NaNO~ with




         20 ml  0.2  N HC1.



   5.10  Chromogenic agent  - Dissolve 1.0 g 1-Naphthol  in 20 ml ethanol.




         Prepare fresh daily.




6.  Calibration



    6.1  To insure  even solvent travel  up the layer,  the tank used for layer




         development must be thoroughly saturated with  developing solvent




         before it  is used.   This  may be  achieved by  lining  the inner walls




         of the tank with chromatography  paper  and introducing the solvent




         1-2 hours  before use.




    6.2  Samples and standards should be  introduced to  the  layer using a




         syringe, micropipet or other suitable  device that permits all the




         spots  to be about  the same size  and as  small as possible.  An air




         stream directed on  the layeTr during spotting will speed solvent




         evaporation and help to maintain small  spots.




    6.3  For qualitative and quantitative work,  spot  a  series representing




         0.1-1.0 pg of a pesticide.   Tables 1 and 2 present  color responses




         and R^ values for several solvent systems.



7.  Quality Control




    7.1  Duplicate  and spiked sample  analyses are recommended as quality control




         checks.  When the routine occurrence of a pesticide is being observed,




         the use of quality  control charts is recommended.

-------
                                    6  -  5




8.  Sample Preparation




    8.1  Blend the sample if suspended matter is present and adjust pH to




         near neutral (pH 6.5-7.5) with 50% sulfuric acid or 10 N sodium




         hydroxide.




    8.2  Quantitatively transfer a one-liter aliquot into a two-liter




         separatory funnel.-




9.  Extraction




    9.1  Add 60 ml of methylene chloride to the sample in the separatory funnel




         and shake vigorously for two minutes.




    9.2  Allow the mixed solvent to separate from the sample, then draw the




         water into a one-liter Erlenmeyer flask.  Pass the organic layer




         through a column containing 3-4 inches of anhydrous sodium sulfate,




         and collect it in a 500 ml K-D flask equipped with a 10 ml ampul.




         Return the water phase to the separatory funnel, and complete the




         extraction procedure a second time.  Perform a third extraction in




         the same manner.




    9.3  Concentrate the extract to 10 ml in a K-D evaporator on a hot water




         bath.  Disconnect the Snyder column just long enough to add 10 ml




         hexane to the K-D flask and then continue the concentration to about




         5-6 ml.   If the need for cleanup is indicated, continue to Florisil




         Column Cleanup (10 below).




    9.4  If further cleanup is not required, replace the Snyder column and




         flask with a micro-Snyder column and continue the concentration to




         0.5-1.0 ml.   Analyze this final concentrate by thin-layer chromato-




         graphy (Section 11).

-------
                                      6  -  6

10.   Florisil Column Cleanup

     10.1  Adjust the sample extract to  10 ml with hexane.

     10.2  Place a charge of activated Florisil (weight determined by

           lauric acid value, see Appendix I) in a Chromaflex chromato-

           graphic column.  After settling the Florisil by tapping the

           column, add about one-half inch layer of anhydrous granular

           sodium sulfate to the top.

     10.3  Pre-elute the column, after cooling, with 50-60 ml of petroleum

           ether.  Discard the eluate and  just prior to exposure of the

           sulfate layer to air, quantitatively transfer the sample extract

           into the column by decantation  and subsequent petroleum ether

           washings.  Adjust the elution rate to about 5 ml per minute and

           separately collect the eluates  in 500 ml K-D flasks equipped

           with 10 ml ampuls.  Perform the first elution with 200 ml of 6%

           ethyl ether in petroleum ether, and the second elution with 200 ml

           of 15% ethyl ether in petroleum ether.   Perform the third elution

           with 200 ml of 50% ethyl ether  - petroleum ether and the fourth

           elution with 200 ml of 100% ethyl ether.

           Eluate Composition - By using an equivalent quantity of any batch

           of Florisil as determined by  its lauric acid value, the pesticides

           will be separated into the eluates indicated below:

                   15% Eluate         50% Eluate         100% Eluate

                   CIPC               Barban (5%)        Neburon (92%)
                   IPC                Linuron            Diuron
                   Barban (95%)       Neburon (8%)       Fluometuron
                                                          Monuron
                                                          Siduron
                                                          Urox (25%)


           CAUTION:   Fenuron and Urab are  not recovered from the Florisil column.

           The recovery of Urox is very  poor.

-------
                                     6  -  7




     10.4  Concentrate the eluates to 6-10 ml in the K-D evaporator in a hot




           water bath.  Change to the micro-Snyder column and continue con-




           centration to 0.5-1.0 ul.




     10.5  Analyze according to 11. below.




11.  Separation and Detection




     11.1  Carefully spot 10% of the extract on a thin layer.  On the same




           plate spot several pesticides or mixtures for screening purposes,




           or a series of 1,2,4,6,8 and 10 yl of specific standards for




           quantitative analysis.




     11.2  Develop the layers 10 cm in a tank saturated with solvent vapors.




           Remove the plate and allow it to dry.




     11.3  Spray the layer rapidly and evenly with about 10-15 ml sulfuric




           acid solution.  Heat the layer in an oven at 110 C for 15 minutes.




     11.4  When the layer is cool, spray it with nitrous acid reagent and




           allow it to dry.  Spray the layer with 1-naphthol reagent and




           allow it to dry again.  The pesticides will appear as purple




           spots (see Table 2).  Identifications are made by comparison of




           colors and R~ values.  Quantitative estimates are made by visually




           comparing the intensity and size of the spots with those of the




           series of standard.




12.  Calculation of Results




     12.1  Determine the concentration of pesticide in a sample by comparing




           the response in a sample to that of a quantity of standard treated




           on the same layer.   Divide the result, in micrograms, by the




           fraction of extract spotted to convert to micrograms per liter.




13.  Reporting Results




     13.1  Report results in micrograms  per liter without correction for




           recovery data.  When duplicate and spiked samples are analyzed

-------
                                     6-8
                                       REFERENCES

(1)   "Handbook  for  Analytical  Quality Control  in Water and Wastewater Laboratories,"
     Chapter 6,  Section 6.4, U.S.  Environmental  Protection Agency,  National
     Environmental  Research  Center,  Analytical Quality Control Laboratory;
     Cincinnati,  Ohio  45268,  1972.

(2)   "Methods for Organic  Pesticides in Water  and Wastewater," U.S.  Environ-
     mental  Protection Agency,  National Environmental  Research Center,  Analytical
     Quality Control Laboratory, Cincinnati, Ohio 45268,  1971.

(3)   Katz, S. E., "Determination of  the Substituted Urea  Herbicides  Linuron,
     Monuron, Diuron,  Neburon,  and Fenuron  in  Surface  Water," Journal of the
     Association of Official Agricultural Chemists,  49, 452 (1966).

(4)   Geissbuhler, H. and Gross, D.,  "Specific  Detection of Urea Herbicide
     Residues by Separation  of Their Amines on Cellulose  Thin-Layer  Plates,"
     Journal of Chromatography, 27,  296 (1967).

(5)   Askew,  J.  et al.,  "Use  of Hydriodic Acid  in the Detection of Pesticides
     After Thin-Layer  Chromatography," Journal of Chromatography, 37, 369 (1968).

(6)   Stahl,  E.,  "Thin-Layer  Chromatography," Academic  Press,  New York,  1969.

(7)   Longbottom,  J. E.  and Lichtenberg,  J.  J., "Determination of Carbamate and
     Urea Pesticides in Surface Waters by Thin-Layer Chromatography." U.S.
     Environmental  Protection  Agency,  National Environmental  Research Center,
     Analytical  Quality Control Laboratory, Cincinnati, Ohio  45268,  1972.

(8)   Steere,  N.  V., editor,  "Handbook  for Laboratory Safety," Chemical  Rubber
     Company, 18901 Cranwood Parkway.  Cleveland,  Ohio  44128,  1971, pp.  250-254.

-------
                                6 -  9
                               TABLE 1
         Rf VALUES OF N-ARYL CARBAMATE AND UREA PESTICIDES
                        IN SEVERAL SOLVENT SYSTEMS

Carbamates              A      B      C      D      E      F      G
Propham               0.49   0.54   0.73   0.48   0.36   0.68   0.69
Chlorpropham          0.57   0.60   0.73   0.49   0.37   0.70   0.73
Barban                0.61   0.59   0.72   0.41   0.28   0.70   0.74
Swep                  0.48   0.44   0.70   0.41   0.28   0.67   0.66
Urea
Fenuron
Urab
Monuron
Urox
Diuron
Linuron
Neburon
Siduron

0.
0.
0.
0.
0.
0.
0.
0.

03
03
04
04
05
40
21
02

0.
0.
0.
0.
0.
0.
0.
0.

04
04
05
06
09
43
28
07

0.
0.
0.
0.
0.
0.
0.
0.

38
36
37
34
38
62
64
68

0.22
0.22
0.24
0.24
0.28
0.39
0.41
0.39

0.
0.
0.
0.
0.
0.
0.
0.

10
10
10
10
13
24
26
25

0.41
0.41
0.47
0.46
0.54
0.66
0.68
0.62

0.30
0.30
0.34
0.34
0.44
0.64
0.65
0.55
Solvent Systems
A.  Methylene chloride
B.  Chloroform
C.  Ethyl Acetate
D.  Hexane/acetone  (2:1)
E.  Hexane/acetone  (4:1)
F. Chloroform/acetonitrile  (2:1)
G.  Chloroform/acetonitrile  (5:1)

-------
                   6-10





                  TABLE 2




COLOR RESPONSES AND DETECTION LIMIT FOR THE




        N-ARYL CARBAMATES AND UREAS
Carbamates
Propham
Chlorpropham
Barban
Swep
Ureas
Fenuron
Urab
Monuron
Urox
Diuron
Linuron
Neburon
Siduron
Color
Red -purple
Purple
Purple
Blue-Purple

Red -purple
Red -purple
Pink-orange
Pink-orange
Blue-purple
Blue-purple
Blue-purple
Red -purple
Detection
Limit
(Mg)
0.2
0.1
0.05
0.2

0.05
0.1
0.05
0.1
0.1
0.1
0.1
0.05

-------
                                6   -  1






                                  APPENDIX I





3.   Standardization of Florisil Column by Weight Adjustment Based on Adsorption




    of Laiiric Acid.




    13.1  A rapid method for determining adsorptive capacity of Florisil is




          based on adsorption of lauric acid from hexane solution (6) (8).




          An excess of lauric acid is used and amount not adsorbed is measured




          by alkali titration.  Weight of lauric acid adsorbed is used to




          calculate, by simple proportion, equivalent quantities of Florisil




          for batches having different adsorptive capacities.




    13.2  Apparatus




          13.2.1  Buret. -- 25 ml with 1/10 ml graduations.




          13.2.2  Erlenmeyer flasks.  -- 125 ml narrow mouth and 25 ml, glass




                  stoppered.




          13.2.3  Pipet. -- 10 and 20 ml transfer.




          13.2.4  Volumetric flasks.  -- 500 ml.




    13.3  Reagents and Solvents




          13.3.1  Alcohol, ethyl.  --  USP or absolute, neutralized to




                  phenolphthalein.




          13.3.2  Hexane.  -- Distilled from all glass apparatus.




          13.3.3  Lauric acid. --Purified, CP.




          13.3.4  Lauric acid solution. -- Transfer 10.000 g lauric acid to




                  500 ml volumetric flask, dissolve in hexane, and dilute to




                  500 ml (1 ml = 20 mg).




          13.3.5  Phenolphthalein Indicator.  -- Dissolve 1 g in alcohol  and




                  dilute to 100 ml.

-------
                             6 - 2
      13.3.6  Sodium hydroxide.  -- Dissolve 20 g NaOH (pellets, reagent




              grade) in water and dilute to 500 ml (IN).   Dilute 25 ml




              1N_ NaOH to 500 ml  with water (0.05N).   Standardize as follows:




              Weigh 100-200 mg lauric acid into 125  ml Erlenmeyer flask.




              Add 50 ml neutralized ethyl alcohol and 3  drops phenol -




              phthalein indicator; titrate to permanent  end point.  Calculate




              mg lauric acid/ml  0.05 N^NaOH (about 10 mg/ml).




13.4  Procedure




      13.4.1  Transfer 2.000 g Florisil to 25 ml glass stoppered Erlenmeyer




              flasks.  Cover loosely with aluminum foil  and heat overnight




              at 130°C.  Stopper, cool to room temperature, add 20.0 ml



              lauric acid solution (400 mg),  stopper, and shake occasionally




              for 15 min.  Let adsorbent settle and  pipet 10.0 ml of




              supernatant into 125 ml Erlenmeyer flask.   Avoid inclusion




              of any Florisil.




      13.4.2  Add 50 ml neutral  alcohol and 3 drops  indicator solution;




              titrate with 0.05N_ to a permanent end  point.




13.5  Calculation of Lauric Acid Value and Adjustment of Column Weight




      13.5.1  Calculate amount of lauric acid adsorbed on Florisil as




              follows:




              Lauric Acid value  = mg lauric acid/g Florisil,= 200 - (ml




              required for titration X mg lauric acid/ml  0.05N_NaOH).




      13.5.2  To obtain an equivalent quantity of any batch of Florisil,




              divide 110 by lauric acid value for that batch  and multiply




              by 20 g.   Verify proper elutiori of pesticides by 13.6.

-------
                             6  - 3







13.6  Test for Proper  Elution Pattern and Recovery of  Pesticides:




      Prepare a  test mixture containing aldrin, heptachlor epoxide,




      p,p'-DDE,  dieldrin,  Parathion and malathion.   Dieldrin and




      Parathion  should elute in the 15% eluate; all  but  a  trace of




      malathion  in  the 50% eluate and the others  in  the  6% eluate.
   us amount*! nuimnoffici an- 759-555/1150

-------
1'   METHOD FOR CHLORINATED PHENOXY ACID HERBICIDES IN INDUSTRIAL EFFLUENTS




 1•  Scope and Application




     1.1  This method covers the determination of chlorinated phenoxy acid




          herbicides in industrial effluents.  The compounds 2,4-dichloro-




          phenoxyacetic acid (2,4-D), 2-(2,4,5-trichlorophenoxy) propionic




          acid (silvex),  2,3-dichloro-o-anisic acid  Cdicamba) and 2,4,5-




          trichlorophenoxyacetic acid (2,4,5-T) may be determined by this




          procedure.




     1.2  Since these compounds may occur in water in various forms  (i.e., acid,




          salt, ester, etc.) a hydrolysis step is included to permit the deter-




          mination of the active part of the herbicide.  The method may be




          applied to additional phenoxy acids and certain phenols.  However,




          the analyst must demonstrate the usefulness of the method for each




          specific compound before applying it to sample analysis.




 2.  Summary




     2.1  Chlorinated phenoxy acids and their esters are extracted from the




          acidified water sample with ethyl ether.  The esters are hydrolyzed




          to acids and extraneous organic material is removed by a solvent wash.




          The acids are converted to methyl esters which are extracted from




          the aqueous phase.  The extract is cleaned up by passing it through




          a micro-adsorption column.  Identification of the esters is made by




          selective gas chromatographic separations and may be corroborated




          through the use of two or more unlike columns.  Detection and measure-




          ment is accomplished by electron capture, microcoulometric or




          electrolytic conductivity gas chromatography (1).  Results are




          reported in micrograms per liter.




     2.2  This method is recommended for use only by experienced pesticide




          analysts or under the close supervision of such qualified persons.

-------
                                  7.2




3.   Interferences




    3.1  Solvents,  reagents,  glassware, and other sample processing hardware




         may yield  discrete artifacts and/or elevated baselines causing




         misinterpretation of gas chromatograms.   All of these materials must




         be demonstrated to be free from interference under the conditions of




         the analysis.   Specific selection of reagents and purification of




         solvents by distillation in all-glass systems may be required.




         Refer to Part  1, Sections 1.4 and 1.5, (2).




    3.2  The interferences in industrial effluents are high and varied and




         often pose great difficulty in obtaining accurate and precise




         measurement of chlorinated phenoxy acid herbicides.   Sample clean-up




         procedures are generally required and may result in loss of certain




         of these herbicides.  It is not possible to  describe procedures for




         overcoming all of the interferences that may be encountered in




         industrial effluents.



    3.3  Organic acids, especially chlorinated acids, cause the most direct




         interference with the determination.  Phenols including chlorophenols




         will also  interfere with this procedure.




    3.4  Alkaline hydrolysis and subsequent extraction eliminates many of




         the predominant chlorinated insecticides which might otherwise




         interfere  with the test.




    3.5  The herbicides, being strong organic acids,  react readily with




         alkaline substances and may be lost during analysis.  Glassware and




         glass wool should be acid-rinsed and sodium  sulfate should be acidi-




         fied with  sulfuric acid to avoid this possibility.

-------
                                   7 -  3




4.  Apparatus and Materials




    4.1  Gas Chromatograph - Equipped with glass lined injection port.




    4.2  Detector Options:




         4.2.1  Electron Capture - Radioactive (tritium or nickel-63)




         4.2.2  Microcoulometric Titration




         4.2.3  Electrolytic Conductivity




    4.3  Recorder - Potentiometric strip chart (10 in.) compatible with




         the detector.




    4.4  Gas Chromatographic Column Materials:




         4.4.1  Tubing - Pyrex  (180 cm long X 4 mm ID)




         4.4.2  Glass Wool - Silanized




         4.4.3  Solid Support - Gas-Chrom-Q (100-120 mesh)




         4.4.4  Liquid Phases - Expressed as weight percent coated on




                solid support.




                4.4.4.1  OV-210, 5%




                4.4.4.2  OV-17-. 1.5% plus QF-1, 1.95%




    4.5  Kuderna-Danish  (K-D) Glassware (Kontes)




         4.5.1  Snyder Column - three ball (macro) and two ball (micro)




         4.5.2  Evaporative Flasks - 250 ml




         4.5.3  Receiver Ampuls - 10 ml, graduated




         4.5.4  Ampul Stoppers




    4.6  Blender   High speed,  glass or stainless steel cup.




    4.7  Graduated cylinders -  100 and 250 ml.




    4.8  Erlenmeyer flasks   125 ml, 250 ml ground glass J 24/40




    4.9  Microsyringes - 10, 25, 50 and 100 yl.




   4.10  Pipets - Pasteur, glass disposable (140 mm long X 5 mm ID).




   4.11  Separatory Funnels - 60 ml and 2000 ml with Teflon stopcock.

-------
                                  7-4





    4.12   Glass  wool  -  Filtering grade,  acid washed.




    4.13   Diazald Kit   recommended for  the generation of diazomethane




          (available  from Aldrich Chemical  Co.,  Cat.  #210,025-2)




    4.14   Florisil -  PR grade (60-100 mesh) purchased activated at 1250F




          and stored  at 130 C.




5.   Reagents, Solvents  and Standards




    5.1   Boron  Trifluoride-Methanol-esterification-reagent,  14 percent




          boron  trifluoride by weight.




    5.2   N-methyl-N-nitroso-p-toluenesulfonamide (Diazald)  - High purity,




          melting point range 60-62 C.   Precursor for the generation of




          diazomethane  (see Appendix I).




    5.3   Ferrous Sulfate - (ACS) 30% solution in distilled  water.




    5.4   Potassium Hydroxide Solution - A  37 percent aqueous solution




          prepared from reagent grade potassium hydroxide pellets and reagent




          water.




    5.5   Potassium Iodide - (ACS) 10% solution in distilled water.




    5.6   Sodium Chloride - (ACS) Saturated solution  (pre-rinse Nad with




          hexane) in  distilled water.




    5.7   Sodium Hydroxide - (ACS) 10 N  in  distilled  water.




    5.8   Sodium Sulfate, Acidified. --  (ACS) granular sodium




          sulfate, treated as follows:   Add 0.1  ml of cone,  sulfuric acid to




          100 g  of sodium sulfate slurried  with enough ethyl  ether to just




          cover  the solid.  Remove the ether with the vacuum.  Mix 1 g of the




          resulting solid with 5 ml of reagent water  and ensure the mixture




          to have a pH  below 4.  Store at 130 C.




     5.9   Sulfuric acid.  -- (ACS) concentrated,  Sp. Gr. 1.84.



     5.9.a.  Carbitol  (diethylene glycol  monoethyl ether).

-------
5.10  Diethyl Ether   Nanograde, redistilled in glass, if necessary.




      5.10.1  Must contain 2% alcohol and be free of peroxides by




              following test:  To 10 ml of ether in glass-stoppered




              cylinder previously rinsed with ether, add one ml of




              freshly prepared 10% KI solution.  Shake and let stand one




              minute.  No yellow color should be observed in either layer.




      5.10.2  Decompose ether peroxides by adding 40 g of 30% ferrous




              sulfate solution to each liter of solvent.  CAUTION:   Reaction




              may be vigorous if the solvent contains a high concentration




              of peroxides.




      5.10.3  Distill deperoxidized ether in glass and add 2%  ethanol.




5.11  Benzene Hexane   Nanograde, redistilled in glass, if necessary.




5.12  Pesticide Standards - Acids and Methyl Esters, reference grade.




      5.12.1  Stock standard solutions - Dissolve 100 mg of each herbicide




              in 60 ml ethyl ether; then make to 100 ml with redistilled




              hexane.  Solution contains 1 mg/ml.




      5.12.2  Working standard - Pipet 1.0 ml of each stock soln into a




              single 100 ml volumetric flask.  Make to volume with a




              mixture of ethyl ether and hexane (1:1).  Solution contains




              10 ug/ml of each standard.




      5.12.3  Standard for Chromatography -  (Diazomethane Procedure) Pipet




              1.0 ml of the working standard into a glass stoppered test




              tube and evaporate off the solvent using steam bath.   Add




              2 ml diazomethane to the residue.  Let stand 10 minutes with




              occasional shaking, then allow the solvent to evaporate




              spontaneously.  Dissolve the residue in 200 ul of hexane for




              gas chromatography.

-------
                                  7  -  6




        5.12.4  Standard for Chromatography -CBoron Trifluoride Procedure)



                Pipet 1.0 ml of the working standard into a glass stoppered



                test tube.  Add 0.5 ml of Benzene and evaporate to 0.4 ml



                using a two-ball Snyder microcoltunn and a steam bath.



                Proceed as in 11.3.1.   Esters are then ready for gas



                chromatography.



6.  Calibration



    6.1  Gas chromatographic operating conditions are considered acceptable



         if the response to dicapthon is at least 50% of full scale when < 0.06



         ng is injected for electron capture detection and < 100 ng is injected



         for microcoulometric or electrolytic conductivity detection.  For all



         quantitative measurements,  the detector must be operated within its



         linear response range and the detector noise level should be less



         than 2% of full scale.



    6.2  Standards, prepared from methyl esters of phenoxy acid herbicides



         calculated as the acid equivalent, are injected frequently as a check



         on the stability of operating conditions.



    6.3  The elution order and retention ratios of methyl esters of chlorinated



         phenoxy acid herbicides are provided in Table 1, as a guide.



7.  Quality Control



    7.1  Duplicate and spiked sample analyses are recommended as quality control



         checks.  When the routine occurrence of a pesticide is being observed



         the use of quality control charts is recommended (3).



    7.2  Each time a set of samples is extracted, a method blank is determined



         on a volume of distilled water equivalent to that used to dilute the



         sample.

-------
                                   7 -  7





8.  Sample Preparation




    8.1  Blend the sample, if suspended matter is present.




    8.2  For a sensitivity requirement of  1 yg/1, when using electron




         capture for detection, take 100 ml of sample for analysis.




         For microcoulometric or electrolytic conductivity detection, take




         1-liter of sample.  Background information on the extent and nature




         of interferences will assist the  analyst in selecting the proper




         sample size and detector.




    8.3  Quantitatively  transfer  the proper aliquot of sample into a two-liter




         separatory funnel,  dilute to one  liter and acidify to approximately




         pH 2 with concentrated sulfuric acid.  Check pH with indicator paper.




9.  Extraction




    9.1  Add  150 ml of ether to the sample in the separatory funnel and shake




         vigorously for one  minute.




    9.2  Allow the contents  to separate for at least ten minutes.  After the




         layers have separated, drain the  water phase into a one-liter




         Erlenmeyer flask.   Then collect the extract in a 250 ml ground-glass




         Erlenmeyer flask containing 2 ml  of 37 percent aqueous potassium




         hydroxide.




    9.3  Extract the sample  two more times using 50 ml of ether each time,  and




         combine the extracts in the Erlenmeyer flask.   (Rinse the one-liter




         flask with each additional aliquot of extracting solvent.)




10.  Hydrolysis




    10.1  Add  15  ml of  distilled water and  a small boiling stone to the  flask




         containing  the  ether extract, and fit the  flask with a 3-ball  Snyder




          column.   Evaporate  the  ether on a steam bath and continue heating




          for  a total  of 60  minutes.

-------
                                  7  -  8




    10.2   Transfer the  concentrate to  a 60 ml separator/ funnel.   Extract




          the  basic solution two times with 20 ml t)f ether and discard




          the  ether layers.   The herbicides remain in the aqueous phase.




    10.3   Acidify the  contents  of the  separatory funnel by adding 2 ml of




          cold (4 C) 25 percent sulfuric acid (5.9).   Extract the herbicides




          once with 20  ml of ether and twice with 10 ml of ether.  Collect




          the  extracts  in a 125 ml Erlenmeyer flask containing about 0.5  g




          of acidified anhydrous sodium sulfate (5.8).   Allow the extract




          to remain in contact  with  the sodium sulfate  for approximately




          two  hours.




11.  Esterification (4,5)



    11.1   Transfer the  ether extract,  through a funnel  plugged with glass wool,




          into a Kuderna-Danish flask  equipped with a 10 ml graduated ampul.




          Use  liberal  washings  of ether.   Using a glass rod,  crush any caked




          sodium sulfate during the  transfer.




          11.1.1  If esterification  is to be done with  diazomethane, evaporate




                  to approximately 4 ml on a steam bath (do not immerse the




                  ampul in water) and  proceed as directed in Section 11.2.




          11.1.2  If esterification  is to be done with  boron trifluoride, add




                  0.5  ml benzene and evaporate to about 5 ml  on a steam bath.




                  Remove the ampul from the flask and further concentrate




                  the  extract to 0.4 ml using a two-ball Snyder microcolumn



                  and proceed as in  11.3.




    11.2   Diazomethane  Esterification




          11.2.1  Disconnect the ampul from the K-D flask and place in a  hood




                  away  from steam bath.   Adjust volume  to 4 ml with ether,  add




                  2 ml  diazomethane, and let stand 10 minutes with




                  occasional swirling.

-------
                             7-9




      11.2.2  Rinse inside wall  of ampul  with several hundred microliters




              of ethyl  ether.  Take  sample  to approximately  2 ml  to




              remove excess diazomethane  by allowing solvent to evaporate




              spontaneously (room temperature).




      11.2.3  Dissolve  residue in 5  ml  of hexane.   Analyze by gas




              chromato graphy.




      11.2.4  If further clean-up of the  sample  is  required, proceed  as




              in 11.3.4 substituting hexane for  benzene.




11.3  Boron Trifluoride Esterification




      11.3.1  After the benzene  solution  in the  ampul has cooled,  add




              0.5 ml of borontrifluoride-methanol  reagent.   Use the




              two-ball  Snyder  micro  column  as an air-cooled  condenser




              and hold  the contents  of  the  ampul at 50  C  for 30 minutes




              on the steam bath.




      11.3.2  Cool and  add about 4.5 ml of  a neutral 5  percent aqueous




              sodium sulfate solution so  that the  benzene-water interface




              is in the neck of  the  Kuderna-Danish  ampul.  Seal the flask




              with a ground glass stopper and shake vigorously for about one




              minute.   Allow to  stand for three  minutes for  phase  separation




      11.3.4  Pipet the solvent  layer from  the ampul to the  top of a  small




              column prepared  by plugging a disposable  Pasteur pipet  with




              glass wool and packing with 2.0 cm of sodium sulfate over




              1.5 cm of Florisil  adsorbent.   Collect the  eluate in a




              graduated ampul.   Complete  the transfer by  repeatedly rinsing




              the ampul with small quantities of benzene  and passing  the




              rinses through the  column until a  final volume of 5.0 ml of




              eluate is obtained.  Analyze  by gas  chromatography.

-------
                                    7  -  10
12.   Calculation of Results
    12.1  Determine the methyl ester concentration by using the absolute
          calibration procedure described below or the relative calibration
          procedure described in Part  I, Section 3.4.2 (2).
          (1)     Micrograms/liter = (A)   (B)   (Vt)


                  A = ng standard
                      Standard area
                  B = Sample aliquot area
                  V,= Volume of extract injected (pi)
                   1
                  V = Volume of total  extract (pi)
                  V = Volume of water extracted (ml)
    12.2  Molecular weights for the calculation of the methyl esters as the
          acid equivalents.
          2,4-D                        222.0      Dicamba               221.0
          2,4-D Methyl ester           236.0      Dicamba methyl ester  236.1
          Silvex                       269.5      2,4,5-T               255.5
          Silvex methyl ester          283.5      2,4,5-T methyl ester  269.5
13.  Reporting  Results
    13.1  Report results in micrograms per liter as the acid equivalent without
          correction for recovery data.   When duplicate and spiked samples are
          analyzed all data obtained should be reported.

-------
                        7-11


                          Table 1

RETENTION RATIOS FOR METHYL ESTERS OF SOME CHLORINATED

       PHENOXY ACID HERBICIDES RELATIVE TO 2,4-D
Liquid Phase
Column Temp.
Argon/Methane
Carrier Flow
Herbicide
2,4-D
silvex
2,4,5-T
dicamba
2,4-D
(minutes absolute)
1.5% OV-17
2.95% QF-1
185 C
70 ml/min
RR
1.00
1.34
1.72
9^0
2.00
5%
OV-210
185 C
70 ml/min
RR
1.00
1.22
1.51
0,61
1.62
 All columns glass, 180 cm X 4 mm ID, solid support
 Gas Chrom Q (100/120 mesh)

-------
                                   7-12
REFERENCES
(1)  Goerlitz, D. G., and Lamar- W. L., "Determination of Phenoxy
     and Herbicides in Water by Electron-Capture and Microcoulometric
     Gas Chromatography", U. S. Geol. Survey Water-Supply Paper
     1817-C (1967).

(2)  "Methods for Organic Pesticides in Water and Wastewater", (1971)
     U. S. Environmental Protection Agency, National Environmental
     Research Center, Cincinnati, Ohio.

(3)  "Handbook for Analytical Quality Control in Water and Wastewater
     Laboratories" (1972), U. S. Environmental Protection Agency,
     National Environmental Research Center, Analytical Quality Control
     Laboratory, Cincinnati, Ohio 45268.

(4)  Metcalf, L. D., and Schmitz, A. A., "The Rapid Preparation of
     Fatty Acid Esters for Gas Chromatographic Analysis", Analytical
     Chemistry, 33, 363 (1961).

(5)  Schlenk, H. and Gellerman, J. L., "Esterification of Fatty Acids
     with Diazomethane on a Small Scale", Analytical Chemistry, 32,
     1412 (1960).                                               ~

(6)  "Pesticide Analytical Manual", U. S. Department of Health, Education,
     and Welfare, Food and Drug Administration, Washington, D.C.

(7)  Steere, N. V., editor, "Handbook of Laboratory Safety," Chemical
     Rubber Company, 18901 Cranwood Parkway, Cleveland, Ohio 44128,
     1971, pp. 250-254.

-------
                                 7 -  1
                               APPENDIX I




Diazomethane in ether (6).




1.  CAUTIONS.  Diazomethane is very toxic.  It can explode under certain




conditions.  The following precautions should be observed.




Avoid breathing vapors.




Use only in well-ventilated hood.




Use safety screen.




Do not pipette solution of diazomethane by mouth.




For pouring solutions of diazomethane, use of gloves is optional.




Do not heat solutions to 100 C (EXPLOSIONS).




Store solutions of gas at low temperatures (Freezer compartment of explosion




proof refrigerators).




Avoid ground glass apparatus, glass stirrers and sleeve bearings where




grinding may occur (EXPLOSIONS).




Keep solutions away  from alkali metals (EXPLOSIONS).




Solutions of diazomethane decompose rapidly in presence of solid material




such as copper powder; calcium chloride, boiling stones, etc.  These solid




materials cause solid polymethylene and nitrogen gas to form.




2.  PREPARATION.




     Use a well-ventilated hood and cork stoppers for all connections.




Fit a 125 ml long-neck distilling flask with a dropping funnel and an




efficient condenser set  downward for distillation.  Connect the condenser




to two receiving flasks  in series   a 500 ml Erlenmeyer followed by a




125 ml Erlenmeyer containing 30 ml ether.  The inlet to the 125 ml Erlenmeyer




should dip below the ether.    Cool both receivers to 0 C.




     As water bath for the distilling flask,  set up a 2-liter beaker on  a




stirplate (hot plate and stirrer), maintaining temperature at 70 C.

-------
                                    7-2
     Dissolve 6 g KOH in 10 ml water in the distilling flask (no  heat).

Add 35 ml Carbitol (diethylene glycol monoethyl  ether),  stirring bar, and

another 10 ml ether.  Connect the distilling  flask  to  the  condenser and

immerse distilling flask in water bath.  By means of the dropping funnel,

add a solution of 21.5 g Diazald in 140 ml ether over  a  period of 20 minutes.

After distillation is apparently complete, add another 20  ml ether and

continue distilling until distillate is colorless.  Combine  the contents  of

the two receivers in a glass bottle (WITHOUT  ground glass  neck),  stopper

with cork, and freeze overnight.  Decant the  diazomethane  from the ice

crystals into a glass bottle, stopper with cork, and store in freezer until

ready for use.  The final solution may be stored up to six months without

marked deterioration.

     The 21.5 g of Diazald reacted in this manner produce  about 3 g of

Diazomethane.
                                                  * Ui. OOrtWMWTPSimiKOfTICtmJ- 759-555/1151

-------
    8-    A METHOD FOR ORGANOCHLORINE SOLVENTS IN INDUSTRIAL EFFLUENTS


 1.  Scope and Application


     1.1  This method describes a direct aqueous-injection  (1) (2} procedure

    M
    t .
          for the determination of gas chromatographable chlorinated hydro-
     •
    -P
    PH     carbons.  The method is specific for hydrocarbons containing chlorine,


    £     iodine, and bromine.  It is sensitive to approximately 1 mg/1.  The
«?  uj  0*     compounds detected are composed of carbon, the above mentioned halo-
 j  Q—  i^-
— '  _   f+
1 1 1  Q-
    -  jJr~-2  The method is useful only  for organo-halide compounds with a water


          solubility exceeding 1 mg/1 @ 22°C.  Many commonly used organo-

    
-------
                                  8-2



    3.3  The sample is  best preserved by protecting it from phase separation.



         Since the majority of the chlorinated solvents are volatile and



         relatively insoluble in water,  it is  important that the sample bottle



         be filled completely to minimize air  space over the sample.  The



         sample must remain hermetically sealed up to the time it is analyzed.



         Refrigeration  or freezing only  encourages phase separation and



         should be avoided.  Acidification will minimize the formation of non-



         volatile salts formed from chloroorganic acids and certain chloro-



         phenols.  However, it may interfere with the detection of acid



         degradable compounds such as chloroesters.   Therefore, the sample



         history must be known before any chemical or physical preservation



         steps can be applied.  To insure sample integrity, it is best to



         analyze the sample within 1 hour of collection.



4.  Interferences



    4.1  The use of a halogen specific detector eliminates  any possibility



         of interference from compounds  not containing chlorine,  bromine, or



         iodine.  Compounds containing bromine or iodine will  interfere with



         the determination of organochlorine compounds.   The use  of two dis-



         similar chromatographic columns helps to minimize  this interference



         and in addition this procedure  helps  to verify all qualitative



         identifications.   When concentrations are sufficiently high,  unequivo-



         cal identifications can be  made using infrared or  mass spectroscopy.



         Though non-specific, the flame  ionization detector may be used for



         known systems  where interferences are not a problem.



    4.2  Ghosting is usually attributed  to the history of the  chromatographic



         system.  Each  time a sample is  injected small amounts of various com-



         pounds are adsorbed on active sites in the inlet and  at  the head of

-------
                                  8,3




         the column.   Subsequent injections  of  water  tend to  steam clean




         these sites  resulting in non-representative peaks or  displacement




         of the baseline.   This phenomenon! normally occurs when  an  analysis




         of a series  of highly concentrated  samples is followed  by  a low




         level analysis.  The system should  be checked for ghost peaks prior




         to each quantitative analysis  by injecting distilled  water in a




         manner identical  to the sample analysis (5).   If excessive ghosting




         occurs, the  following maintenance should be applied,  as required,




         in the order listed:




         1)  Multiple flushes with distilled water




         2)  Clean or replace the glass injector liner




         3)  Replace  the chromatographic column




5.   Apparatus and Materials




    5.1  Gas Chromatograph   Equipped with programmed  oven temperature




         controls and glass-lined injection  port.  The oven should  be equipped




         with a column exit port and heated  transfer line for  convenient




         attachment to the halogen specific   detector.




    5.2  Detector Options:




         5.2.1  Microcoulometric Titration




         5.2.2  Electrolytic Conductivity




         5.2.3  Flame lonization




    5.3  Recorder - Potentiometric strip chart recorder (10 in)  compatible




         with the detector.




    5.4  Syringes - 1 yl,  10 yl, and 50 yl.




    5.5  BOD type bottle or 1 quart bottle with Teflon lined screw  cap.




    5.6  Volumetric Flasks - 500 ml, 1000 ml.




    5.7  Syringe   Hypodermic Lur-lock  type  (30 mi).




    5.8  Filter glass fiber filter - Type A  (13 mm).

-------
                                  8  -4





    5.9    Filter holder  -  Swinny  type hypodermic adapter (13  mm).



    5.10   Glass stoppered  ampuls  -  10 ml



          5.10.1   Non-Polar Column  -  12 ft x 0.1 in ID x 0.125 in OD



                  stainless steel column #304 packed with 5%  OV-1 on chromo-



                  sorb-W (60-80 mesh).



          5.10.2   Moderately-Polar  Column - 23 ft x 0.1 in ID x 0.125 in



                  OD stainless  steel  column #304 packed with  5% carbowax



                  20 M on  Chromosorb-W (60-80 mesh).



          5.10.3   Highly-Polar  Column - 23 ft x 0.1 in ID x 0.125 in OD



                  stainless steel #304 packed with 5% l,2,3-Tris-(2-cyano-



                  ethoxy)  propane on  Chromosorb-W (60-80 mesh).



          5.10.4   Porous Polymer  Column - 6 ft x 0.1 in ID x  0.125 in OD



                  stainless steel #304 packed with Chromosorb-101 (60-80 mesh).



6.  Reagents



    6.1   Chlorinated hydrocarbons  reference standards



          6.1.1    Prepare  standard  mixtures in volumetric flasks using con-



                  taminant free distilled water as solvent.   Add a known



                  amount of the chlorinated compounds with a  microliter syringe.



                  Calculate the concentration of each component as follows:




            mg/1  = (Density of  Compound) (yl injected) |>-. ,   .	^—.	7—rJ
                                 r                  (Dilution Volume (ml)J



7.  Quality Control



    7.1   Duplicate quantitative  analysis on dissimilar columns should be



          performed.   The  duplicate quantitative data should  agree within



          experimental error (±6  percent).   If not, analysis  on a third



          dissimilar column should  be performed.  Spiked sample analyses



          should  be routinely performed to insure the integrity of the method.

-------
                                  8 -  5





8.   Selection Gas Chromatographic Column




    8.1   No single column can efficiently resolve all chlorinated hydrocarbons.




          Therefore, a specific column must be selected to perform a given




          analysis.  Columns providing only partially or non-resolved peaks




          are useful only for confirmatory identifications.  If the qualitative




          nature of the sample is known then an efficient column selection can




          be made by reviewing literature (4).  In doing this,  one must remember




          that injection of large volumes of water can cause two serious




          problems not normally noted using common gas chromatographic tech-




          niques :




          1)  Water can cause early column failure due to liquid phase




              displacement.




          2)  Water passing through the column causes retention times and




              orders to change when compared to common sample solvent media,




              ie., hexane or air.




              For these reasons column life and the separations obtained by




              direct aqueous injection may not be identical to  those suggested




              in literature.




    8.2   If nothing is known about the sample, a thermally stable non-polar




          column such as the OV-1 column is a good first choice.  Temperature




          programming this column from room temperature to its  upper limit




          will provide a wide molecular weight range analysis.   Following this




          with a moderately polar column and a highly polar column provides




          efficient separations and corroborative identifications for a wide




          variety of common chlorinated solvents.  The unique low molecular




          weight separations achieved on porous polymer columns is extremely




          useful when the samples contain a mixture of such compounds.

-------
                                   8 -  6

 9.   Sample  Preparation

     9.1    If the sample is  turbid it  should be filtered or contrifuged to

          prevent syringe plugging or excessive  ghosting problems.   Filtering

           the sample is accomplished  by filling a 30 ml hypodermic syringe

           with sample and attaching the Swinny type hypodermic filter adaptor

           with a glass  fiber filter "Type A" installed.  Discard the first

           5 ml of sample then collect the filtered sample in a glass stoppered

           ampule filled to  the top.   (One should occasionally analyze the

           non-filtered sample to insure that the filtering technique does not

           adversely effect  the sample).

10.   Method of Analysis

     10.1  First, analyze the filtered sample of unknown composition  by in-

           jection of a 3 to 10 ul into  the gas chromatograph.   The injection

           volume and detector sensitivity is recorded.

     10.2  Prepare a standard mixture  consisting of the  same compounds in con-

           centrations approximately equal to those detected in the sample.

           Chromatograph the standard  mixture under conditions identical to the

           unknown.

11.   Calculation of Results

     11.1  Measure the area  of each unknown peak and each reference standard

           peak as follows:

           Area = [Peak  Height][Width  of Peak at 1/2 Height]

     11.2  Calculate the concentration of each unknown as follows:

       ,j _  (Area of Sample  peak)(yil of Standard Injected) (Cone1 n of  Standard)
      g   ~         (ul of Sample Injected)(Area of Standard Peak)

12.   Reporting Results

     12.1  Report results in mg/1.  If a result is negative, report the minimum

           detectable limit, ie. <1 mg/1.  When duplicate and spiked  samples

           are analyzed, all data obtained should be reported.

-------
                                  8 -  7






References




1.  "Tentative Recommended Practice for Measuring Volatile Organic Matter in




    Water by Aqueous - Injection Gas Chromatography", D2908-70T, 1971




    Annual Book of ASTM Standards, Part 23, Water; Atmospheric Analysis,




    American Society for Testing and Materials, 1916 Race Street, Philadelphia,




    Pennsylvania  19103.




2.  Bellar, T. A. and Lichtenberg, J. J., "Method for the Determination of




    Chlorinated Organic Solvents by Direct Aqueous   Injection Gas Chromatography",




    U. S. Environmental Protection Agency, National Environmental Research




    Center, Cincinnati., Ohio  45268  (March 1973).




3.  "Handbook of Chemistry and Physics", 48th Edition, The Chemical Rubber




    Company, 18901 Cranwood Parkway, Cleveland, Ohio  44128.  (1967-1968)




4.  "Gas Chromatography Abstracts", Knapman, C.E.H., Editor, Institute of




    Petroleum, 61 New Cavendish Street, London W1M8AR, Annually 1958 to date,




    since 1970, also includes Liquid Chromatography Abstracts.




5.  Dressman, R. C., "Elimination of Memory Peaks Encountered in Aqueous-




    Injection  Gas Chromatography", Journal of Chromatographic Science, 8^




    265  (1970).

-------
                          I  I I  1 I 1 I I I  I  I
                              16
24
32
                      RETENTIOH TIME IN MINUTES
       Figure 1. Column: Chromosorb-101, Temperature Program: 125C
       for 4 min then 4C/min  up to 280 C., Carrier Gas: Nitrogen at
       36 ml/min,  Detector: Microcoulometric.
ft u.s. IIMMCITMMTUBomct 1973- 759-555/1152

-------
                           ANTIMONY
                                                   STORE! NO:
                    (Standard Conditions)             TOTAL   : 01097

Optimum Concentration Range:   1-40 mg/1 using a wavelength of 217.6 nm

Sensitivity:  0.3 mg/1

Detection Limit:   0.2 mg/1

Preparation of Standard Solution:

     1.  Stock Solution:  Carefully  weigh  2.7426 grams of antimony

         potassium tartrate (analytical reagent grade) and dissolve

         in distilled water.   Dilute to 1  liter with distilled water.

         One ml equals 1 mg Sb (1000 mg/1).

     2.  Prepare dilutions of the  stock solution to be used as cali-

         bration standards at the  time of  analysis.

Sample Preparation:

     1.  The procedure for the determination of total metals as given

         in "Methods for Chemical  Analysis of Water and Wastes, 1971"

         (p 88, 4.1.3) has been found to be satisfactory.

Instrumental Parameters (General):

     1.  Antimony hollow cathode lamp.

     2.  "Wavelength:  217.6 nm

     3.  Type of burner:  Boling.

     4.  Fuel:  Acetylene

     5.  Oxidant:  Air

     6.  Type of flame:  Fuel rich

     7.  Photomultiplier tube:  R106

                                        NAT!P:'M.  POLLUTANT

                                      n"?' ;•• •  "   . ' :.:-;;uATION
                                      \j i *.' •* i ' * •

                                        SYSFEffi,  APPENDIX  A

                                  Fed.  Res-,  38-  No-  75»

-------
                      ANTIMONY (continued)


Interferences:

     1.   The presence of high dissolved solids in the sample may

         result in an interference from non-atomic absorbance such

         as light scattering.  If background correction is not avail-

         able,  a non-absorbing wavelength should be checked.
                                                  i
     2.   In the presence of lead (1000 mg/1), a spectral interference

         may occur at the 217.6 nm resonance line.  In this case the

         231.1  nm antimony line should be used.

     3.   Increasing acid concentrations decrease antimony absorption.

         To avoid this effect, the acid concentrations in the samples

         and in the standards should be matched.

Notes:

     1.   Where  the sample matrix is so complex that viscosity, surface

         tension, and components cannot be accurately matched with

         standards, the method of standard addition must be used.

     2.   Data to be entered into STORET must be reported as yg/1.
                                                                     3/73

-------
                           BARIUM
                                                      STORE! NO:
                    (Standard Conditions)               TOTAL   :  01007

Optimum Concentration Range  0.5-20 mg/1 using a wavelength of 553.6 ran

Sensitivity  0.2 mg/1

Detection Limit  0.03 mg/1

Preparation of Standard Solution

     1.  Stock Solution:  Dissolve 1.7787 g barium chloride (BaCl2'2H-20,

         analytical reagent grade) in distilled water and dilute to 1

         liter.  One ml equals 1 mg Ba.

     2.  Potassium chloride solution:  Dissolve 95g potassium chloride,

         KC1, in distilled water and make up to 1 liter.

     3.  Prepare dilutions of the stock barium solution to be used as

         calibration standards at the time of analysis.  To each 100 ml

         of standard and  sample alike add 2.0 ml potassium chloride

         solution.

 Sample  Preparation

     1.  The procedure  for the determination of total metals as given  in

         "Methods for Chemical Analysis of Water and Wastes", 1971  (p  88

         4.1.3) has been  found to be satisfactory.

 instrumental Parameters  (General)

     1.  Barium hollow  cathode lamp

     2.  Wavelength:  553.6 nm

     3.  Type of burner:  Nitrous oxide

     4.  Fuel:  Acetylene

     5.  Oxidant:   Nitrous oxide

     ti.  Type of  flame:   Fuel rich

     7.  PhctomultLplier  tube:  1P28

-------
                       BARIUM (continued)



Interferences




     1.   The presence of high dissolved solids in the sample may




         result in an interference from non-atomic absorbance




         such as light scattering.  If background correction is




         not available, a non-absorbing wavelength should be




         checked.




     2.   The use of a nitrous oxide-acetylene flame virtually




         eliminates chemical interference; however, barium is




         easily ionized in this flame and potassium must be added




         (1000 mg/1) to standards and samples alike to control




         this effect.




     3.   If the nitrous oxide flame is not available and acetylene-




         air is used, phosphate, silicon, and aluminum will severely




         depress the barium absorbance.  This may be overcome by the




         additon of 2000 mg/1 lanthanum.




Notes




     1.   Where the sample matrix is so complex that viscosity, surface




         tension, and components cannot be accurately matched with




         standards, the method of standard addition must be used.




     2.   Data to be entered into STORET must be reported as ug/1.
                                                                        3/73

-------
                            BERYLLIUM
                                                         STORE! NO:
                      (Standard Conditions)                TOTAL   :  01012

Optimum Concentration Range:  0.02-1.5 mg/1 using a wavelength of  234.9 niii

Sensitivity:  0.007 mg/1.

Detection Limit:  0.002 mg/1.

Preparation of Standard Solution:

     1.  Stock solution:  Dissolve 11.6586 g beryllium sulfate,

         BeSO., in distilled water containing 2 ml cone, nitric acid

         and dilute to  1 liter.  One ml equals 1 mg Be.

     2.  Prepare dilutions of the stock solution to be used as

         calibration standards at the time of analysis.  Maintain

         an acid strength of 0.15% nitric acid in all calibration

         standards.

Sample Preparation:

     1.  The procedure  for the determination of total metals as given

         in "Methods for Chemical Analysis of Water and Wastes, 1971"

         (p 88, 4.1.3)  has been found to be satisfactory.

Instrumental Parameters  (General):

     1.  Beryllium hollow cathode lamp

     2.  Wavelength:  234.9 nm

     3.  Type of burner:  Nitrous oxide

     4.  Fuel:  Acetylene

     5.  Oxidant:  Nitrous oxide

     6.  Type of fl.-imc:  Fuel rich

     7.  Photomultiplier tube R 106

-------
                       BERYLLIUM (Continued)




Interferences:



     1.  The presence of high dissolved solids in the sample may



         result in an interference from non-atomic absorbance such



         as light scattering.  If background correction is not



         available, a non-absorbing wavelength should be checked.



     2.  Sodium and silicon at concentrations in excess of 1000 mg/1



         have been found to severely depress the beryllium absorbance.



     3.  Bicarbonate ion is reported to interfere, however, its effect



         is eliminated when samples are acidified to a pH of 1.5.



Notes:



     1.  Where the sample matrix is so complex that viscosity, surface



         tension, and components cannot be accurately matched with



         standards, the method of standard addition must be used.



     2.  Data to be entered into STORE! must be reported as yg/1.
                                                                  3/73

-------
                          BORON
                                                    STORE! NO:
                     (Curcumin Method)'                TOTAL  :  01022

1.  -Scope and Application

    1.1  This colorimetric method finds maximum utility for waters

         whose boron content is below 1 mg/1.

    1.2  The optimum range of the method on undiluted or unconcen-

         trated samples is 0.1-1.0 mg/1 of boron.

2.   Summary of Method

    1.1  When a sample of water containing boron is acidified and

         evaporated in the presence of curcumin, a red-colored product

         called rosocyanine is formed.  The rosocyanine is taken up

         in a suitable solvent, and the red color is compared with

         standards either visually or photometrically.

3.   Comments

    3.1  Nitrate nitrogen concentrations above 20 mg/1 interfere.

    3.2  Significantly high results are possible when the total of

         calcium and magnesium hardness exceeds  100 mg/1 as CaCO^.

         Passing the sample through a cation exchange resin eliminates

         this problem.

    3.3  Close control of such variables as volumes and concentrations

         of reagents, as well as time and temperature of drying, must

         be exercised for maximum accuracy.

4.   Precision and Accuracy

    4/1  A synthetic unknown sample containing 240 pg/1 B, 40 yg/1 As,

         250 ug/1 Be, 20 yg/1 Se, and 6 pg/1 V in distilled water was

-------
                        BORON (continued)





         determined by the curcumin method with a relative standard




         deviation of 22.8% and a relative error of 0% in 30




         laboratories.




5.  Reference




    5.1  The procedure to be used for this determination is found




         in:  Standard Methods for the Examination of Water and




         Wastewater, 13th Edition, p 69, Method 107A (1971).




6.  Data to be entered into STORE! must be reported as ug/1.
                                                                       3/73

-------
                           COBALT
                                                       STORE! NO:
                    (Standard Conditions}                TOTAL   : 01037

Optimum Concentration Range:  0.2-7 mg/1 using a wavelength of 240.7 nm

Sensitivity: TJ.05 mg/1

Detection Limit;  ILD2 mg/1

Preparation of Standard Solution

     1.  Stock Solution:  Dissolve 4.037 grams of cobaltous chloride,

         CoCl2 • 6H?0  (analytical reagent grade) in distilled water.

         Add 10 ml of concentrated nitric acid and dilute to 1 liter

         with distilled water.  One ml equals 1 mg Co  (1000 mg/1).

     2.  Prepare dilutions of the stock cobalt solution to be used as

         calibration standards at the time of analysis.  Maintain an

         acid strength of 0.15% nitric acid in all calibration

         standards.

Sample Preparation

     1.  The procedure for  the determination of total metals as  given

         in "Methods for Chemical Analysis of Water and Wastes", 1971

         (p 88, 4.1.3) has been found to be satisfactory.

Instrumental Parameters  (General)

     1.  Cobalt hollow cathode lamp.

     2.  Wavelength:   240.7 nm

     3.  Type of burner:  Boling

     4.  Fuel:  Acetylene

     5.  Oxidant:  Air

     6.  Type of Flame:  Stoichiometric

     7.  Photomultiplier tube:  R-106

-------
                       COBALT  (continued)




Interferences




     1.  The presence of high dissolved solids in the sample may




         result in an interference from non-atomic absorbance




         such as light scattering.  If background correction is




         not available, a non-absorbing wavelength should be




         checked.




     2.  Interference from high concentrations (1000 mg/1) of




         calcium,  aluminum, potassium, magnesium, phosphate,




         sulfate,  nitrate and silicate amy be observed.   The use




         of the nitrous oxide-acetylene flame will lessen this




         interference with some loss of sensitivity.




Notes




     1.  Where the sample matrix is so complex that viscosity,




         surface tension, and components cannot be accurately




         matched with standards, the method of standard  addition




         must be used.




     2.  With the exception of certain samples and/or effluents




         containing high levels of extractable metals, the APDC-MIBK




         extraction technique should be used for concentrations




         below 20 ug/1.




     3.  Data to be entered into STORET must be reported as ug/1.
                                                                      3/73

-------
                         MOLYBDENUM

                                                     STORE! NO:

                      (Standard Conditions)            TOTAL   :   01062



Optimum Concentration Range  0.4-20 mg/1 using a wavelength  of  313.3 nm .,



Sensitivity  0.1 mg/1



Detection Limit  0.03 mg/1



Preparation of Standard Solution



     1.  Stock Solution:  Dissolve 1.840 grams of  ammonium molybdate



         (NH.), Mo_00.-4H_0  (analytical reagent grade)  in distilled
            4 D    /  Z*4   2.


         water and dilute to 1 liter.  One  ml  equals 1  mg Mo (1000 mg/1).



     2.  Prepare, dilutions of  the  stock molybdenum solution  to  be  used



         as call br.-itKji: standards  at  the  time  of analysis.



Sample Preparation



     1.  The procedure  for the determination of  total metals as given



         in "Methods for Chemical  Analysis  of  Water and Wastes", 1971



         (p 88, 4.1.3)  has been  found to  be satisfactory.



Instrumental Parameters  (General)



     J.  Molybdenum  hollow cathode lamp



     2.  Wavelength:  313.3  nm



     3.  Type of burner:   Nitrous  oxide



     4.  Fuel:  Acetylene



     5.  Oxidant:   Nitrous Oxide



     (i.  Type of  flame:   Fuel  rich



     7.  I'hutomultiplier  tube:   1P28



 ; n i i •rJ'ej'ijiCAV-:



      1.   The  presence of  high dissolved solids in  the  sample may  result



          in an interference  from non-atomic absorbance such as  light



          scdLtcring.  If  background correction is  not  available,  a

-------
                    MOLYBDENUM (continued)




         non-absorbing wavelength should be checked.




     2.  With the recommended nitrous oxide-acetylene flame,




         interferences may be suppressed by adding 1000 mg/1




         of a refractory metal such as aluminum.  This should




         be done to both samples and standards alike.




Notes




     1.  Where the sample matrix is so complex that viscosity,




         surface tension, and components cannot be accurately




         matched with standards, the method of standard addition




         must be used.




     2.  For low levels of molybdenum an oxine extraction procedure




         may be useful.  (Ref:  Chau et.al.,  Anal. Chem.  Acta ^




         205, 1969).




     3.  Data to be entered into STORE! must  be reported  as ug/1.
                                                                         3/73

-------
                           NICKEL
                                                      STORE! NO:
                     (Standard Conditions)              TOTAL   :  01067

Optimum Concentration Range  0.2-7 mg/1 using a wavelength of 232.0 nm

Sensitivity  0.05 mg/1

Detection Limit  0.01 mg/1

Preparation of Standard Solution

     1.  Stock Solution:  Dissolve 4.953 grams of nickel nitrate,

         Ni(NO ) '6H_0  (analytical reagent grade) in distilled

         water.  Add 10 ml of concentrated nitric acid and dilute

         to 1 liter with distilled water.  One ml equals 1 mg Mi

         (1000 mg/1).

     2.  Prepare dilutions of the stock nickel solution to be used

         as calibration standards at  the time of analysis.  Main-

         tain an acid strength of 0.15% nitric acid in all calibration

         standards.

Sample Preparation

     1.  The procedure  for the determination of  total metals as given

         in "Methods for Chemical Analysis of Water and Wastes", 1971

         (p 88, 4.1.3)  has been  found  to be satisfactory.

Lnstrumental Parameters  (General)

     1.  Nickel hollow  cathode lamp.

     2.  Wavelength:  232.0 nm

     3.  Type of burner:  Boling

     4.  Fuel:  Acetylene

     5.  Oxidant:  Air

     (S.  Tvpe of Flame:  Oxidizing

     '/-  r 1101 omul L iplier Tube:   K 106

-------
                     NICKEL (continued)




Interferences




     1.   The presence of high dissolved solids in the sample may result




         in an interference from non-atomic absorbance such as light




         scattering if background correction is not available, a




         non-absorbing wavelength should be checked.




     2.   The 352.4 nm wavelength is Less susceptible to non-atomic




         absorbance and may be used.  The calibration curve is more




         linear at this wavelength; however, there is some loss of




         sensitivity.




     3.   Interference from high concentrations (1000 mg/1) of calcium,




         aluminum, potassium, magnesium, phosphate, sulfate, nitrate




         and silicate may be observed.  The use of the nitrous oxide-




         acetylene flame will lessen this interference with some loss




         of sensitivity.




Notes




     1.   Where the sample matrix is so complex that viscosity, surface




         tension, and components cannot be accurately matched with




         standards, the method of standard addition must be used.




     2.   With the exception of certain samples and/or effluents con-




         taining high levels of extractable metals, the APDC-MIBK




         extraction technique should be used for concentrations below




         20 yg/1




     3.   Data to be entered into STORET must be reported as ug/1.
                                                                          3/73

-------
                           SILVER
                                                    STORE! NO:
                    (Standard Conditions)             TOTAL   :  01077

Optimum Concentration Range  0.1-20 mg/1 using a wavelength of 328.1 nm.

Sensitivity  0.05 mg/1

Detection Limit   0.01 mg/1

Preparation of Standard Solution

     1.  Stock Solution:  Dissolve 1.575 g of AgNO_ (analytical reagent

         grade) in distilled water, add 10 ml HNO~ and make up to 1

         liter.  One ml equals 1 nig of silver (1000 mg/1).

     2.  Prepare diltuions of the stock solution to be used as calibra-

         tion standards at the time of analysis.  Maintain an acid

         strength of 0.15% HNO  in all calibration standards.

Sample Preparation

     1.  The procedure  for the determination of  total metals  as given

         in "Methods for  Chemical Analysis of Water and Wastes", 1971

         (p 88, 4.1.3)  has been found  to be satisfactory.  The residue

         must be taken  up in dilute nitric acid  rather than hydrochloric

         to prevent precipitation of AgCl.

Instrumental Parameters  (General)

     1.  Silver hollow  cathode lamp

     2.  Wavelength:   328.1 nm

     3.  Type of burner:   Boling

     4.  Fuel:  Acetylene

     5.  Oxidant:  Air

     6.  Type of Flame:   Oxidizing

     7.  i'ho tomu 11 i plier  tube:  1P28

-------
                      SILVER (continued)





Interferences




     1.  The presence of high dissolved solids in the sample may result




         in an interference from non-atomic absorbance such as light




         scattering.  If background correction is not available, a




         non-absorbing wavelength should be checked.




     2.  Interference from high concentrations (1000 mg/1) of calcium,




         aluminum, potassium, magnesium, phosphate,  sulfate, nitrate




         and silicate may be observed.  The use of the nitrous oxide-




         acetylene flame will lessen this interference with some loss




         of sensitivity.




Notes




     1.  Where the sample matrix is so complex that  viscosity, surface




         tension, and components cannot be accurately matched with




         standards, the method of standard addition  must be used.




     2.  Silver nitrate standards are light sensitive.  Dilutions  of




         the stock should be discarded after use  as  concentrations




         below 10 mg/1 are not stable over long periods of time.




     3.  The 338.2 nm wavelength may also be used.   This has a relative




         sensitivity of 3.
                                                                      3/73

-------
                          THALLIUM
                                                      STORE! NO:
                    (Standard Conditions)               TOTAL   :  01059

Optimum Concentration Range  1-20 mg/1 using a wavelength of 276.8 nm

Sensitivity    0.2 mg/1

Detection Limit  0.05 mg/1

Preparati-jn of Standard Solution

     1.  Stock Solution:  Dissolve 1.303 grams of thallium nitrate,

         T1NO~ (analytical reagent grade) in distilled water.  Add

         10 ml of concentrated nitric acid and dilute to 1 liter with

         distilled water.  One ml equals 1 mg Tl  (1000 mg/1).

     2.  Prepare dilutions of the stock  thallium  solution to be used

         as calibration standards at  the time of  analysis.  Maintain

         an acid strength of 0.15% nitric acid in all calibration

         standards.

Sample Preparation

     1.  The procedure  for the determination of total metals as given

         in "Methods  for Chemical Analysis of Water and Wastes", 1971

         (p 88, 4.1.3)  has been  found to be satisfactory.

Instrumental Parameters  (General)

     1.  Thallium hollow cathode lamp.

     2.  Wavelength:  276.8 nm

     3.  Type  of burner:  Boling

     4.  Fuel:  Acetylene

     5.  Oxidant:  Air

     6.  Type  of flame:  Stoichiometric

     7.  Photomultiplier tube: R 106

-------
                     THALLIUM  (continued)




Interferences




     1.  The presence of high dissolved solids in the sample may




         result in an interference from non-atomic absorbance




         such as light scattering.  If background correction is




         not available, a non-absorbing wavelength should be




         checked.




     2.  Interference from high concentrations (1000 mg/1) of




         calcium, aluminum, potassium, magnesium, phosphate, sulfate,




         nitrate, and silicate may be observed.  The use of the nitrous




         oxide-acetylene flame will lessen this interference with some




         loss of sensitivity.




Notes




     1.  Where the sample matrix is so complex that viscosity, surface




         tension, and components cannot be accurately matched with




         standards, the method of standard addition must be used.




     2.  Data to be entered into STORET must be reported as ug/1.
                                                                         3/73

-------
                             TIN
                                                        STORET NO:
                    (Standard Conditions)                 TOTAL   :  01102

Optimum Concentration Range:  10-200 mg/1 using a wavelength of 235.5 run.

Sensitivity:  2 mg/1

Detection Limit:  0.4 mg/1

Preparation of Standard Solution:

     1.  Stock Solution:  Dissolve 1.000 gram of tin metal (analytical

         reagent grade) in 100 ml of concentrated HC1 and dilute to 1

         liter with distilled water.  One ml equals 1 mg Sn (1000 mg/1).

     2.  Prepare dilutions of the stock tin solution to be used as

         calibration standards at the time of analysis.  Maintain an

         acid concentration of 10% HC1 in all solutions.

Sample Preparation:

     1.  The procedure for the determination of total metals as given

         in "Methods for Chemical Analysis of Water and Wastes, 1971"

         (p 88, 4.1.3) has been found to be satisfactory.

Instrumental Parameters (General):

     1.  Tin hollow cathode lamp

     2.  Wavelength:  235.5 nm

     3.  Type of burner:  Boling

     4.  Fuel:  Acetylene

     5.  Oxidant:  Air

     6.  Type of flame:  Fuel rich

     7.  Photomultiplier tube:  R 106

-------
                      TIN (Continued)




Interferences:



     1.   The presence of high dissolved solids in the sample may



         result in an interference from non-atomic absorbance such



         as light scattering.  If background correction is not



         available, a non-absorbing wavelength should be checked.



     2.   Interference from high concentrations (1000 mg/1) of



         calcium, aluminum,  potassium, magnesium, phosphate, sulfate,



         nitrate and silicate may be observed.  The use of the



         nitrous oxide-acetylene flame will lessen this interference



         with some loss of sensitivity.



Notes:



     1.   Where the sample matrix is so complex that viscosity, surface



         tension, and components cannot be accurately matched with



         standards, the method of standard addition must be used.



     2.   Data to be entered  into STORET must be reported as yg/1.
                                                                 3/73

-------
                          TITANIUM
                                                       STORE! NO:
                     (Standard Conditions)               TOTAL   :  01152

Optimum Concentration Range  2-100 mg/1 using a wavelength of 364.3 nm

Sensitivity  1.0 mg/1

Detection Limit  0.3 mg/1

Preparation of Standard Solution

     1.  Stock Solution:  Dissolve 4.008 grams of titanium sulfate

         (Ti   (SO.) ) in dilute HC1 and make up to 1 liter with

         distilled water.  One ml equals 1 mg Ti (1000 mg/1).

     2.  Potassium chloride solution:  Dissolve 95g potassium

         chloride, KC1 in distilled water and make up to 1 liter.

     3.  Prepare dilutions of the stock titanium solution to be

         used as calibration standards at the time of analysis.

         To each 100 ml of standard and sample alike, add 2 ml of

         potassium chloride solution.

Sample Preparation

     1.  The procedure for the determination of total raetals as

         given in "Methods for Chemical Analysis of Water and Wastes",

         1971  (p 88, 4.1.3) must be modified by the addition of  3 ml

         of concentrated sulfuric acid in addition to the nitric acid.

         This is necessary to keep any titanium that may be present

         in solution.

Instrumental Parameters (General)

     1.  Titanium hollow cathode lamp

     2.  Wavelength:  365.3 nm

     3.  Type of burner:  Nitrous Oxide

-------
                    TITANIUM (continued)




     4.  Fuel:   Acetylene




     5.  Oxidant:   Nitrous Oxide




     6.   Type of flame:  Fuel rich




     7.   Photomultiplier:  1P28




Interferences




     1.   The presence  of high dissolved solids ir. the ?3-ple ~ay




         result in  an  interference  fror:. r.on-ato~ic j-bsorbar.ee




         such as  light scattering.   If background correction is




         not available,  a  non-absorbing wavelength should be




         checked.




     2.  Titanium is easily ionized in the flame and potassium




          (1000  mg/1) must  be added  to standards and samples alike




         to  control this effect.




Notes




     1.  Where  the  sample  matrix  is so complex that viscosity,




         surface  tension,  and components cannot be accurately




         matched  with  standards,  the method of standard addition




         must be  used.




     2.  Data to  be entered into  STORET must be reported as pg/1.
 
-------
                             CYANIDE,  Total







   ,                     "  -_                                STORET NO.  00720





1.   'Scope and Application




    1.1  This method is applicable to the determination of cyanide in surface




         waters, domestic and industrial wastes, and saline waters.



    1.2  The titration procedure using silver nitrate with p-dimethylamino-




         benzalrhodanine indicator is used for measuring concentrations of



         cyanide exceeding 1 mg/1 (0.2 mg/200 ml of absorbing liquid).




    1.3  The colorimetric procedure is used for concentrations below 1  mg/1



         of cyanide and is sensitive to about .02 mg/1.



2.   Summary of Method



    2.1  The cyanide as hydrocyanic acid (HCN) is released from metallic



         cyanide complex ions by means of a reflux-distillation operation



         and absorbed in a scrubber containing sodium hydroxide solution.



         The cyanide ion in the absorbing solution is then determined by



         volumetric titration or colorimetrically.




    2.2  In the colorimetric measurement the cyanide is converted to



         cyanogen chloride, CNC1, by reaction with chloramine-T at a pH



         less than 8 without hydrolyzing to the cyanate.  After the reaction




         is complete, the CNC1 forms a red-blue dye on the addition of  a



         pyridine-barbituric acid reagent.   The absorbance is read at 578 run.




         To obtain colors of comparable intensity, it is essential to have



         the same salt content in both the sample and the standards.



    2.3  The titrimetric measurement uses a standard solution of silver nitrate



         to titrate cyanide in the presence of a silver sensitive indicator.

-------
                                                               (Cyanide)



3.   Definitions



    3.1  Cyanide is defined as cyanide ion and complex cyanides converted



         to hydrocyanic acid (HCN) by reaction in a reflux system of a



         mineral acid in the presence of cuprous ion.



4.   Sample Handling and Preservation



    4.1  The sample should be collected in plastic bottles of 1 liter or



         larger size.  All bottles must be thoroughly cleansed and



         thoroughly rinsed to remove soluble material from containers.



    4.2  Samples must be preserved with 2 ml of 10 N sodium hydroxide per



         liter of sample (pH > 12) at the time of collection.



    4.3  Samples should be analyzed as rapidly as possible after collection.



         If storage is required, the samples should be stored in a refrig-



         erator or in an ice chest filled with water and ice to maintain



         temperature at 4°C.



    4.4  Oxidizing agents such as chlorine decompose most of the cyanides.



         Test a drop of the sample with potassium iodide-starch test paper



         (KI-starch paper); a blue color indicates the need for treatment.



         Add ascorbic acid, a few crystals at a time, until a drop of



         sample produces no color on the indicator paper.  Then add an



         additional 0.6 gram of ascorbic acid for each liter of sample



         volume.



5.   Interferences



    5.1  Interferences are eliminated or reduced by using the distillation



         procedure described in Procedure (8.1 through 8.5).



    5.2  Sulfides adversely affect the colorimetric and titration



         procedures.  If a drop of the sample on lead acetate test

-------
                                                                (Cyanide)






6.   Apparatus




    6.1  Reflux distillation apparatus such as shown in Figure 1 or Figure




         2.  The boiling flask should be of 1 liter size with inlet tube




         and provision for condenser.  The gas absorber may be a Fisher-




         Milligan scrubber.




    6.2  Microburet, 5.0 ml  (for titration).




    6.3  Spectrophotometer suitable for measurements at 578 nm with a 1.0




         cm cell or larger.




7.   Reagents




    7.1  Sodium hydroxide solution.  Dissolve 50 g of NaOH in distilled




         water, and dilute to a liter with distilled water.




    7.2  Cadmium carbonate.




    7.3  Ascorbic acid.




    7.4  Cuprous Chloride Reagent - Weigh 20 g of finely powdered Cu2Clo



         into an 800-ml beaker.  Wash twice,  by decantation, with 250-ml




         portions of dilute sulfuric acid (l^SC^, 1 + 49) and then twice




         with water.  Add about 250 ml of water and then hydrochloric acid




         (HC1, sp gr 1.19) in 1/2-ml portions until the salt dissolves




         (Note 1).   Dilute to 1 liter with water and store in a tightly




         stoppered bottle containing a few lengths of pure copper wire or




         rod extending from the bottom to the mouth of the bottle (Note 2).




              Note 1:   The reagent should be  clear; dark discoloration




         indicates the presence of cupric salts.




              Note 2:   If it is desired to use a reagent bottle of smaller




         volume, it should be kept completely filled and tightly stoppered.




         Refill it from the stock solution after each use.

-------
                                                            (Cyanide)






     paper indicates the presence of sulfides, treat 25 ml more of the



     stabilized sample (pH 2.12) than that required for the cyanide



     determination with powdered cadmium carbonate.  Yellow cadmium



     sulfide precipitates if the sample contains sulfide.  Repeat this



     operation until a drop of the treated sample solution does not



     darken the lead acetate test paper.  Filter the solution through



     a dry filter paper into a dry beaker, and from the filtrate,



     measure the sample to be used for analysis.  Avoid a large excess



     of cadmium and a long contact time in order to minimize a loss



     by complexation or occlusion of cyanide on the precipitated material.



5.3  Fatty acids will distill and form soaps under the alkaline titration



     conditions, making the end point almost impossible to detect.



     Fatty acids are removed by extraction as suggested by Kruse and



     Mellon.  Acidify the sample with acetic acid ( 1 + 9) to pH 6.0



     to 7.0.  (Caution—This operation must be performed in the hood



     and the sample left there until it can be made alkaline again



     after the extraction has been performed.)  Extract with iso-octane,



     hexane, or chloroform (preference in order named) with a solvent



     volume equal to 20 percent of the sample volume.   One extraction



     is usually adequate to reduce the fatty acids below the interference



     level.   Avoid multiple extractions or a long contact time at low



     pH in order to keep the loss of HCN at a minimum.  When the



     extraction is completed, immediately raise the pH of the sample



     to above 12 with NaOH solution.

-------
                                                             (Cyanide)






 7.5  Sulfuric acid, concentrated.




 7.6  Sodium dihydrogenphosphate, 1 M.  Dissolve 138 g of NaH2P04.H20



      in one liter of distilled water.  Refrigerate this solution.




 7.7  Stock cyanide solution.  Dissolve 2.51 g of KCN and 2 g KOH in




      one liter of distilled water.  Standardize with 0.0192 N AgNOs.



      Dilute to appropriate concentration so that 1 ml = 1 mg CN".




 7.8  Standard cyanide solution, intermediate.  Dilute 10 ml of stock




      (1 ml = 1 mg CN) to a liter of distilled water (1 ml = 10 yg).




 7.9  Standard cyanide solution.  Prepare fresh daily by diluting 100 ml




      of intermediate cyanide solution to a liter of distilled water




      and store in a glass stoppered bottle.  One ml = 1.0 ug CN (1.0




      mg/1).




7.10  Standard silver nitrate solution, 0.0192 N.  Prepare by crushing




      approximately 5 g AgNC>3 crystals and drying to constant weight at




      40°C.  Weigh out 3.2647 g of dried AgNO,, dissolve in water, and




      dilute to 1.0 liter (1 ml = 1 mg CN).




7.11  Rhodanine indicator.  Dissolve 20 mg of p-dimethylamino-benzal-




      rhodanine in 100 ml of acetone.




7.12  Chloramine T solution.  Dissolve 1.0 g of white water soluble




      Chloramine T in 100 ml of distilled water and refrigerate until




      ready to use.  Prepare fresh weekly.




7.13  Pyridine-Barbituric Acid Reagent.  Place 15 g of barbituric acid




      in a 250-ml volumetric flask and add just enough water to wash the




      sides of the flask and wet the barbituric acid.  Add 75 ml of




      pyridine and mix.   Add 15 ml of HC1 (sp gr 1.19), mix, and cool




      to room temperature.  Dilute to 250 ml with water and mix.

-------
ALLIHN CONDENSER —

AIR INLET
- CONNECTING TUBING
ONE LITER	
BOILING FLASK
                                    SUCTION
                   GAS ABSORBER
                 FIGURE 1
   CYANIDE DISTILLATION APPARATUS

-------
   COOLING WATER
   INLET TUBEv
SCREW CLAMP
     I
                                  TO LOW VACUUM
                                     SOURCE
                                 ABSORBER
                           DISTILLING FLASK
        HEATER-
                  O
             FIGURE 2
CYANIDE DISTILLATION  APPARATUS

-------
                                                                (.Cyanide)




8.   Procedure




    8.1  Place 500 ml of sample, or an aliquot diluted to 500 ml in the




         1-liter boiling flask.  Add 50 ml of sodium hydroxide  (7.1) to




         the absorbing tube and dilute if necessary with distilled water




         to obtain an adequate depth of liquid in the absorber.  Connect




         the boiling flask, condenser, absorber and trap in the train.




    8.2  Start a slow stream of air entering the boiling flask by adjusting




         the vacuum source.  Adjust the vacuum so that approximately one




         bubble of air per second enters the boiling flask through the  air




         inlet tube.  (Caution:  The bubble rate will not remain constant




         after the reagents have been added and while heat is being applied




         to the flask.  It will be necessary to readjust the air rate




         occasionally to prevent the solution in the boiling flask from




         backing up into the air inlet tube).




    8.3  Slowly add 25 ml concentrated sulfuric acid (7.5) through the  air




         inlet tube.  Rinse the tube with distilled water and allow the




         airflow to mix the flask contents for 3 min.  Pour 10 ml of CuoC^




         reagent (7.4) into the air inlet and wash down with a stream



         of water.




    8.4  Heat the solution to boiling, taking care to prevent the solution




         from backing up into the overflowing from the air inlet tube.



         Reflux for one hour.  Turn off heat and continue the airflow for




         at least 15 minutes.  After cooling the boiling flask, disconnect




         absorber and close off the vacuum source.




    8.5  Drain the solution from the absorber into a 250 ml volumetric




         flask and bring up to volume with distilled water washings




         from the absorber tube.

-------
                                                              (Cyanide)

  8.6  Withdraw 50 ml of the solution from the volumetric flask and

       transfer to a 100-ml volumetric flask.   Add 15 ml of sodium phos-

       phate solution (7.6) and 2.0 ml of Chloramine T solution (7.12)

       and mix.  Immediately add 5.0 ml pyridine-barbituric acid solution

       (7.13), mix and bring to mark with distilled water and mix again.

       Allow 8 minutes for color development.

  8.7  Read absorbance at 578 nm in a 1.0 cm cell within 15 minutes.

  8.8  Prepare a series of standards by diluting suitable volume? of

       standard solution to 500.0 ml with distilled water as follows:

           ml of Standard Solution        Cone., When Diluted to
             (1.0 ml = 1 y g CN)              500 ml, mg/1 CN

                   0 (Blank)                       0
                   5.0                             0.01
                  10.0                             0.02
                  20.0                             0.04
                  50.0                             0.10
                 100.0                             0.20
                 150.0                             0.50
                 200.0                             0.40

8.8.1  Standards must be treated in the same manner as the samples, as

       outlined in 8.1 through 8.7 above.

8.8.2  Prepare a standard curve by plotting absorbance of standards vs.

       cyanide concentrations.

8.8.3  Subsequently, at least two standards (a high and a low)  should

       be treated as in 8.8.1 to verify standard curve.  If results

       are not comparable (±10%) , a complete new standard curve must

       be prepared.

8.8.4  To check the  efficiency of the sample distillation, add an

       increment of  cyanide from either the intermediate standard (7.8)

       or the working standard (7.9) to insure a Icv.-l of 10 ug/1 or a

-------
                                                            (Cyanide)






         significant  increase  in  absorbance  value.   Proceed  with  the




         analysis  as  in  Procedure (8.8.1)  using  the  same  flask  and




         system from  which  the previous  sample was just distilled.




    8.9  Alternatively,  if  the sample  contains more  than  1 mg of  CN



         transfer  the distillate, or a suitable  aliquot diluted to




         250 ml, to a 500-ml Erlenmeyer  flask.   Add  10-12 drops of




         the benzalrhodamine indicator.




   8.10  Titrate with standard silver  nitrate to the first change in




         color from yellow  to  brownish-pink.  Titrate a distilled water




         blank using  the same  amount of  sodium hydroxide  and indicator




         as in the sample.




  .8.11  The analyst  should familiarize  himself  with the  end point of




         the titration and  the amount  of indicator to be  used before




         actually  titrating the samples.   A  5 or 10  ml microburet may




         be conveniently used  to  obtain  a  more precise titration.




9.   Calculation




    9.1  Using the colorimetric procedure, calculate concentration of




         CN, mg/1, directly from  prepared  standard curve.




    9.2  Using the titrimetric procedure,  calculate  concentration of




         CN as follows:
         CN,  mg/1  =
(A-B)x  1000	 Y  	250
                    Vol.  of  original  sample    Vol.  of aliquot  titrated



         where:




         A =  volume  of  AgN03  for  titration of  sample.




         B =  volume  of  AgN03  for  titration of  blank.

-------
                                                                  (Cyanide)
                                References
1.   Bark, L. S., and  Higson,  H. G.  Investigation  of  reagents for the




    colorimetric determination of small amounts  of cyanide.   Talanta,




    2:471-479  (1964).




2.   Elly, C. T.  Recovery of  cyanides by modified  Serfass distillation.




    Journal Water  Pollution Control Federation,  40:848-856 (1968).
                                                           it IT, GOVtBNMENT PH'NTING OfFICF 19')-  7-

-------
                CHEMICAL ANALYSIS

                       FOR


                 DEMAND, NUTRIENT

                       AND

                  OIL AND GREASE
                       By

                    Ho Young
                    EPA, Region IX
                    San Francisco CA
Presented at the Workshop on Sampling, Monitoring and Analysis
 of Water and Wastewater, March 6-12, 1974, Honolulu HI.

-------
      The primary purpose of the chemistry lectures is
 to describe briefly the analytical methods in recent or
 current use in EPA laboratories for determining oxygen
 demand, nutrients, oil and grease, metals and pesticides.
 Secondarily, they are to point out the advantages and the
 limitations of these methods.

      The first three parameters measure the various commonly
 existing materials present in water and the waste discharges,
              ORGANIC CONTENT OF WASTES
 I.  Definition of organic materials.
 II. Properties of organic materials.
     A.  Organic compounds are usually combustible.
     B.  Organic compounds, generally, have lower melting
         and boiling points.
     C.  Organic compounds are usually less soluble in water.
     D.  Reactions of organic compounds are usually molecular
         rather than ionic.

            2C2H6~f- 7O2 —> 4C02 -f- 6H20

     E.  Molecules are usually larger and heavier than
         those in inorganic substances.
     F.  Most organic compounds can serve as a source of
         food for microorganisms.
III. Types of organic substances in wastes.
     A.  Simple naturally-occurring organic compounds.
         1.   Hydrocarbons
         2.   Carbohydrates
     B.  Complex naturally-occurring organic compounds.
         1.   Organic nitrogen compounds—amino acids, protein,
         2.   Organic phosphate compounds—nucleic acid,
             ADP, ATP*, etc.
                          0-
                                        OH
                                        /
                     CH-CH-CH-CH-CH2-0-P —0
                                         )H

        adenine       ribose          phosphate

-------
        3.  Organic sulfate compounds—amino  acids  with  a
            sulfur group, e.g. cystine, cysteine, mathionine.
CH-NHo
I
COOH
                                cysteine
        4.  Organic halogen compounds—thyroxine,  thyroid
            hormone.
                          — 0-
                          thyroxine
                     I—CH0-CHOOH
                       I 2
                       NH,,
    C.  Synthetic organic compounds.
        1.  Chlorinated organic compounds—polychlorinated
            biphenyls
        2.  Pesticides—organochlorinated Pesticides such as
            aldrin, dieldrin, DDT; o-aryl carbamate pesticides;
            organophosphorus pesticides; and triazine
            pesticides.
        3.  Chlorinated phenoxy acid herbicides.
IV. Analytical methods for organic substances in wastes.
    A.  Direct measurements
        l.i;-. Total organic phosphate and orthophosphate.
        2.  Total organic carbon.
        3.  Total Kjeldahl nitrogen, ammonia, and nitrates.
    B.  Indirect measurements.
        1.  Biochemical oxygen demand.
        2.  Chemical oxygen demand.
Prepared by Bo Lee Young,
Chief, Chemistry Section
Laboratory Support Branch
EPA, Region IX
March 1, 1974
        Ph.D,
                          -2-

-------
            ANALYSIS OF ORGANIC COMPOUNDS
I.   Oxygen Demands:  It is a determination of the decrease
    of dissolved oxygen in receiving water when waste is
    discharged into water.

    A.  Objective:  This is an indirect method to measure
        the oxidizable organics of the waste.

    B.  Measurement Techniques

        1.  Oxygen Probe
        2.  Winkler - Azide Method

    C.  Biological Oxygen Demands (BOD) .

        1.  Principle:  It is an estimate of the biodegradeable
            organic materials of the sample by establishing
            the amount of decrease of dissolved oxygen.  The
            BOD test gives an indication of the amount of
            oxygen needed to stabilize or biologically oxidize
            the waste.

        2.  Types of biodegradable organic materials measured
            in BOD test.

            a.  Organic Carbon:  carbohydrates (common sugars
                and their metabolic by-products)  and hydrocarbons,

            b.  Nitrogenous compounds:  amino acids, nitrates,
                ammonia and some complex nitrogenous compounds
                such as nucleic acid, nucleotides and
                nucleosides.

        3.  Types of BOD measurement

            a.  Dynamic measurement:  to measure the change
                or the rate of change in oxygen utilization with
                respect to time.

                1.  Manometric method:  Warburg Apparatus
                2.  Electrolysis BOD devices:  Hach Apparatus

            b.  Static measurement:   to measure the amount of
                oxygen used at a fixed time interval.

                1.  Ultimate BOD:  to measure the amount of
                    oxygen required to oxidize the entire
                    amount of the biodegradeable materials.
                           -3-

-------
14 —
                    second- -stage
  0 1
                          23   456789M
                          Time  in  Days
            First Stage BOD -  mainly oxidation of
            carbon compounds.
            Second stage  BOD - low rate oxidation
            of most resistant  compounds and/or
            nitrogenous compounds.

        2.  6005:  An emperical bioassay type procedure.
            It, in general, with  the corrected dilu-
            tion, measures the oxygen consumption from
            oxidation of  carbon sources.

            It measures the dissolved oxygen consumed
            by microorganisms  while assimilating and
            oxidizing the organic matter present during
            incubation.   The incubation period is five
            days.  Incubation  conditions are 20°C in
            the dark, and pH near neutrality.  Seeds
            used are common sewage bacteria or
            commercial septic  activator.

4.  Advantage and limitations

    a.  Advantage:  it measures only the organic
        compounds which are oxidized by the micro-
        organisms, mainly bacteria.

    b.  Limitations

        1.  The difficulty in  obtaining consistent
            and repetitive values - variations in
            the time lag  between sampling and results
            of analysis.

        2.  The actual environmental conditions of
            temperature,  biological population and
            seed acclimation,  water movement, sunlight
            and oxygen concentration cannot be accur-
            ately reproduced in the laboratory.
-4-

-------
            3.  Results obtained in the laboratory may
                or may not represent the oxygen demands
                at the effluent site.

            4.  Accumulation of C02 influences the test
                results.

            5.  Lack of seed acclimation results in
                erroneously low readings.

            6.  Sea water interferes because of salinity
                differences due to dilution.

D.  Chemical Oxygen Demand (COD)

    1.  Principle:  It is an estimate of that proportion
        of the sample matter which is susceptible to
        oxidation by a strong chemical oxidant.

    2.  Types of substances oxidized by dichromate in
        50% sulfuric acid.

        a.  Sugars, branched and straight chain aliphatics
            and substituted benzene rings.

        b.  Straight-chain acids, alcohols and amino acids
            can be completely oxidized in the presence of
            the silver sulfate catalyst.

            Benzene, pyridine and toluene are not oxidized
            by this method.

    3.  Procedures

    4.  Advantages and limitations

        a.  Advantages as compared to BOD

            1.  Time, manipulation, and equipment costs
                are lower.

            2.  COD oxidation conditions are effective for
                a wider spectrum of chemical compounds.

            3.  COD test conditions can be standardized
                more readily to give more precise results.

            4.  COD results are available in few hours.

            5.  The COD results plus the oxygen equivalent
                for ammonia and organic nitorgen is a
                good estimate of the ultimate BOD for many
                municipal wastewaters.

                      -5-

-------
             b.  Limitations

                 1.  Certain inorganic substances, such as
                     sulfides, sulfites, thiosulfates, nitrites
                     and ferrous iron are oxidized by dichromate,
                     creating an inorganic COD, which is mis-
                     leading when estimating the organic
                     content of the wastewater.

                 2.  The COD test may not include some volatile
                     organics such as acetic acid and ammonia.

                 3.  Dichromate in hot  50% sulfuric acid
                     requires close control to maintain safety
                     during manipulation.

                 4.  Because of chloride interference, it is
                     not advisable to expect precise COD results
                     on saline water.

                 5.  Requires a large quantity of mercuric
                     sulfate which is a pollutant.

II.  Total Organic Carbon  (TOC)

     A.  Principle:  all carbon atoms of organic molecules
         are oxidized to C02 at high temperature; the amount
         of C02 produced is measured by an infra-red analyzer.

                 C6H1206 + 6 °2 —>  6 C02 + 6 H20

     B.  Methods

         1.  Direct injection:  carbon atoms are combusted at
             950° C.

         2.  Indirect digestion:  carbon atoms are oxidized
             in acid at 166° C in the presence of pure oxygen.

     C.  Advantages and Limitations

         1.  Advantages

             a.  Speed, direct injection method takes 2 minutes.

             b.  To measure the total carbon of all forms.

         2.  Limitations:  carbonate and bicarbonate interfers
             with the analysis.


                            -6-

-------
III.   Relationships between BOD, COD and TOC's

           BOD is not the most useful test of waste load
      because of the long incubation time required to obtain
      a meaningful result.  It is, therefore,  important to
      develop a correlation between BOD, COD and TOC.
                        Table 1
           Comparison of BOD, COD and TOC Tests
  Test temp °C
  Oxidation
    system
  Measurement
   Variables
  Equipment
  Cost
BOD

 20
  Reaction time  5 days
Biol. prod.
Enz. Oxidn.
 COD

145


2 hrs.

50% H2S04
I^C^OY
May be catalyzed
                                    TOC

                                   950° or 166°
                                   with pressure

                                   minute or hours

                                   oxygen, atmos-
                                   phere, catalyzed
Dissolved       Chemical oxidation
oxygen com-     susceptibility of
pound, environ- the test sample to
ment biota, time  the specified
numbers, metabo-  oxidation
lie acceptability
etc.
                                   Infra-red C02
                                   comparable to
                                    theoretical for
                                   carbon only.
Bottles
Incubator

$150
                heater
                glassware

                $500
                   TOC
                   Analyzer

                   $8000
                            -7-

-------
                      Table 2
        COD-TOG and BODs-TOC Relationships
Substance

Acetone
Ethanol
Phenol
Salicylic
Methanol
Benzoic Acid
Sucrose
Benzene
Pyridine
COD/TOG

2.44 (3.56)*
3.35 (4.00)
2.96 (3.12)
2.83 (2.86)
3.89 (4.00)
2.90 (2.86)
2.44 (2.67)
0.84 (3.34)
nil  (3.33)
BOD5/TOC
Waste                Raw      Effluent    Raw     Effluent

Domestic             4.15      2.20       1.62       0.47
Chemical             3.54      2.29
Refinery-Chemical    5.40      2.15       2.75       0.43
Petrochemical        2.70      1.85
*Values in parenthesis are the theoretical values.
                          -8-

-------
     IV.  Nutrients:  These include nitrogenous and phosphorus
          compounds.

          A.  Nitrogenous compounds:  ammonia  (NH3) nitrite  (N02),
              nitrate (N03)  and total organic nitrogen  (TKN)

              1.  Procedures:  Table 3
Sample
 Preparation
Detection
Range
distilled from
alkaline sol,
absorbed in
borate buffer
1. colorime-
tric method
by Nesslariza-
tion 400-425nm

2. titration
with acid
0.05-1 mg/1
1-25 mg/1
Interferences Cyanates
              alcohols
              aldehydes and
              ketones
  N02

formation of
diazonium
compound with
diazotatin of
sulfanilanide,
coupled with
N-(l-naphtyl)
-ethylene
diamine-red-
dish purple

spectrophoto-
metric, at
540 nm.
0.05-1 mg/1
                  strong oxi-
                  dizing or
                  reducing
                  agents
 NO 3

reaction
with
brucine
sulfate
in
spectro-
photome-
tric at
410nm.
  TKN

acid diges-
tion, dis-
tilled and
absorbed in
borate
buffer
0.1-2
mg/1

strong
oxidizing
and reduc-
ing agents
Fe-W-, Fe-H-
Mn-H, Cl~
organic
matter
1. colori-
metric method
by Nessler-
ization 400-
425 nm.

2. titration
with acid.

0.05-1 mg/1
1-25 mg/1
              2.  Other analytical methods usable for samples con-
                  taining high salt concentration - to be discussed.

      V.   Phosphorate Compounds:  Phosphorus is usually present as
          orthophosphate, polyphosphate, and organically bound
          phosphorus.
                                -9-

-------
A.  EPA Spectrophotometric Method  (p. 252) .

    1.  Principle:  It is an analysis of the total phos-
        phorus by the formation of antimony-phospho-
        molybdate complex.

    2.  Methods

        a.  Polyphosphates are rapidly hydrolized into
            orthophosphate in boiling water at low pH.

        b.  Organic forms of phosphorus are converted
            to orthophosphates by wet oxidation.

        c.  Orthophosphate reacts with ammonium molybdate
            and potassium antimonyl tartrate in acid
            medium to form antimony - phosphomolybdate
            complex.

        d.  The complex is reduced to an intensely blue-
            colored complex by ascorbic acid.

        e.  The blue color which is proportional to the
            concentration of phosphorus is measured at
            880 nm.

    3.  Interference

        a.  Cl~ concentration below 50 mg Cl/1 interferes.

        b.  High iron concentration causes precipitation
            of phosphorus.

        c.  Arsenic at sea water level does not interfere.

B.  Phosphate classification
                     -10-

-------
                                      Table  4
                      PHOSPHORUS COMPOUNDS CLASSIFIED  BY
                             ANALYTICAL  METHODOLOGY
      Desired P Components
    Technique
                                               (1)
                               Incidental P Included
                                                  (2)
    1.  Ortho phosphates
No treatment on clear
samples
Easily hydrolyzed
(a) poly phosphates -
(b) organic -P,
(c) Mineral -P, + or
    2.  Polyphosphates
       (2)-(l) = poly P
        (hydrolyzable)
       i
acid hydrolysis on clear
samples,  dilute
   (a) H2S04

   (b) HC1
heated
(a) ortho-P    +
(b) organic -P  + or
(c) mineral -P + or
    3. Organic phosphorus
         (3)   (2) + orgP
         (hydrolyzable)
acid + oxidizing hydrolysis
on whole sample,  dilute

  (a) HS0
  (b) H2S04

  heated
(a) ortho P   +
(b) poly P    +
(c) mineral P + or -
                                               (NH4)2S208
    4.  Soluble phosphorus
       (preferably classified
       by clarification method)
clarified liquid following
filtration,  centrifugation
or subsidence
generally includes
(a) 1. 2, or 3

(b) particulates. not
    completely separated
    5.  Insoluble phosphorus
       (residue from clari-
       fication)
Retained residues separated
during clarification
       See  (6)
(a) generally includes
    sorbed or complexed
    solubles.
    6.  Total phosphorus
Strong acid + oxidant
digestion

 (a) H,SO  + HNO
      24      3
 (b) H_SO, + HNO, +HC10.
      24      3        4

 (c) H O   + Mg(NO  )  fusion
      & £1         O
all components in
1.  2. 3, 4,  5 in the
whole sample
    (1)  Determinative step by phospho molybdate colorimetric method.

    (2)  Coding:  + quantitative yield
                -  a small fricHoa of the amount present
                +  or  - depends upon the individual chemical and sample history
38-4
                                          -11-

-------
  VI.   Oil and Grease

       A.   Sources

           1.  Industrial waste:  petroleum product, lubrication
               oil.

           2.  Decomposition of planktons and higher forms of
               aquatic life-

       B.   Principle:  dissolved, emulsified or adsorbed oil
           or grease is extracted by intimate contact with
           various organic solvents.

       C.   Types of extractions:  liquid-liquid and soxhlet
           extractions as discussed below:

Table 5. Summary of oil and grease analysis
   Sample

   Sample
    Preservation

   Solvent
   Sample
    Preparation
   Extraction temp

   Extraction time
   Extractable
    Material

   Interferences
Aqueous

acidification
Trichlorotrifluoro-
 ethane  (Freon)

Acidified - H2SO4
Room temperature

Two minutes vigor-
ously.  Shaking in
separatory funnel.

Oils, lubrication
oil, fats
Sediment tissues

Freezing


Hexane


Acidified-HCl to pH
2.0 dehydration with
magnesium sulfate
monohydrate

70°C

4 hrs. (80 cycles)
soaps, fats, waxes,
and oil
Evaporation of low     elementary S, organic
boiling oils, kerosene dye, and oxidation of
                       oil.
                             -12-

-------
VII.  References
      1.  Standard Methods for the Examination of Water and
          Wastewater, 13th Edition, American Public Health
          Association, 1015 18th Street, N.W. Washington, D.C.

      2.  Methods for Chemical Analysis of Water and Wastes,
          EPA, National Environmental Research Center, Analytical
          Quality Control Laboratory, Cincinnati, Ohio, 1971.

      3.  J.D.H. Strickland and T.R. Parsons.  A Practical
          Handbook of Seawater Analysis, Fisheries Research
          Board of Canada, Ottawa, 1968.
  Prepared by
  Ho Lee Young, Ph.D.
  Chief, Chemistry Section
  Laboratory Support Branch
  EPA, Region IX
  March 1, 1974
                           -13-

-------
AMOUNT OK DISSOI.VKO OXYGEN, ix WATER AT DIFFERENT TEMPKRATURES
WHEN EXI-OSKD  TO AN  ATMOSI'IIKHK CONTAINING 20.9  PKK CF.NT or
OXYGRN UNIIKK A PltESSUItK OF 7rtO MM.  INCLUDING PuKSbUHE OK WATKIl
                             VAPOR*
Temp.
•c.
0
1
2
3
4
5
6
7
" 8
9 .
10 '
11
12
13
H
15
Parti per
Million
14.62
14.23
13.84
' 13.48
13.13
12.80
12.48
12.17
11.87
11.59
11.33
11.08
10.83
10.60
10.37
10.15
Ce. per liter
(at 0* C. and
760 imn.)
' 10.23
9.9(5
9.68
9.43
9.19
8.96
8.73
• 8.52
. 8.31
. 8.11
7.93
7.75
7.58
7.42
7.26
' 7.10
•TCT//P.
•c.
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30

Pamper
Million
9.95
9.74
9.54
9.35
9.17
8.99
8.83
8.68
8.53
8.38
8.22
8.07
7.92
7.77
7.63

Ce. per liter
(atO' C.aiiJ
760 ;//»;/.)
6.96
6.82
6.68
6.54
. 6A2
6.29
6.18
6.07
5.97
5.86
5.75
5.65
5.54
5.44
5.34
%

-------
                                                        Page 1 of: 2


                D. 0. MEASUREMENT WITH OXYGEN PROBE


 1.   Oxygen probe with the stirrer attached should be kept in a
     moistened BOD bottle at all times.

 2.   Meter on off position.

 3.   Switch to check position - point should read "check"

 4.   0-15 range, warm up for 10 minutes.

 5.   Remove probe gently and carefully.  If the probe sticks in the
     bottle's neck, turn the probe slowly to free it.
 6-   Cover and shake the BOD bottle vigorously for 15 seconds.

 7.   Remove the stirring bar (at the bottom) from the probe.

 8.   Wipe the probe very carefully to remove moisture.

 9.   Shake the bottle again.

10.   Insert the probe into the bottle.

11.   Set function switch at temp, note the temperature.

12.   Read the solubility  of oxygen in fresh water at that temper-
     ature from Table I  (on top of the dissolved Oxygen meter)  for
     example at 23°C = 8.7 ing/liter.

13.   Turn function switch to 0 - 15 range.
14.   Adjust calibration knob until the meter reads the proper dis-
     solved Oxygen at that temperature.   (e.g. 8.7)

15.   Repeat steps 8 to 14.

16.   Turn the function switch to temperature or check.

]7.   Assemble the stirring bar assembly to the probe.  Probe is
     ready to be used for measuring D.O. in sample.

18.   Insert Oxygen probe into the sample bottle very carefully.  If
     the BOD bottle is too small, do not force the probe in, just
     discard the sample.
19.   Place the sample bottle on a magnetic stirrer.

20.   Be sure no air bubble(s)  trapped on the surface of the membrane
     (look at the bottom of the probe through the sample bottle).
     Remove air bubble by raising the probe above the water surface
     and insert the probe again.

21.   Turn on the magnetic stirrer.

22.   Turn function switch to 0 - 15.range.

23.   Allow sufficient time for the probe to equilibriate with the
     sample - up to 2 minutes.

-------
                                                      Page 2 of 2
24.  Record the dissolved oxygen reading'e.g. 2.1 mg/1.
25.  Turn the function switch to temp.
26.  Turn probe around to loosen the water seal, then remove the probe,
27.  When the measurement is finished, return the probe into the
     moistened BOD bottle.
28.  Turn off the meter.
29.  Turn off the magnetic stirrer.
                               -16-

-------
                                                  Page 1 of 4

      Winkler - Azide Method for Dissolved Oxygen
I.    Reagents

      1.   Alkali - iodide - azide reagent (NaOH - Nal - NaNo or
           KOH - KI - NaN3) .

      2.   Manganese sulfate solution (MnSO^) .

      3.   0.0375 N Potassium biiodate standard [KH(I03)2].

      4.   Potassium Iodide  (KI crystals).

      5.   Sodium thiosulfate titrant

      6.   Starch Solution.

      7.   Sulfuric acid  (concentrated

      8.   10% Sulfuric acid (10% H2S04).


II.   Standardization of Na2S203 with Primary Standard, 0.0375 N
      potassium biiodate  [KH (103)2].

      1.   Dissolve 2 g of KI crystals (two level scoops full)
           with 100 - 150 ml of distilled water in a wide-mouth
           500 ml Erlenmeyer flask.

      2.   Add 10 ml of 10% H2S04 into the KI solution and mix
           well.

      3,   Add 20 ml of 0.0375 N KH  (I03)2 and mix well.

      4.   Place in dark for five minutes.

      5.   Fill a 25 ml buret with Na2S203.

      6.   Bring KI - H2S04 - KH(I03)2 mixture to a total volume
           of approximately 300 ml.

      7.   With the magnetic stirrer set at moderate speed, titrate
           the mixture with Na2S203 until the color of the solution
           turns to a pale straw color.

      8.   Add 1 ml of starch solution  (2 droppers full)  to the
           the mixture, and mix well.

      9.   Continue the titration until the mixture turns colorless
                               -17-

-------
                                                   Page 2 of 4



      10.  Record the amount of Na2S2C>3 used for titration.

      11,  Compute the normality of
Normality of Na2S203 =  ml of KH(I03)2 x Normality of KH(I03)2
                                 ml of Na2S203

III.  Titration of dissolved oxygen of a sample.

      1.   Fill the BOD bottle up to the top with sample.
           Note:  for a predicted BOD 5 mg/1 , pipet a proper
           amount of sample* into a BOD bottle and fill the
           bottle with oxygenated dilution water (Dilution water:
           Distilled water aerated overnight containing 1 ml
           CaCl2 solution, 1 ml FeCl3 solution, 1 ml MgS04
           solution and 1 ml phosphate buffer per liter) .

      2.   Add 2 ml of MnS04 and 2 ml of alkali - iodide -
           azide reagent.  Make sure the tip of the pipet is
           immersed well below the surface of the sample to
           prevent formation of bubbles.

      3.   Immediately stopper the bottle, with care to exclude
           air bubbles .

      4.   Mix the solution well by inverting the bottle at least
           five times.

      5.   When the precipitate settles, leaving a clear supernatant
           above the manganese hydroxide floe, mix the solution
           again.

      6.   When settling has produced at least 100 ml of clear
           supernatant on the upper portion, carefully remove the
           stopper and immediately add 2 ml of concentrated
           H2S04 to the mixture.

      7.   Stopper the bottle and invert the bottle until the
           iodide is uniformly distributed throughout the bottle.

      8.   Transfer the entire sample into a 500 ml wide-mouth
           Erlenmeyer flask.

      9.   Titrate the sample with the standardized Na2S2O3
           solution as described in items 7-9 of part II.
                               -18-

-------
                                                     Page 3 or A
      10.  Record the quantity of Na2S203 used for the titration.

      11.  Compute the dissolved oxygen in the sample.

DO, mg/1 =  ml of ^328203 x Normality of Na2S2C>3

                            Normality of KII(I03)2

         =  ml of Na2S203 x Normality of Na2S203
                                 0.0375 N
REFERENCE:  Dissolved Oxygen:  Methods For Chemical Analysis Of
            Wastes, 1971, Environmental Protection Agency,
            pp 53-59

            BOD:  IBID pp 15-16

-------
                                                   Paqe 4 of 4
                          FOOTNOTE
*Sample size for a BOD5 5 mg/1  when  using  300 ml BOD bottle and
 dilution water (The initial  dissolved  oxygen of the mixture
 should be  7.0 mg/1).
  Sample Size                      mg/1 BOD Range Covered

    1 ml                               300  - 1800 mg/1
    2 ml                               150  -   900 mg/1
    3 ml                               100  -   600 mg/1
    4 ml                               75  -   450 mg/1
    5 ml                               60-360 mg/1
    .6 ml                               50  -   300 mg/1
    7 ml                               43  -   257 mg/1
    8 ml                               38  -   225 mg/1
    9 ml                               33  -   200 mg/1
   10 ml                               30  -   180 mg/1
   15 ml                               20  -   120 mg/1
   20 ml                               15  -   90 mg/1
   25 ml                               12  -   72 mg/1
   30 ml                               10  -   60 mg/1
   40 ml                               7.5  -   45 mg/1
   50 ml                                6  -   36 mg/1
   75 ml                                4  --  24 mg/1
  100 ml                                3-18 mg/1
  150 ml                                2-12 mg/1
  200 ml                               1.5-9 mg/1
  300 ml                              '1-6 mg/1
                             -20-

-------
          Defini15on of Terms Used  in EPA Methods








 Range  of  Applicability;  This is intended to state the




 upper  and lower  concentration (or other appropriate




 characteristics  of the parameter) for which the method




 is  applicable.   When only an upper or lower limit is  given




 in  the reference source, that information is entered.   When




non-quantitative  information is given in the reference source,




that information  may also be entered under this heading.



Sensitivity:  In  general, sensitivity is used synonymously with




"Detection Limit",  to indicate  the lowest concentration of a




pollutant  (or lowest value of some other parameters)  that




a given method can consistently measure.  In a very few cases,




the source reference or  reviewer distinguishes between "Detection




Limit"  (as the lowest measurable value) and "Sensitivity" (as




the magnitude of  signal  needed  to obtain a reliable measurement,




taking into account the  noise level of the measurement system).




When this distinction is made in the reference source, it is




reflected in the  Method  Summary.



    Sensitivity,  in either of the senses discussed above,




may be  considered to be  either  a statistical characteristic




or a limitation of the method.  Sensitivity information was




available lor a relatively small proportion of the methods




summarized.  The  "sensitivity" heading is not included on the




Method SummnrD'es  when data are not available.  When sensitivity




information is reported, it is usually under the category of




"Limitations".




                           -21-

-------
                                   \    •


Accuracy and P_re c I s ' oil :  There  is  considerable diversity in the

use of these terms among the several reference sources from

which the Method Summaries were derived.  Rather than impose

rigorous statistical definitions of these and related terras,

the compilers of this  compendium chose to accept the statistical

characteristics of the method as stated. in the reference source,

and to fit this information as  well as  possible under the headings

Accuracy and Precision, as  these terms  were used more or less

consistently in most of the EPA source  documents^  In tliese

sources, the implied approximate definitions are as follows:

     "Accuracy" — the average  of  the deviations  of a set  of

     replicate measurements of  a given  variable  from the  "known"

     value of that  variable.
     "Relative accuracy" (or "relative  error", or "bias")

     — the difference between average  value of a set of

     replicate measurements and the "known" value of the

     variable expressed as  a proportion or percentage of

     the known value.                              [~  .           \ i /
                                                     V      ~  2\ 2
                                                        ~  —
     "Precision" — either the standard deviation
                                                        ]
                                                        J
or the standard error of the mean  / Z-AXl - xj  i   i   of
                                  [I   n(n - 1)
a set of n replicate measurements (Xi) of a given variable.

"Relative precision" (or "relative standard deviation",

or "coefficient of variation")  — the standard  deviation

of a set of replicate measurements, expressed as  a pro-

portion or percent of the average value of the  set.
                            -22-

-------
     DEFINITIONS,  CONVERSION FACTORS, AND EQUIVALENTS
   %  = parts per 100 parts
       1 part per 100 parts = 1%
       1 pound in 100 pounds = 1%
 ppm = parts per million parts
       1 pound per million pounds = 1 ppm
       1 gram per million grams = 1 ppm
          1 pound (English) = 453.6 grams (metric)
       1 milligram per 1000 grams = 1 ppm
          1 milligram = .001 gram
       1 microgram per gram = 1 ppm
          1 microgram = .000001 gram


ml/1 = milliliters per liter
          1 liter = 1000 milliliters (ml.)
          1 milliliter (ml) = .001 liters (1)
          1 liter = 1.057 quarts
          1 quart (U.S.-Liquid) = 0.946 liters (metric-liquid)
          1 ml = 1.000027 cm3
rag/1 = milligrams per liter = ppm
       1 milligram =  .001 gram  (weight)
       1 liter =  1000 grams (weightof  water  at  standard  conditions)
       1 milliliter = 1 ml  (volume) or  .001  liters
       1 milligram =  1 mg (weight)  or  .001 grams
       1 milliliter of water weighs 1  gram (at  standard  conditions)

-------
LENGTH (cont.)
1

1
inch

foot

=
=
2
2
1
5
.
2
= 30

1


1
1


1

1

yard


fathom
league
(land)

rod

chain
=
0
.
= 36
=
=
=
=
=
=
=
=
9
.
6
5
3
4
1
5
1
9

2

.
6
.
= 66
.4
54
in
.48
304
in
.44
144
ft.
80
mi .
828
.5
092
mm
cm
.

.

cm .
8
.
m .
(3 feet)
cm .
meters

yd


.

1 micron «* 1
1 mm = 0 .
= 0.
1 centimeter = 0 .
= 10
1 meter = 39
= 1.
X 1
0"
03937
3 mm
in .
1 cm .
393
mm
.37
093
= 1000
= 1


X 1


7
,
i
6
mm
0~


in .

n .
yds .

3 km


km .
ft
10
•
meters








feet
AREA


1 sq .  inch
1 sq.  foot

1 sq.  yard

1 acre


1 sq.  mile



1 sq.  centimeter

1 hectare
1 sq. kilometer
      cm,
       2
6.452
144 in
929.0 cm
9 ft. 2
0.8361 m
43,560
4046.9
0.4Q47
640
258
       m 2
       ft.2
         2
       m
       hectare
    acres
    99 hectares
  590 km.2
  section  (of land)
  1550 in.2
  X 10~4 hectare meters2
  471 acres
107,640 ft.2
1 X 104 m.2
0.01 km.2
100 X 100  meters
247.1 acres
0.3861 sq. miles
1 X 106 m.2
100 hectares
1000 X 1000 meters
                         (1X10"8  ha)
                            -25-

-------
VOLUME
1 cubic inch
1 cubic foot
 1  cubic  yard
 1  ounce  (fluid)
 1  pint  -  -  -  -
 1  quart-


 1  gallon
 1  cubic foot
 1 acre inch
 1 acre foot
 1 hectare centimeter
 1 cubic centimeter -
 1 milliliter

 1' liter  - -
 1 cubic meter
 1000 cubic meters
 1000 cubic meters/hectare-

 1 cubic foot/sec.   - - - -
 1 million gallons/day- - -
 1 inch of rain ------
   barrel (oil) ______
= 16.39 cm-5
= 7.481 U.S. gallons
= 1728 cu.  inches
= 28.32 liters
= .0283 m3
= 27 cubic feet
= 0.7645 m3
= 29.57 ml.
= 0.50 quarts
= 0.125 gallons
= 0.473 liters
= 0.25 gallons
= 946.25 milliliters
= .946 liters
= 0.1337 cubic feet
= 231  cubic inches
= 3.785 liters
= 0.83267  Imperial
= 8.337 Ibs. water
= 7.481 gallons
= 62.37 pounds
= 28.32 liters
= .0283 m3
= 3630 cubic feet
= 27,150 gallons
= 226000 pounds
= 102.8 cubic meters
= 43,560 cubic feet
= 325,900  gallons
= 2.716 million  pounds
= 1233.4 m3
= 12.173 ha cm
= .9728 acre in.
= 0.06102  cubic  inches
= 0.99997  milliliters
= 1.000027  cm3
= .001 liters
= 1.05680  quarts
= 0.2642 gallons
= 1000 milliliters
= 1000.027  cm3
= .99997X10'3 m3
= .03531 cubic feet
= .001 m3
= .2201 Imperial  gallon
= 264.2 gallons
= 35.32 cubic feet
= 1.308 cubic yards
= 1000 liters
= 35320 cubic feet
= 0.81084  acre feet
 = 10  ha cm (.1 ha mm =  1
 = 0.32814  acre feet/acre
 = 10  ha cm/ha
 = 1.98 acre  feet/day
 = 3.069 acre feet/day
 = 27,150 gallons/acr0
 = 42  gallons
                                                gallons
                                                @ 62°F (8.345
Ibs.  @4°C)
                                                       i3 =  1 metric ton)

-------
FLOW RATE
1 gallon per minute
 1 million  gallons  per  day-  -  -  =
 1 cubic  foot per  second  (cfs)
   second  foot, or  CUSEC
 1  acre-foot per  day

 1  liter per second
                                   minute
                                               (1699.3  lit/min.)
                                               meters/sec.
 1  cubic meter per  second  -  -  -  =
        (CUMEC)

 1  cubic meter per  hour  -  -  -  -  =

 miners inch  (N.  Calif)  -  -  -  -  =


 miners inch  (S.  Calif.)'  ~  ~  ~  =
.002228 cfs
.05304 ac.  inches/day
13860  inch3/hr.
96.25 ft.2  covered 1"
0.06308 lit/sec.
694 gpm
1.55 cfs
3.07 AF/day
43.7 1./sec.
448.83 U.S. gallons per
0.99 acre  inches/hr.
1.98 AF/day
28.32 l./sec
.02832 cubic
226 gpm
14.2 1./sec.
15.852 gallons/min.
0.0353 cfs
.03495 acre inches/hr.
3.6 cubic  meters/hour
0.36 mm ha/hr-
1.58 X 104  gpm
35.314 cfs
1000 l./sec.
0.278 liters/sec.
4.403 gallons/min.
1/40 cfs
11.25 gpm
0.6 ac. inch/day
1/50 cfs
9.0 gpm
0.48 ac. inch/day
                                                        deep  in  1  hr
 WEIGHT
 1  grain
 1  ounce
 1  pound  -  -  -

 1  short  ton  -


 1  metric ton
 1  picogram
 1  iianogram
 1  microgram
 1  milligram
 1  centigram
 1  gram   - -
    0.0648 gms
    0.0625 Ibs
    28 . 3495 gms
    453.5924 gms
    0.4536 kg.
    2000  Ib.
    907. 1849 kg.
    0 . 9072 m.  ton
    2204.6 Ib.
    1000  kg.
    1.1023 short ton
    10-12 gms
    10-9
- = 10
      -6
         gms
 1  ki1ogram
         gms
    10~  gms
    10~2 gms
    0.03527 oz
    0.001 kg.
    35 .27 oz .
    2 . 205 Ib.
    1000 gm.

-------
WEIGHT (continued)
1 lb./ac.- - -
1 ton/acre - -
1 kg/ha- - - •
1 met. ton/ha-
     = 1.12085 kg/ha
     = 2.24169 metric tons/ha
     = .89235 Ibs/ac.
     = .44597 tons/ac. (892.8 Ibs./acre)
PRESSURE
1 psi- -

1 kg/cm2
1 atmosphere
TEMPERATURE

°F
0
10
20
30
32
45
50
60
65
70
75
80
85
90
95
100
105
110
115
120
212
°C
°C
-17.78
-12.22
- 6.67
- 1.11
0.00
7.22
10.00
15.56
18.33
21.11
23.89
26.67
29.44
32.22
35.00
37.78
40.56
43.33
46.11
48.89
100.00
       .07031 kg/cm2
       7.031 kg/m2
       14.223 psi
       .9678 atmospheres
       32.81 feet of water
       28.96 inches mercury
       14.696 lbs/in2
       1.033 kg/cm2
       101.33 centibars
       29.921 inches mercury (at 32 °F)
       33.95 feet of water (at 62PF)
       1033 cm of water (at 62°F)
       76 cm of mercury (at 0°C)
9/5 (°C)  + 32
5/9 (°F - 32)
0
5
10
15
20
25
30
35
40
45
50
100
32
41
50
59
68
77
86
95
104
113
122
212
                           -28-

-------
        SELECTED FIELD AND LABORATORY BIOLOGY METHODS
                             By

                       Milton Tunzi
                       EPA, Region IX
                       San Francisco CA
Presented at the Workshop on Sampling, Monitoring and Analysis
  of Water and Wastewater, March 6-12, 1974, Honolulu HI.

-------
            U.S. ENVIRONMENTAL PROTECTION AGENCY

                    Region IX Laboratory

                     620 Central Avenue

                 Alameda, California  94501

        SELECTED FIELD AND LABORATORY BIOLOGY METHODS

                      Table of Contents


  I.  Sample Collection

 II.  Sample Collection Forms

III.  Algal Bioassays

      A.  Laboratory Bioassay Directions

      B.  Cell Mass Measurement

      C.  Measuring Dry Weight

      D.  Measuring Algal Chlorophyll

      E.  Maintaining Algal Cultures in the Laboratory

 IV.  Statistical Procedures

  V.  Fish Bioassays

 VI.  Use of Random Numbers

VII.  Appendix

          Tables of Useful Data
Prepared by Milton G. Tunzi, Ph.D., EPA Laboratory
620 Central Avenue, Alameda, California 94501
(Comments and corrections would be appreciated)
First Edition October 1973
Second Edition February 1974

-------
I. Sample Collection

        Representative samples from any body of water
   are difficult to take.  Directions can be giyen for a
   completely statistically valid approach  (e.g., random
   sampling), but these would probably be beyond the
   resources of most laboratories.  Furthermore, the
   approach should be determined in relation to the
   purposes of the study.  This may preclude a random-
   sampling approach or make it unnecessary.

        One of the best ways to assure that a sample is
   representative of a site (whether that site be chosen
   randomly as indicated above or arbitrarily as in this
   section) is to composite 3 or 4 or more equal-volume
   samples from each site.  These can be put into a
   plastic bucket, mixed, and a container filled from
   this bucket.

   A.   Routine Sampling

        1.   Generally, the specific sampling sites are
             chosen because they are accessible, equally
             distant from each other, traditional
             sampling sites, or in locations of importance
             near dischargers or in areas of water use.
             Samples may be taken above and below a dis-
             charge pipe, or they may be taken in the
             receiving water near the discharge pipe.
             Many times the location selected is one
             where a water quality standard may be
             exceeded.

        2.   Rivers, streams,  estuaries

             Sites can be sampled from different depths,
             from different locations around the sides
             of a relatively-stationary large boat,
             and from different locations if a small boat
             is allowed to drift.  Water moving past an

-------
anchored boat can be sampled every half-
minute, or longer time interval depending
on time limitations at the station.  A
stream should also be sampled in this way
from the bank, i.e., with samples taken from
the stream throughout a given time period
and composited.  A wide-month, liter,
plastic container attached to a pole can
be used to reach further from shore so that
flowing water can be more easily sampled,
or so that the moving part of a stream can be
reached.  A wide stream which is not above
boot-top in depth can be sampled by sub-
samples from 4 or 5 locations in a transect
across the stream.  The subsamples must be
taken upstream so that they will not be
contaminated by the stream bottom stirred
up from walking.

Compositing samples is suggested because
then fewer samples would have to be analyzed.
However, if variations within time or a
small space are desired, then the samples
could be kept discrete, i.e., not composited.
Spatial or temporal variations at one one or
more stations can then be used in statistical
comparisons between the stations.

Lakes, reservoirs, and ponds

If five individual samples for nitrate
analysis are to be taken from five-acre
Lake X whose water is being mixed thoroughly
(e.g., because of fall turnover), then one
might choose locations so that all parts of
the Lake would be represented.   (Figure  1).

-------
          Figure 1.  Lake X, Divided into Sections,
          Five Stations Shown.

          In fact, one could take several samples in
          the area where he was to sample the single
          station and composite the several samples.
          For example, four or five subsamples could
          be taken in an area near sampling Station 1
          (indicated on Figure 1 by circle), and the
          composited sample would represent Station 1.
          The compositing can be done from sample
          taken at different lake depths.  This com-
          positing approach is very useful where little
          time can be allotted to the execution of the
          sampling or where the project does not
          require a more sophisticated approach.

B.   Random Sampling

     Because of such variations as density, light
     penetration differences and the like, most
     waters would require a stratified random sampling
     approach.

     1.   Random Sampling, Spatial Approach

          First the water body is arbitrarily divided
          into areas which are physically or geogra-
          phically distinct.  Then each area is divided
          into a grid pattern, and each section is
          numbered.  The sections to be sampled in each
          area are selected by using a random numbers
          table.  Only 2 or 3 locations are sampled in
          each physically or geographically distinct
          lake area.  The same number of sections can
          be sampled in each area if the areas are
          approximately of the same size.  The total
          number of samples would depend on resources
          and availability.

          If a long channel is to be randomly sampled,
          it can be divided into separate sections each

-------
                                                      4.
          with its own subsections.  The difficulty
          with the subsection approach is that the
          areas are hard to delimit on the water, as
          there are no lines marked on the water surface.

     2.   Other random sampling approaches.  As an
          alternative to a spatial design, temporal
          considerations also may be important.  Time
          intervals also can be selected randomly with
          a new set of sections again selected by means
          of a random number table each time samples
          are taken.

          A given approach may be suitable for one type
          of measurement and not for another.  Such
          factors as water movement, animal migration,
          and diurnal fluctuations cannot be overlooked
          in designing proper sampling.

C.   Tests for Random Distribution

          There are several ways in which a biological
     parameter can be tested to see if it is randomly
     distributed.  The simplest way would be to compare
     the variance and means of samples taken from an
     area.  Table 1 shows the significance of three
     such comparisons: S2 = X; s2^>x; S2«=CX.  These
     comparisons are valid if the distribution in only
     one location is being considered and if perhaps
     five or more samples have been taken in different
     sites of that specific location, and their mean
     and variance calculated  (see Statistical Calcu-
     lations) .

          The areal size, time span, and number of
     samples taken require further consideration.  A bio-
     logical parameter might be distributed randomly in
     one location and not in another, so the presumption
     that the results at one location apply to all
     locations in a water body is not valid.

-------
                                                           5.
               The main advantages of a random distribution in
          a comparison of samples or in making statements
          about parameters is that confidence limits can
          be set which will delimit the true mean.  However,
          if just a "yes or no" answer is required about
          whether there is a difference between samples,
          then non-parametrical statistical approaches can
          be used for both randomly and non-randomly
          distributed parameters.

     D.   Non-parametric Statistics

               These procedures are very useful because
          no presumptions are made about sampling techniques,
          analytical methods, time of collection and, of
          course, distribution of the parameter.  Further-
          more, they are usually simpler to calculate than
          the parametric tests.  At the selected probability
          level, the results of the test give an answer
          to the question of whether or not there is a
          difference among the compared sampled means.
          Confidence limits containing the true population
          mean cannot be calculated using these tests.
          This is one of their main drawbacks.  Suggested
          tests are below.  The Kruskal-Wallis test is
          given in the Statistical Section.  The others
          can be found in the references.
Test
Mann-Whitney U - Test
Kruskal-Wallis Test
An alternative to the t - test
Sample numbers do not have to
be equal

Comparable to a one-way analysis
of variance.  Two or more
samples can be compared.  As
above sample numbers do not
have to be equal

-------
                                                            6.
Wilcoxon's Signed Rank Test    Use for detecting differences
                               in paired samples

Spearman Rank - Correlation    This is the alternative to
  Coefficient  (rs)             calculating the correlation
                               coefficient for bivariate
                               normal distributions  (r)
                          References

Elliot, J. M., 1971.  Some Methods  for the Statistical
     Analysis of samples of Benthic Invertebrates.
     Scientific Publ. No. 25, Freshwater Biological
     Association.  Ambleside, Westmorland, England.
     144 pp.

Snedecor, G. W. and W. G. Cochran.  1962.  Statistical
     Methods.  Iowa State Univ. Press. Ames, Iowa.  534 pp.

Steel, R. G. D. and J. H. Torrie.   Priciples and Procedures
     of Statistics.  McGraw-Hill Book Co., Inc., New York.
     481 pp.

Woolf, C. M.  1968.  Principles of  Biometry.  D. Van Nostrand
     Co, Inc., Princeton, N. J.  359 pp.

-------
Table 1 Significance of the variance and mean.
measurement as indicated.
                            Transform by converting each X
Conditions of
the Samples
   = x
S2x
Distribution

Random (Poisson
Uniform, Regular
(Underdispersion or
evenly spaced)

Contagious, Overdispersion
(clumped or aggregated)
     ;2 _ ^
 1) S  = x means approximately equal
Statistical
Approach

Use parametic
statistics
Use non-parametric
statistics
Use non-parametric
statistics or
transform so than
S2 = x
Transformation

If numbers are low
transform each value
x = \/ x  or x =
 
-------
II. Sample Collection Forms

         On subsequent pages are sample collection forms,
    including directions for sample preservation.  The
    sample preservation information is mostly from the
    EPA "Methods for Chemical Analysis of Water and
    Wastewater"; however, the determinations for which
    the same preservatives are used are placed conti-
    guously.

         The forms are suggestions only and can be modi-
    fied according to needs.

-------
B.
                     SAMPLE  COLLECTION FORM

                          INSTRUCTIONS

     Project Director -  Indicate  measurements,  location,  date,
     sample points,  sampler,  time,  samples to be taken and
     whether composite or grab.   Indicate composite frequency.
     Check off  these items  on Sample Collection Form (one per
     sample).

     Sampler -  On  same form fill  in field data, correct date,
     and time of sampling if  this information is different from
     that entered  by Project  Director (I).
                          X  X  X X  X X X
Location
                                                Date

                                                Time
Sample Point_

Sampler(s)	
Field Measurements

	  Flow	

     DO
     Settleable Solids
     Specific Conductance
                                         Clarity_

                                         PH	
                                         Chlorophy11	
                                         (mis filtered)
     Gz.
          BOD
          COD
          Coliform, Total
          Nitrogen, Total
          NH3-N
          N03-N
          N02~N
          Odor
          Oil and Grease
          Phenol
          Phosphorus, Total
          Cyanide
C
G
    Composite
    Grab
Remarks:  I.  Project Director

         II.  Sampler
                                    Suspended  Solids
                                    Total  Solids
                                    Volatile Solids
                                    Sulfide
                                    Total  Organic
                                      Carbon
                                    Turbidity
                                    Pesticides
                                    Oil  Spill  Sample
                                    Fish Bioassays
                                    Algal  Bioassays
                                    Benthic Sample
container r
Heavy Metals
  Arsenic
  Chromium
  Copper
  Cadmium
  Iron
  Lead
  Mercury
  Nickel
  Zinc
Specific
  Conductance
Others
                  *"* att3Ched pages for sainPle size< Preservative and

-------
                                                              3.
           Check Equipment to be Taken for Sampling


(Always take - distilled water or deionized water, jugs, cubi-
tainers, preservatives, rubber bulbs, Van Dorn Bottler or
Kemmerer Sampler, pipettes, squeeze bottles, plastic bucket,
rope, thermometer, towels, record book and/or Sample Collection
Forms.)


	  Flow - flow meter, weir apparatus
          DO - DO meter, buret, reagents, thiosulfate soln.,
          starch, beaker, BOD bottles

          Specific conductance - meter, 2 cells  (constants of
          2x and lOx)

          Clarity - Secchi disk and line

          Benthic Samples - dredges, container, formalin

          Chlorophyll - GF/C filters, filter flask, vacuum
          pump  (hand or electric), desiccant jars, styrofoam
          container, ice

          Algal count - container, formalin
Other Samples                  Number of Containers

	  Glass jugs           	

          Cubitainers          	

	  Wide mouth plastic   	
          jars
Preservatives, etc.
          Styrofoam container and ice
          Mailing container
          H2SO4
          10 N NaOH
          HNOo
          CuS04 + H3P04
          HgCl2 (Saturated solution)
          2N Zn acetate
          1:1 HNO3

-------
                                                                4.
Container*
NA
Glass
NA
Gl.
Gl.
Gl.
       Parameter

       Dissolved
       Oxygen

       Dissolved
       Oxygen
        (by
       titration)

       pH, tempera-
       ture, Settable
       solids, clarity
Preservative

Determine on
   site

2 ml MnS04 +
2 ml ALK-I
Determine on
   site
 Holding
 Period

   NA
4-8 hours
   NA
               Metals,         Filtrate: 3 ml    6 months
               Dissolved       1:1 HN03 per
                                 liter
               (With arsenic HNOs interferes with
               reduction method; preserve arsenic
               samples with HC1)
       Total organic
         carbon
2 ml H2S04 per
liter (pH 2)
       Chemical Oxygen 2 ml H2S04
                 Demand
                       per leter
       Oil and Grease  2 ml 113804
                       per liter-4°C
               Petroleum
               Products
                       None required
Volume
Needed

  NA
300 ml
  NA
Cub.
or
Gl.



Metals, Total 5 ml HNO3
(one or all per liter
can be analyzed
from same
sample)


6 months (For all
parameters:
1 quart for
individual
analyses
unless indi-
cated differ-
ently. One
gallon for
combinations |
                                                            preservative)
*Cub.
 Gl.
 NA
Cubitainer or polyethylene jar
glass
not applicable
 7 days
                 7 days
                 24 hours    1 gallon
                 Bring to    1 quart
                 Lab as soon
                 as possible

-------
                                                              5.
Container
Gl.
Gl.
Gl.
Cub. or Gl
Parameter

Pesticides,
   PCB
Organo
Phosphates

Chlorinated
Hydrocarbons

Phenolics
Cyanide

Sulfide



Turbidity


Solids
               Acidity-
               Alkalinity ,
               Color, Thres-
               hold Odor

               Biochemical
               Oxygen Demand

               Sulfate, Odor
               Fish Bioassays
Preservative
Maximum
Holding
Period
Volume
Needed
None required
(Put teflon or
aluminum foil
under cap)
                 12 hours
                  2 days
                             2 gallons
                             2 gallons
                             2 gallons
                                1.0 g CuSO4/l +  24 hours    1 gallon
                                Cone. H3PO4 to
                                pH 4.0 -  4°C
                                (Use methyl orange indicator.  At pH 4
                                it turns  pink upon additions of HsPO^.
                                Use 2 drops indicator/100 ml of sample
                                or use pH meter)
2 ml ION NaOH/1  24 hours

                  7 days
                             1 gallon
2 ml 2N Zn
acetate per
liter

None
Available

Refrigerate at
    4°C

Refrigerate at
    4°C
                Refrigerate at
                    4°C

                Refrigerate at
                    4°C

                Refrigerate at
                    4°C
                  7 days


                  7 days


                 24 hours
                                                             1 gallon
                  6 hours


                  7 days


                  6 hours
                             20 gallons of
                             sample.  20
                             gallons of
                             receiving
                             water

-------
                                                                6.
Container
Cub. or Gl,
Wide-mouth
  Jar
Parameter
Preservative
Algal Bioassays Refrigerate
                  at 40°C
               Chloride,
               Hardnedd,
               Specific
               Conductance,
               Fluoride/
               Calcium

               Algal Count
                None Required
                4% formalin
Benthic Sample  10% formalin
               Kjeldahl
               Nitrogen
               Ammonia,
               Nitrate-
               Nitrite,
               Phosphorus
                1 ml/1 of sat
                urated
                4°C
Maximum
Holding
Period

12 hours
                  7 days
                 Indefinite

                 Indefinite


                 Unstable



                  7 days
Volume
Needed

-------
                                                           1.
III.  Algal Bioassays

           There are two general approaches in carrying out
      algal bioassays:   (1) Using indigenous algae found
      naturally in a water sample (indigenous); or (2) adding
      a laboratory-grown single culture of algae.  It is
      sufficient to say here that the use of indigenous
      algae in a bioassay is much easier than adding laboratory
      cultures.  However, if the results of a bioassay are to be
      expressed as the dry weight of algae, this parameter can
      be more easily derived from single-specied bioassays.
      (There are many more advantages and disadvantages, to
      both approaches.  These are discussed at length elsewhere
      [Tunzi, 1972]).

           Directions to follow in carrying out algal bioassays
      will be divided into several sections:

           Laboratory Bioassay directions
           Cell Mass Measurement
           Measuring Dry Weight
           Measuring Algal Chlorophyll
           Maintaining Algal Curtures in the Laboratory

      A.   Laboratory Bioassay Directions
           (Bioassays utilizing Indigenous Algae)

           Materials

                Glass or polyethylene containers  (e.g.,
                     cubitainers) for sample collection.
                Ice chest, ice.
                Filtration apparaters, vacuum pump.
                Erlenmeyer flasks (250 or 500 ml each),
                     acid rinsed  (0.1NH Cl), then rinsed with
                     tap water and distilled water; water
                     volume marks should be indicated on
                     side of flask.
                Waterproof labeling pens; black ink
                     Examples:  Sanford's Sharpie #49;
                     Scientific Products #P1226, Fine Tip
                     Marker.

-------
                                                2.
     Foam rubber stoppers for erlenmeyer flasks;
          rubber stoppers for same.
     Light box capable of 400 ft. candle
          illumination at 20°C; check uniformity
          of light with light meter.

Method

1.   Collect samples in glass or polyethylene
     containers.  Collapsible cubitainers are the
     most convenient.  If the samples are from
     eutrophic water, about 1 quart is sufficient.
     Otherwise collect 1 gallon.  If spiking of
     samples with nutrients or effluent is antici-
     pated, then collect 1 gallon.

2.   Keep the samples out of the sunlight.  If
     necessary, surround samples by ice, but do not
     freeze.  It is usually not necessary to
     ice if transport time is less than 1 hour.

3.   If the samples are to be shipped a long dis-
     tance, they can be put into styrofoam containers
     and surrounded by ice.  The algae in the samples
     will remain cool and viable for about 12 hours
     during transit.

4.   At the laboratory filter part of each sample
     for dry weight or chlorophyll determination
     (50 to about 400 ml is needed for eutrophic
     and 1 to 2 liters for oligotrophic waters).

5.   Choose sample concentration.  Suggested
     additions of effluent to receiving water are
     1%, 5%, 10%, 50% of total volume.  When pre-
     paring nutrients, prepare high concentrations
     so that additions will be   5 ml/liter of
     sample.  Otherwise the distilled water used
     to dissolve the nutrients will dilute the
     sample so that comparisons with a control or
     with other nutrient additions is difficult
     (see Table 1, components of Macronutrient
     Medium for Algal Cultures, Section III-E.

-------
                                                  3.
 6.    Prepare about 1 liter of each concentration
      and mix well before adding the water to the
      replicates.

 7.    Prepare at least 4 replicates per sample type.
      Use black ink, waterproof pens for labeling.

 8.    Number the sample containing Erlenmeyer flasks
      with a waterproof marker pen.  Replicates
      should be numbered; e.g., 1-1, 1-2, 1-3, 1-4;
      2-1, etc.  Number the flasks permanently on
      the frosted parts.  If a flask has consistent
      erratic results compared to replicates of the
      same series, discard it.

 9.    If samples are to be incubated without addi-
      tions, the flasks can be filled directly from
      the sample container.  First shake sample
      well; then put 125 ml into the 250 ml flasks
      or 150 ml into 500-ml flask.  Add the water
      to the volume marks on flasks.  Extreme
      accuracy is not important.

10.    Cover the flasks with foam rubber stoppers.

11.    Take an initial cell-mass measurement on 2
      of the 4 replicates  (see Cell Mass Measure-
      ments) .

12.    Incubate the samples under 400 ft. candles
      of light at 20°C.  If higher temperatures
      are used, the cultures grow too rapidly.
      There does not appear to be much advantage
      in intermittent lighting.  The main point is
      uniform light.  A light meter should be used
      to check that all areas of the incubation
      shelf are receiving approximately equal
      light ( + or -10%).

13.    Measure the algal mass at about the same time
      every day.

-------
                                                     4 .
    14.   Before measuring the mass, plug the flask
          with a rubber stopper and shake it vigorously.
          This promotes aeration and lessens the possi-
          bility of attached growth.

    15.   Expected growth curve, calculations, and
          reporting forms are shown in Figures 1 and
          2.  A completed reporting form is shown in
          the Statistical Section.

B.   Cell Mass Measurement

     Direct Cell Counting

     Materials

          Whipple micrometer reticule
          Stage micrometer
          At least 4 Sedgwick-Rafter Chambers
          Pasteur pipette or automatic volume delivery
               pipette.

     Procedure

     1.   Calibrate the microscope and Whipple disc
          (see Section 301 C, page 731, Standard
          Methods, 13th Edition), using a stage micro-
          meter.

     2.   Fill each Chamber with water from one of the
          replicates by means of a Pasteur pipette or
          automatic volume pipette.  Let the chambers
          settle for 5 minutes  (Chamber volume is 1 ml).

     3.   Usually 2 strips are counted in each chamber
          and a factor is used to convert the number of
          cells counted to cells per ml for the sample.
          Make two counts of the cells in each chamber
          and record average.

-------
                                                5.
4.   Dilute aliquots from the flasks  (with distilled
     water) if the cell concentration becomes too
     high.  Serial dilutions may also be made to
     check accuracy of counting technique.

Turbidimetry by Turbidity Meter

Materials

     Hach 2100 turbidimeter or equivalent.
     Tubes for reading in Hach 2100.

Procedure

1.   Calibrate the Hach Turbidimeter by means of
     the standard  (the one supplied with the machine
     is adequate).  The machine is set at the
     value indicated on the standard tube (usually
     50-80 JTU's).

2.   Read turbidity in each sample.

3.   Obtain an average reading by watching the
     needle for 10-15 seconds.  Fluctuations in
     readings are  to be expected.

4.   Use the same  sample tube for each of the
     replicates of the same samples.  It is not
     necessary to  rinse the tube with distilled
     water between replicates of a single sample;
     however, the  same tube must be well-rinsed or
     even washed between different samples.

5.   When the maximum turbidity reading is reached
     (after incubation of sample), combine the
     water from the replicates, mix, and use for
     dry weight measurements  (see Section on
     Weighing).  (Maximum growth is reached when
     readings are  approximately the same for
     2-3 days [see Statistical Procedures for
     approach to evaluating differences in the
     samples].)

-------
                                                6.
6.   The lower limits of the detectable turbidity
     is about 2000 cells/ml, but 10 fold increases
     changes turbidity only about 2 units.

Absorbance by Spectronic 20

Materials

     B & L Spectronic 20; Spec 20 tubes

Procedures

1.   Set the wave length at 600 nm.

2.   Using special Spec 20 tubes, read absorbance
     for each sample.  Many tubes are required,
     since the sub-samples have to be poured back;
     generally 20-30 ml volume is utilized at
     each reading, and discarding this would
     deplete the incubating sample too drastically.

3.   Take readings daily at about the same time.

4.   When maximum value is reached and stabilized,
     express terminal values as dry weight.  The
     water from the replicates can be combined to
     give enough volume to yield weight differences
     and the individual reading used for statistical
     comparisons  (see sections on Weighing and on
     Statistical Treatment).

5.   Depending on the size of the algal counts, the
     Spectronic 20 is good starting at about the
     100,000/ml level.  It is an instrument rather
     insensitive to any but large cell number changes.

In Vivo Fluorescence

Materials

1.   Turner Model III Fluorometer (or equivalent) with
     an ultra-violet light source F4T5, the red-
     sensitive R-126 photomultiplier, Corning 5-60

-------
                                                7.
     primary filter and 2-64 emission filter.  The
     general purpose photomultiplier can be used
     for dense cultures {«1C)4 cells/ml and up) .  The
     R-126 photomultiplier is sensitive down to
     1000/ml.  In contrast to the turbidity meter,
     10 fold increases in cell number changes
     fluorescence 50-100 units.

Procedure

1.   Zero the machine using the black plastic tube
     which comes with the machine.  Check the zero
     calibration when changing from slit to slit
     or after reading every 3 or 4 samples.  There
     are slits on the machine - 1 X, 3 X, 10X, 30X,
     the latter allowing the most light to pass
     through.  Do not use the 1 X slit, as response
     of the machine is not linear with this slit.  On
     top of the machine a dial reads from 0-100.
     Record both slit and dial values for each
     reading taken.

2.   Establish a calibration factor for converting
     readings from one scale to another.

3.   Follow sample incubation, etc. under 1-14 of
     the Laboratory Bioassay Directions, IIIA.

4.   Shake the flasks thoroughly immediately
     prior to reading as clumping of algae can
     cause fluctuations in the readings.

5.   Pour 5 ml of water directly into cuvette
     and take reading.  Each reading only takes
     about 5 ml of sample, so that once the
     aliquot is read the water used can be
     thrown away.

6.   Rinse tube as follows:  for replicates of
     the same sample, rinse the tube with a
     subsample from the next replicate; between
     different samples, rinse the tube with dis-
     tilled water.

-------
                                                     8.
     7,   Wipe the outside of the cuvette dry before
          inserting it in the holder.

     8.   Shake the next replicate, then take the
          reading of the tube in the machine.  This
          should give about a 10-15 second period
          between readings.  It is important that the
          time span be consistent.

     9.   When growth reaches a plateau (i.e., the
          amount of fluorescence does not seem to
          increase), combine the replicates for either
          chlorophyll a or weight measurement.  Since
          algal chlorophyll fluorescence is the primary
          cause of sample fluorescence, chlorophyll a^
          determination is the more reasonable one to
          make (see Measuring Algal Chlorophyll, begin-
          ning with Filtration, D-2).  There is usually
          not enough sample for both measurements.

C.   Measuring Dry Weight

     1.   Wash 4.25 cm GF/C Whatman filters by placing
          them in a pan of distilled water.  Loose
          fibers will separate from the filters.

     2.   Place filters on a towel to partially dry.

     3.   Place them separately on a sheet of aluminum
          foil.

     4.   Dry them for three hours at 90°C.   (Put
          into a desiccator if filters are to be
          stored for more than 5-10 minutes before
          weighing.)

     5.   Number them lightly on their edges with
          a soft lead pencil.

     6.   Weigh filters to nearest hundredth milligram.
          Handle the filters with tweezers, grasping
          the edges.

-------
                                                      9.
     7.   Put them into small envelopes with their
          weights and number written on the outside
          of the envelope.

     8.   When needed, filter as much sample as will
          go through the filter in about two minutes at
          low vacuum, less than 5 inches of mercury.

     9.   Record volume filtered in liters.

    10.   Double the filter, algal side inward.

    11.   Place filter on aluminum foil and dry for
          at least three hours at 90°C.

    12.   Remove them from oven and, using forceps to
          transfer, weigh them after they have cooled
          for about five minutes.  Cooling in a desiccator
          may be desirable but appears to be of limited
          advantage.

     13.  Subtract original weight of dried filter
          from final weight and express results as mg/1
          of dry weight.

D.   Measuring Algal Chlorophyll

     Introduction

          There are two practical approaches to measuring
     the concentration of indigenous algae in water.
     They can be counted directly or may be enumerated
     indirectly by determining the chlorophyll content
     of a sample of water (or performing some other
     mass measurement).  The following method details
     procedures for measuring chlorophyll concentration.

     Materials and Equipment for Laboratory Analysis

          Whatman GF/C glass fiber filters, 4.25 cm
               diameter

-------
                                                10.
     Filter-holding apparatus: either
          Millipore or Gelman
     Covered small glass jars containing desiccant
     Freezer
     Scissors
     Tissue horaogenizer with teflon pestle: either
          Kontes Glass Co. No. 885-380-0023; or
          A. H. Thomas Co. No. 4288B
     Acetone,  (90% acetone, 10% water) spectro-
          photometric grade
     Centrifuge tubes (if Kontes tissue homogenizer
          not used)
     Centrifuge adapters (necessary only if Kontes
          tissue homogenizer used)
     Pasteur pipettes
     Beckman DU Spectrophotometer, or equivalent
     Cuvettes  (for Spectrophotometer), 1 cm or
          small volume 5 cm ones
     Hydrochloric acid,  IN

Method

1.   Sample Collection

          It is best not to collect a single
     grab sample.  Instead an integrated sample
     should be taken by collecting small (about
     250 ml) equal-volume sub-samples at a given
     site and depth over a time period, such as
     10 minutes.  These sub-samples should be mixed
     together in a plastic busket and transferred
     to a transport container (e.g., a 1-gallon
     cubitainer).

     Consult section on Sample Collection for further
     considerations on representative samples.

2.   Filtration

     1.   As soon as possible after collection
          (the sooner the filtration the more
          valid the data; e.g., a sample stored
          in the dark on ice should be filtered

-------
                                                 11
          within 4-5 hours, if possible), filter
          under low vacuum as much water as will
          go through a Whatman GF/C filter within
          about two minutes.

     2.    If possible, prepare 4-5 filtrations of
          water from the same sample.

     3.    Record the water volume that has been
          put through each filter.

     4.    Double the filters, algal side inward
          and put them into a small jar with
          dessicant and then into the freezer
          for storage.

     5.    Extract for chlorophyll within three
          weeks of filtration.

3.    Extraction Methods
     1.
Figure 1.  Folded Filter

  Refer to Figure 1.  Using scissors,
  carefully trim off the white border to
  the edge of the green algae-contining
  section.  Discard white section.  Cut
  each.trimmed filter into smaller pieces,
  putting these directly into tissue
  grinder homogenizer.
      2.    Add 5 ml of acetone.

      3.    Grind filter with teflon pestle.

-------
                                            12.
 4.    Ground-filter-and-acetone mixture should
      only get slightly warm to touch during
      the process.   Move the tube slowly up and
      down while grinding, ceasing the grinding
      whenever the  tube becomes warm to touch.

 5.    Keep ground-filter-and-acetone mixture
      out of strong light by covering it with a
      towel.

 6.    Pour the mixture into a centrifuge tube,
      cover with parafilm (or other cover),  and
      shake well.  If Kontes tissue grinder is
      used, the container may be centrifuged
      directly (if  adapters are present),  thus
      making it unnecessary to transfer the
      ground mixture to a centrifuge tube.

 7.    Let set in the dark for 20 minutes at
      least.

 8.    Shake the centrifuge tube well again after
      the 20-minute waiting period.

 9.    Centrifuge the tubes for 10 minutes at
      2500 - 5000 RPM, preferably at the higher
      RPM's.  Tap the tubes to bring particulate
      matter to the bottom.   Recentrifuge.

10.    By means of a Pasteur pipette, carefully
      draw off enough of the supernatant to fill
      a 1 cm cuvette  (about 4 ml).

11.    Take absorbance readings at 750 nm,  blanking
      against 90% acetone.  If above 0.005-0.008
      Optical Density (O.D.), re-centrifuge.

12.    Take absorbancy at 663 nm, blanking against
      90% acetone.

-------
                                                 13.
    13.   Add 2 drops of IN HC1 to each cuvette and
          re-read absorbancy after 2 minutes at 663 nm.
          One cm cuvettes do not have to be shaken to
          disperse the acid, but it is necessary to
          shake those of larger dimension.

    14.   If the absorbancy of the sample is too
          high for the spectrophotometer scale,
          the sample can be diluted.  Keep an
          accurate measure of the total amount of
          acetone used, as this volume is necessary
          for calculations.

4.   Calculation of Results

     1.   The amount of chlorophyll ig phaeophytin per liter = 26.7 [1.7 (ODa) - ODbl x Ac
                                               W x cm

     This value may be 0 or negative, indicating no phaeo-
     phytin in sample.

3.   Total chlorophyll a_ per liter in any sample is the
     sum of the values obtained in 2 and 3.

-------
                                                     14.
     4.   If more than one filter was prepared,  the chlorophyll
          concentration values from the 4 or  5  filters can be
          used to establish the standard deviation/ standard
          error, and 95% confidence limits of the chlorophyll
          values for the sampling site  (see Statistical
          Procedures).

Discussion

     As was mentioned in the introduction, there are numerous
ways to determine the amount of algae present in a sample.
These include direct counting, weight measurement (biomass),
trubidity determinations, and chlorophyll measurement.

     Counting is a slow process.  Its principal drawback
however is that algae vary greatly in size, so  that to get an
estimate of the mass of algae in water each separate species
has to be measured and the total volume obtained by multi-
plying the number of each species times its volume.    (This
value can be converted to mg/1 of algae by assuming a
specific gravity of about 1.0 for the algae.)

     An extraction of algal chlorophyll is one  of the
standard methods of estimating standing crops in water
(Strickland and Parsons, 1965).  This is true because
chlorophyll is a necessary constituent of green plants,
serving as a catalyst in the initial carbon fixation process.
One problem in determining concentration, though, is that
the ratio of chlorophyll to cell mass can be  changed,
especially by varying the light intensity.  The chlorophyll
a to cell carbon ratios are in the range 1:40 to 1:100.

     Biomass might also be determined.  One disadvantage of
this procedure is that the volume of material available for
filtration is usually small so that the resulting weight of
the cells retained by the filter is not too much greater
than the weight of the filter itself.   This causes a wide
variation in results.  If one wishes to determine cell
weight, though, the method can be employed.   Empirically it
has been observed that the cell dry weight is approximately
equal to two times the cell carbon (Maciolek, 1962).

     A summary of the advantages of chlorophyll as a measure
of mass would include the following points:

-------
                                                     15.
     1.   The amount of chlorophyll  is determined
          spectrophotometrically-  The precision of
          this determination  is greater than that  for
          any cell-count method.

     2.   Large volumes of water can be filtered to
          determine chlorophyll.  Only one ml at most
          is used in direct counts  (and hence is not
          too representative).

     3.   The green chlorophyll color of algae is  the
          substance seen when one looks at algae in
          water; therefore, measuring chlorophyll  in
          water usually is a  direct way of quantifying
          the size of an algal bloom.

E.   Maintaining Algal Cultures in the Laboratory

          The EPA Report Algal Assay Procedure -
     Bottle Test  (1971) available from Thomas Maloney,
     EPA, NERC, Corvallis, Oregon gives useful infor-
     mation on culturing algae.  Pure cultures of
     algae can be obtained from NERC, Corvallis or
     from the Culture Collection of Algae, Dept, of
     Botany, Indiana Univ., Bloomington, Indiana.
     Direction are available  in the  Indiana University
     listing for media for specific algae.

General Directions

     These are applicable for Selenestrum, Scenedesmus and
mixtures.

     1.   Stock cultures can be kept viable for months if
          they are kept out of direct light.  They may be
          stored at normal room temperature in a shelf of
          the laboratory where the light is constantly
          subdued or at least off at night.  Cultures kept
          under constantly high light will go through a
          growth phase, exhaust nutrients, and usually die.

-------
                                                 16.
2.   Use aseptic techniques for transferring uni-
     algal cultures.  Pasteur pipettes, flasks and
     stoppers (or other covers) can be autoclaved or
     heated to 90°C if an autoclave is not available.

3.   Table 1 shows a simple mixture of nutrients
     which will promote growth.  Stock culture can
     be kept in polyethylene or glass bottles.  Micro-
     nutrients are not needed as there appears to be
     ample present as contaminants in the chemicals.
     If they are desired, utilize those given in the
     EPA Corvallis publication (or add to 1 liter
     macronutrients 1 ml of a solution prepared by
     adding a small amount of bouillon cube to 100
     ml water) .

     Table 1.  Components of Macro-nutrient Medium for
     Algal Cultures

     Component                     Amount in g/1

     NaNO3                             6 g/1
     CaCl2                             0.6
     MgS04                             1.8
     NaCl                              0.6
     KH2P04                            0.875
     K2HP04                            0.375
     NaHC03                           10.0
     Fe(S04)2 (NH4)2   '  12 HOH      860 mg  Dilute in
         EDTA  •  2 HOH              660 mg  one liter
4.   Add 10 ml of each chemical except the iron solution
     above to a two liter flask and bring volume up to
     1 liter with ion-free or distilled water.

5.   Cover the flask with a beaker and heat to 90 °C or
     autoclave for 20 minutes.

6.   Autoclave the iron solution or heat to 90 °C.  The
     iron solution should be kept in a screw-cap flask.
     Loosen caps when heating or autoclaving and tighten
     when cool.

-------
                                                            17.
           7.   Add 1 ml of iron solution to liter of the macro-
                nutrients when the latter has cooled.

           8.   The nutrient solution can then be dispensed to
                sterile smaller flasks  (250-ml ones are suitable)

           9.   Inoculate the small flasks with the stock algae.
                Put under constant light of about 400 ft-candles.
                Solutions should be densely green in about five
                to seven days and ready for use.

                              References

Anonymous, 1971.  Algal Assay Procedure.  Bottle Test, NERC
      Environmental Protection Agency-  Corvallis.  82 pp.

Maciolek, J. A. , 1962.  Limnological Organic Analyses by
      Quantitative Bichromate Oxidation.  Res. Rept. 60. U.S.
      Fish and Wildlife Service.  61 pp.

Tunzi, M. G., 1972.  Algal bioassays:  Examples, advantages,
      and limitations of current approaches, 173-197 pp. in
      Proceedings of Seminar on Eutrophication and Biostimu-
      lation.  California Dept. of Water Resources.  Sacramento.
      229 pp.

Strickland, J. D. H., and T. R. Parsons.  1965.  A Manual of
      Sea Water Analysis.  Fisheries Research Board of Canada.
      Bull, No. 125.  Ottawa, Canada.

-------
Algal Cell
Concentration
(any type of
 measurement;
  cell count,
  optical
   density)
  fluorescence,
  chlorophyll
   concentration)
                10
                                                         Days
Algal Growth Data
Sheet Parameters
Initial chlorophyll
Concentration
Peak chlorophyll
Concentration
Increase in chlorophyll
Concentration
Days to reach peak
Maximum Growth rate
J, day -1
Value from Figure
(A) 10
(E) 90
(E minus A) 80
4 days
£.*(-£.) - !$).!.«
t i
              x0 •  cell concentration at beginning of maximum growth

              x,  «  cell concentration at end of maximum growth

              t  -  time
              A
              u  «  maximum specific  growth rate, (day "1)

    Maximum growth rate is derived from the  steepest part of the  growth curve,
    utilizing the log of the cell concentration at the beginning  and end of
    the curve.
    Fig.  1.   Typical algal growth response.   The  values  are the
    means of  the replicates,  whose range are indicated by  the
    vertical  lines.

-------
                        Figure  2
ALGAL GROWTH DATA SHEET

Sample
Location



^


-





Rubber











Average Initial
Chlorophyll
Concentration
•-








	 .....
jig Chi a/1
Average Increase
In Chlorophyll
Concentration

»









Average Maximum
Chlorophyll
Concentration










)
Average Maxima
Growtn Kace
/» . - -,
V day* x











NO. Of
n»«« »A
usys to
Beach Peak










The results below connected by underlining are not different  from each other at the 95Z confidence level.
                                          on the reaulta of  four replicates.
                                                        Average baaed
    Concentration
 Increase jig Chi a/1
        le Huaber
 Concentration Maximum
     ^g Chi  a/1
    SIITBTT le Number
 Maximum Growth Rate
 Fig. 2.   Data reporting  sheet with multiple range section on  the lower part.
 for elaboration.
                                            See  statiscal section

-------
                                                          1.
IV.    Statistical Procedures

       A,  Introduction
                Statistics is a scientific method involving
           collection, analysis, and interpretation of
           numerical data.  An understanding of basic statis-
           tical principles and procedures is helpful to both
           field and laboratory workers.  The mathematics
           involved is simple except for advanced procedures
           which are infrequently used.

                The data collected for statistical treatment
           are measurements or observations of a characteristic
           of a population.  The population can be the cells
           in a series of flasks, the nitrate ions in a lake,
           the oligochaetes in the sediments of a bay, etc.
           Thus the population can have discrete physical
           boundaries or ones which the planner sets himself.

                Almost without exception we cannot make all
           the desired measurements of a population, so that
           instead we take a sample from the population.   From
           the sample mean, predictions can be made about the
           same characteristic in the entire population.   By
           sample  is meant a series of measurements, although
           in reality we would have to take a separate sample
           for each measurement.

                Greek letters are used for population statis-
           tical terms and English letters for sample ones.
           The measurements from a population are called
           parameters and those of the sample called statistics.

                Assuming that we have measurements from a
           population, the above can be clarified by the  following
           table.

                                  Population         Sample
                                  Parameter          Statistic

                Mean                u (Mu)               x
                                     2                   o
                Variance            ©•                   s

                Standard
                Deviation           0- (Sigma)            S

-------
                                                 2.
B,
Definitions
    1.
    2.
     Mean (x).   The average value calculated by
     dividing the sum of the measurements by the
     number  (n) of measurements.

     Variance  (s2).  The variability or spread of
     the data about the mean (See Figure 1).
          u
          c
          0)
          3
          er
          a;
                  Mean
                                Mean
         Figure 1.  Two sample measurements with
         equal means but differing variances.
    3.
    4.
     The standard deviation  (s).
     of the variance.
The square root
     The standard error or the standard error of
     the mean (S^).  The standard deviation of the
     sampling distribution of means.
    5.   Degrees of freedom - usually equal to N -1.

    Calculations

         The calculations for the above statistics
    are very simple.

         Given the data below collected from a
    population with individual measurements listed
    under x.
X
11
12
15
16
11
x - x
-2
-1
2
3
-2
(x - x)2
4
1
4
9
4
x2
121
144
225
256
121
    65
                               22
         867

-------
                                              3.
  (x-x)2 is the sum of the squared deviations which
is called the sum  of the squares (SS).

x2 is calculated because it is used in the working
formula for the variance.
The mean being: n
               £
       X =      1
=  65  =
   5~
                               13
The variance is:
               n
       s2 =
               n-1
                        =  22  =  5.5
                           4~
The working formula is simpler because the sum of
the squares does not have to be calculated.
               'n   \
                7x   2
                z_xi /
                  n       =
        n'
                                867 -
            n-1
Standard deviation
                                            = 5.5
Standard error
                                  2.35
                   =  2-35  =  1.05
                      2.24
By use of the standard deviation confidence limits
for the measurements can be set:
      x  + IS includes 68% of the sample measurement
      etc. (see below).
A*-
s r~
/
•
-------
                                                  4 .

    Confidence limits of the population are much
    more important.  That is we want to set limits
    which bracket the true population mean or average
     ( /i  ) -

    This can be done by using the standard error S~
    and  t   table values for the degrees of freedom
    in our sample.

    x  +  s~    will include the true population mean
       ~~   .A.
     (p. ) 68 out of 100 times.

    x  ±  (t  Q> Q5) sx will include^ 95 out of 100 times

    *  ±  (t  0/6Jl) sx wiH include ju 99 out of 100 times
    The difference between sample confidence limits
    and population confidence limits must be clearly
    understood.

    Using the  last formula in the above data:

    x +   (4.60) (1.05) - 13± 4.83

    The t(Q.05) and fc(0 01) are found  in a t table for
    a range of n-1 values  (a few values are given below)
                          Degrees of Freedom  (n-1)

              6 DF   5 DF    4 DF    2 DF      1 DF

    t(0.05)   2.45   2.57    2.78    4.30      12.71

    t(0.01)   3.71   4.03    4.60    9.93      63.66
    By utilizing the t values  for various degrees  of
    freedom, we can see the  importance  of high  numbers
    of replicates in sampling.

D.  Group Comparison of Two  populations

    1.   A comparison consist  of two  steps.

         a.  At test to determine  if the sample means
             come from one or  two populations.

         b.  Confidence limits can  be set for  the  sample
             means if the t  test is significant.

-------
                                                   5.
x  •

..2.
     Special formulas for group comparisons

                                 2
     a.   p  =  x  +  t (0.05)   p
                   ""             n

          Note:  If the t test is not significant then
          both samples come from the same population.

          The confidence limits for the population means
          may overlap slightly even when the t test
          is significant.

     b.   Polled variance

                 n   0   / n   \ „     n
                                 +   1
                          Si                  n2
          These formulas can be used whether or not
          nl = n2 '
 3 .   Example of a group test
X
32
31
52
44
159
625
39.75
6625 -
X2
17
35
22
24
98
2574
24.50
(159)2 2574 - (98)2
4 + 4
                      (4-1) +  (4-1)

-------
s 2 =  6625 - 6320  +  2574  - 2401
                    6

s 2 =  478   =  79.7
 ir      /-
       39.75  -  24.50        15.25
                                     =  2.42
       79.7(1/4+1/4)          39.85
For 6 degrees of freedom t[0.05] = 2.45.  Therefore,
there is no difference between the set of data.

-------
                                                    7.
Comparison of Two Groups by Pairing

1.   If two samples are not independent, then a pairing
     test can be used to compare them.  Of course,
     nl = n2*  Generally, high values in one sample are
     associated with high values in another.

2.   Example of the data from a pairing test:

                       X! - x2
       X;L     X2      difference      d^






I
X
f n
10
9
8
11
14
12
64
10.64
\
19
18
17
19
22
18
113
18.83

-9
-9
-9
-8
-8
-6
-49
xd= -8.17

81
81
81
64
64
36
407


                                        n
                                            d2 = 407
                                   2
     Variance of the difference  sd  =
       2
            407 _ (49) 2      407 _  2401
                                               n - 1
     sd  =  •  -     6     _ _ 6        =  1.4

-------
          The standard error is:
          s        7
           xd   - / -l-4    -  ./ 0.233 =  0.48
                 V   6        v

          t  =   I *d I    =   8.17    =   17.0
                 s_          0.48
                  xd

          Since the t value of 17.0 is greater than the t
          value for 5 degrees of freedom (0.05) , there is a
          significant difference between the sample means.

          The 95% confidence limits for the difference
          between the samples can be calculated by using the
          following formula:
                           +   t(0.05)
                                       ;
                                          n

F.   Comparison of data from more than two groups

     1.   Analysis of variance is the technique used to
          compare one characteristic from three or more
          populations.

          Three or more populations cannot validly be
          compared by the sequential use of a t test.  It
          is especially bad to single two groups out of a
          large number and subject them to a t test to see
          if they differ.

          The completely randomized design is used for
          comparing the means from three or more populations.

     2.   The following calculations for unequal sample size
          can also be used when the same number of measurements
          are made for all samples:

-------
                                               9.
Sample






r
E




	 i_
10
11
12
13

x TiT
x2 534
n 4
~ 11.50
(£x)2 529
n

	 2_
6
12
14


—
376
3
10.67
341.3

(133)2
	 3_
10
12
10
9
14 	
~5lT ^_ = 133
621 ^_= 1531
3l- "
11.00
605 51 = 1475.3


Correction Factor =   12     =  1474.1

Total Sum of Squares = 1531 - 1474.1 =56.9

Treatment Sum of Squares = 1475.3 - 1474.1 = 1.2


              Analysis of Variance Table

Source of Variation     DF    SS    Mean Square     F

            Total       11   56.9

            Treatment    2    1.2      0.6        0.097

            Error        9   55.7      6.2

      Treatment mean square
F  =  Error mean square

F values for 2 degrees of freedom in the numerator
and 9 in the denominator for the 0.05 probability
level is 4.3, much higher than our F value.  There-
fore, there is no significant difference between
our sample means.

-------
                                                        10.
     3.   If the F value is significant, a multiple range
          test must be used to determine which samples
          actually differ.  Duncan's new multiple range
          test for equal replication (p. 107) and unequal
          replication  (p 114) are good ones to use  (Steel
          and Torrie,  1960).

G.   Non-parametric methods

          These approaches are useful when it is not certain
     that the normality of the population distribution and
     its means and variance are the same as that of the sample,

          One of the more useful tests is the Kruskal-Wallis
     test which compares medians from 2 or more populations.
     An example of this ranking test is given below:
Data Set I
6
8
8
12
14
30
23
Rank
2
3.5
3.5
6
7
11
10
Data Set II
21
15
4
11



Rank
9
8
1
5



      Median - 12       n]_ =7    Median = 13     n2 =4
                        Rl =43                   R2 =23
     Median-the middle value for odd number of values, and
     the mean of the two middle values for even number of
     values

                 11 + 15   =26   =13
                    2          2
     An H value is then calculated where-

        k = number of samples

        T = total number of measurements in all sets

-------
                                                    11.
   k-^ = degrees of freedom

   R = sum of the rank

   n = number of measurements
H =
  12 = a constant

       12
     T (T+l)
                          - 3  (T+l)
For the above data:

      12        432
H   11 (12)

H = 0.035
                         232
                                -3  (12)
     The hypothesis made is that the populations are
identical.  When the H value is greater that the chi-
square value for k-1 degrees at the 0.05 probability
level, then there is a difference between the sample
medians.  For 1 degree of freedom this =3.84.  So the
hypothesis is accepted.  (Chi-square tables are found
in most mathematical handbooks and statistic books.)

-------
       Sample   3/17/70
TABLE 1 - BIOASSAY DATA WITH MULTIPLE RANGE STATISTICAL PRESENTATION

                       ALGAL GROWTH DATA SHEET

                          yg Chi a/1
Location
Redwood City Sewage
Treatment Plant


downstream location



San Francisco Bay
Number
12
1
2
3
4
5
6
15
Average Initial
Chlorophyll
Concentration
7.2
2.0
2.0
2.1
2.1
2.5 \
2.8
3.0
Average Increase
In Chlorophyll
Concentration
2.0
33.6
0.0
66.6
69.8
40.3
26.1
6.8
Average Maximum
In Chlorophyll
Concentration
9.2
35.6
2.0
68.7
71.9
42.8
28.9
9.8
Average Maximum
^Growth Rate
Jib. days -1
0.13
0.94
0.00
1.09
0.61
1.67
1.57
0.93
No. of
Days to
Reach Peak
6
6
0
3
3
3
3
2
The results below connected by underlining are not different from each other at the 95 percent confidence level.   Average
based on the results of four replicates.
Sample Number
Concentration
Increase >ig Chi a/1
Sample Number
Concentration Maximum
jig Chi a/1
Sample Number
Maximum Growth Rate
ufe, day'1
2
0.0

2
2.0
2
0.00
12
2.0

12
9.2

12
0.13
15 6 1
575 267! 3375"

15 6 l
978 2oT9 3575

4 15 1
0710" 0.93 0794"

4"0?3
*A
1.09
5575
5877

6
1.57
4
4
71.9

df

-------
                                                           1.
V.  Fish Bioassays

    Sources of Fish

    1.   Collection
              Test fish may be collected from a large body of
         water using a seine; from a small slough, one can use
         dip nets.  Usually a collection permit is required
         (get this from state fish and game departments).

              Since collection of suitable and adequate  number
         of fish is somewhat uncertain, it should be done only
         if time is of little importance, if the locations of
         the desired fish are well-known, and if there is no
         other way to obtain fish.

              After collection, place fish in a suitable
         transport container.  A five-gallon plastic bucket
         with a snap-on lid can hold 100 small (up to 2  inches)
         fish for short distances.

              If one collects his own fish, he must have
         several good battery-operated aerators (e.g., the
         Jorgensen portable aerator - $7.00; Lewis Air Pump -
         $3.50).  Take extra batteries and check at least
         every hour to see that they are not run down.  Aerate
         fish on the way back to the laboratory.

              Aeration is accomplished by connecting aerator
         to a flexible line with an airstone at the end.  The
         airstone should be weighted or it will float to the
         water surface.  A large  (No. 10) rubber stopper with
         a hole in it  (to put the tube through) will hold the
         aerator under water.
    2.   Purchase
              Commercial aquarium and fish stores generally
         charge too much to make them a reasonable source of
         fish.  Names of dealers who supply fish for bioassay
         are generally available from agencies such as State
         Water Resources Control Boards and fish and game
         agencies or from other persons who carry out fish

-------
                                                  2.
bioassays.  The price per fish delivered is
usually 20-50 cents, depending upon the species.
This is normally the most economical way to get
fish.  Be sure not to acquire more fish than
needed.  It is usually easier to purchase fish in
lots as required than it is to maintain fish for
many weeks in an expectation that they might be
needed.

     Possible sources of fish from agencies include:
(Normally the hatcheries supply fish only to other
public agencies)

     Striped Bass
          Bureau of Reclamation, Tracy
          Phone  209-935-3122
     Rainbow Trout
          American River Hatchery
          Phone  916-351-0314
     Salmon, Steelhead Trout
          Nimbus Fish Hatchery
          Phone  916-351-0383
     Black Bass, Blue Gill, Shad, Catfish
          Elk Grove Hatchery Phone  916-685-9555
    Two    commercial fish dealers in the San
Francisco Bay area are:
     William Putman
          5449 Modoc St. Richmond, CA   94804
     Alex Fish Company
          2235 Juniperberry Drive
          San Rafael, CA
A list of commercial fish dealers in California is
available from the California Fish and Game Department,

-------
                                                        3.
 Recommended Species

      Ideally,  the best fish to use are the most abundant
 or economically significant young small ones found in the
 receiving water area.   However, this may be impractical or
 impossible to  carry out as they would be too difficult
 to catch or only available during specific seasons.

      A standard test species available throughout the
 year would make comparisons between tests more meaning-
 ful.* Fish most commonly used in California are:

                euryhaline

      3-spine stickleback    Gasterosteus aculeatus
      Threadfish shad        Dorosoma petenese
      Killifish              Fundulus parvipinnis
      Striped bass           Roccus saxatilis

                Fresh Water

      Golden shiner          Notemigonus chrysoleucas
      Channel catfish        Ictalurus punctatus

 Maintenance of Fish

 1.    Disinfection

      a.    A new group  of fish should be disinfected by
           putting them (for about  5-10  minutes) in
           water containing both .025 ml/1  of formalin and
           0.05 mg/1 malachite green  (Leteux  and Meyer,
           1972).

      b.    Fifty fish can be put into approximately two
           gallons  of water.

      c.    Watch them carefully,  and  remove them immediately
           if they  show signs  of distress  (floating up
           slightly sideways).

 2.    Aeration

      a.    Before adding  fish  to water,  aerate water for
           12-24 hours.
*Table 3 lists animals suitable for bioassay in Hawaii

-------
                                                       4.
     b.   A twenty-gallon aquarium can hold 100 small fish
          if it has two activated charcoal filters and
          one to two airstones running constantly.  Some-
          times it is better to replace the water or part
          of it every three or four days, but aerate the
          water for 12-24 hours before adding it to the
          tank.

     c.   One example of an activated charcoal filter
          is the large-size Halvin which attaches on
          the side of the aquarium.  Examples of
          electric air pumps are the Silent Giant
          ($15), Oscar and Star ($8).  Activated
          charcoal should be changed every two days.
          The charcoal can be reused if fired in an
          oven at 450°C for an hour.  Less heat will
          not destroy the organics absorbed in the
          charcoal surfaces.

     d.   If one uses a compressor as an air source,
          the air should first be passed through one
          tube:  the first half holding non-absorbant
          cotton and the second half holding activated
          charcoal.  The cotton and charcoal should be
          changed every month.

     e.   A large sand filter fiberglass system is shown
          in Figure 1.  This can be used for 200-300 fish.
          The pump can be run constantly-  Its size should
          be sufficient to circulate the water in the tank
          once per hour.

          Back-flush the sand filter every 3 weeks.  Turn
          off the pump when feeding the fish.

3.    Temperature for Fish Maintenance

     a.   Cold-water fish should be kept at 13-14°C in
          order to remain disease-free.  Warm-water fish
          also are usually less apt to contact disease
          when kept at these cool temperatures.

-------
                                                       5.
     b.    There are several ways to maintain these cool
          temperatures.   One of these is a water bath with
          a refrigerant system.  Another is a walk-in box
          with a refrigerant system.

4.    Feeding Fish During Maintenance Period

     a.    When feeding fish, turn off the aerators and
          any filtration system (including activated
          charcoal).

     b.    Throw in food slowly until fish cease eating;
          this usually takes 10 to 15 minutes.

     c.    Look at the individual fish and remove any
          that have any discoloration and, of course,
          any dead ones.  Generally only 1 or 2 fish
          will die out of a hundred, and these in the
          first days after delivery.

     d.    Fish should be fed 3 times a week; however,
          they can do without feeding on the weekends.

     e.    Do not over feed.

     f.    Fish food may be purchased as pellets or in
          frozen form.  Fish food is available in bulk
          in pellet form of various sizes.  No. 2 is
          suitable for small fish, but larger pellets
          can be ground in a mortar if only one size is
          available.   Brine shrimp can be purchased
          frozen.  Chunks can be broken off as needed.
          Put the frozen chunks into a beaker of water
          until they melt apart.  Stir them and let the
          shrimp settle to the bottom.  Pour off the
          supernatant water, add more water and repeat the
          process.  In this way, less debris is added
          along with the shrimp.  (If one is feeding fish in
          large tanks, the brine shrimp chunks can be
          thrown in directly).

     f.    Fish are not to be fed 2 days before the
          commencement of any test.

-------
                                                       6.
5.    Holding and Dilution Water

          Most fish are either marine or fresh-water, but
     some fish can live in water of varying salinity.
     These are termed euryhaline fish.  If freshwater
     discharges into a freshwater receiving water are being
     tested for their toxicity, then a freshwater species
     can be used and a marine species for saline discharges
     into the ocean.

          However, when low-salinity water is discharged
     into an estuary or the ocean, then a euryhaline
     species is the appropriate one to use.  The euryhaline
     species can be kept in a 1:1 mixture of marine and
     tap water.  It will withstand without great stress
     transferral from this mixture into both the effluent
     and sea water.  These extremes in salinity would
     be present in the test waters because the concentra-
     tions used would include both sea water and effluent
     and mixtures of the two.

          Fresh water holding water and the dilution
     water can be tap water that has been aged or
     aerated for 12 hours.  For some of the reasons
     given above, there are usually two controls, one
     the holding water and the other the dilution
     water.  If the fish are kept in the holding water
     within the laboratory-maintained temperature range,
     then the fish left in the holding water can be
     considered controls.

          When both the receiving water and the effluent
     from the discharger are suspected to be toxic, a
     double control can be made.  Dilution water could be
     river water upstream of the effluent in which a
     double control should be used.  Control 1 being
     the river dilution water; control 2 the aged tap
     water.  Sometimes results will vary if you use
     existing receiving water as a diluent instead of

-------
     using tap water as diluent.  For example, when
     salts are high in receiving waters, this may have
     a positive or negative effect on effluent toxicity.
     If one has two controls (river water and tap water)
     and there is mortality due to the receiving water
     (river) rather than the effluent, use of the second
     control, tap water, will make this obvious.

Bioassay Procedure

Materials Required

     Bioassay containers.  These may be five-gallon
          (19-liter) aquaria, pickle jars or battery
          jars which are available in sizes up to one
          gallon; the size depends upon size of fish -
          one gm fish per one liter water; fish nor-
          mally require 10-15 liters per test sample.
     Container-cleaning facilities (large thick rug;
          garden hose).
     Aluminum foil or lids for bioassay containers.
     Temperature controllers.   (Capable of maintaining
          20°C + 2°C for warm-water fish and 15°C + 2°C
          coldwater ones).
     Aeration device
     Dissolved oxygen meter.  DO can be measured by
          siphoning but then large-volumed containers
          are required.  See Fig. 2.
     Thermometer.   (Either a recording thermometer or
          a small thermometer in a jar of water).
     Optional:  devices for measuring pH, conductivity,
          turbidity, and hardness.
Data recording sheets (See attachment, Figure 3)
Bioassay organisms  (e.g. fish)   [fish must be held at
     experimental temperatures for 10 days prior to
     commencement of bioassay for legal purposes].

Method

1.   Scrub bioassay glass containers clean and rinse
     them well with tap water.   If the containers are
     large,  it is safer to do this on a large thick
     rug, using a light garden hose for rinsing the
     jugs.  This is best done out of doors on a cement
     platform.

-------
2.    Let the containers drain for about one hour, then
     let them air dry inside the laboratory.  After
     they are dry, cover them with aluminum foil or
     lids to keep dust-free, or store upside down.

3.    Normally ten fish are added to each container.  The
     weight of the fish cannot exceed 1 gram per liter of
     water.  If fish are too large for 1 container, put
     five fish into each of two separate containers
     containing the same sample solution.

4.    There are several ways to increase the reliability of
     the tests:

     a.   Increase the number of fish from 10 to 20 per
          container (remaining consistent with the
          weight to volume restriction above).

     b.   Prepare replicates of each concentration so
          that there would be two or more of each test
          solution (with 10 fish per container).

     c.   Prepare concentrations with closer increments of
          toxicants e.g., instead of 10%, 20%, 30% there
          would be 10%, 15%, 20% etc. additions.

5.    a.   Preparation of concentrations

          The graph shown in Figure 4 is a standard plot
          of log of concentration versus regular arithmetic
          increments.   This plot is based upon experimental
          results which show that effect of a toxicant
          upon an organism is logarithmic rather than
          arithmetic.   That is to say that, in general,
          if one doubles the concentration one does
          not double the mortality.

          Actual additions of toxicant are in logarithmic
          increments.   An excerpt from Standard Methods
          is given in 5b.  It includes Table 1 which
          shows some log increments; a more complete

-------
                                                       9.
          range is expressed in Figure 4.  Figure 4 shows
          concentrations ranging from 100% to 10%.  If
          a wider range of concentrations were to be
          employed, then several-cycle semilog paper
          would be used - e.g., a range of 100% to
          0.1% would require four-cycle semilog paper -
          or divide values in Fig.  4 by 10 or multiples
          of 10.

          If possible,  the actual concentrations chosen
          would be based on a preliminary test of 12-24
          hours with toxicants added in concentrations
          covering a wide range of  values.  For an
          unknown substance this might be 100%, 50%, 10%,
          1%, and 0.1%.  Regardless of the preliminary
          results, if possible, always include a 100%
          full strength test sample because many
          toxicity standards are based on percent
          survival in the pure test sample.

     With experience and a preliminary test,  the concen-
trations can be selected so that containers very close to
the TLso value will be the most numerous.  A preliminary
test using 2-4 fish per concentration can be carried out
if the test material does not degrade.  For example, if
the preliminary test using two fish per liter showed the
following results

     Concentration of

       Test Solution               Survival

          100%                        0
           50                         0
           25                         1
           10                         2
            1                         2

     Then the following concentrations could be set up:

                    100% (Optional)
                     56
                     32
                     24
                     18
                     10

-------
                                                  10.
b.   Excerpt from Standard Methods, 13th Ed., p. 565
     "Although a TL5Q may be determined by testing any
     appropriate series of concentrations of the sub-
     stance or waste assayed, the geometric series
     of concentration values given in Table 1 is often
     most convenient and has been widely used.  These
     values can represent concentrations expressed
     as percent by volume or as milligrams per liter,
     etc.; they may all be multiplied or divided, as
     necessary, by any power
   TABLE 1:  GUIDE TO SELECTION OF EXPERIMENTAL
  CONCENTRATIONS, BASED ON PROGRESSIVE BISECTION
         OF INTERVALS ON LOGARITHMIC SCALE

Col. 1  Col. 2     Col. 3     Col. 4     Col. 5

10.0
                                          8.7
                               7.5
                                          6.5
                    5.6
                                          4.9
                               4.2
                                          3.7
         3.2
                                          2.8
                               2.4
                                          2.1
                    1.8
                                          1.55
                               1.35
                                          1.15
 1.0
of 10.  For example, the two values in the first
column may be 10.0 and 1.0 as shown, or they may be
100 and 10, or 1.0 and 0.1, with the values in the
other columns changed accordingly.  The values of

-------
                                                        11.
      the series 10.0,  5.6, 3.2, 1.8, and 1.0  (i.e.,
      Cols.  1-3),  or 10.0, 7.5, 5.6, 4.2, 3.2, etc.
      (Cols.  1 through 4), are evenly spaced when
      plotted on a logarithmic scale."

 6.    At the beginning of the bioassay, measure dissolved
      oxygen (DO)  in each container.  If it is below
      4  mg/1, aerate that container until the DO is
      above 4 mg/1.

 7.    Additional optional measurements (in order of
      importance)  include pH, conductivity, turbidity and
      hardness (titration, expressed as EDTA as CaCOs).
      Figure 3 shows a blank data sheet.   Figure 5 shows a
      typical data sheet with observations recorded.

 8.    Record the temperature daily  (on Data Sheet,
      Figure 5,  range of temperature is recorded following
      reading on 7-day recording thermometer).

 9.    Keep room semidark and do not let people wander need-
      lessly in to frighten fish.

10.    When transferring fish, do so gently so as not to
      harm them.

11.    Add fish in groups of two to the jugs.   Random
      placement of jugs and random addition of fish is
      recommended (see section on Random Sampling).

12.    Using data sheet, record mortality and D.O. at
      least every 24 hours along with any other information
      about the bioassay that may be subsequently of
      interest.   Remove dead fish as soon as they are
      observed.

 Calculation of Results

 1.    The TLso (concentration of toxicant killing 50% of
      the fish)  at 96 hours should be calculated by
      plotting toxicant concentration on the ordinate
      scale  of semilog  graph paper and survival on the
      abscissa (normal  scale axis).

-------
                                                       12
     For example,  if the  96-hour  results were obtained
     from a toxicity test as below  (in Table 2)  the
     TLso can be  seen  from  Inset  in Figure  5 to  be
     68%.

          Table 2.  Survival of Fish vs. Toxicant,
          Typical Data

              Survival
           (per 10 fish total)          % Toxicant

                  0                       100
                  3                       75
                  6                         65
                  9                         56
                10                         42
                10                         24
                10                         10

Statistical Treatment  of  Fish  Bioassay Results
     The TLso value  can  also  be  calculated by using the
Reed-Muench Method  (Woolf ,  1968) .  This method also
allows one to calculate  the 95%  confidence limits which
contain the true  TL5Q value.
     Utilizing  the data  given on the  sample record
sheet for the "Northwest STP", the calculations are
given in Figure 6.   Natural logs can  be used in place
of logs to the  base  10,  if  this  is more convenient.
If the lowest dose in mg/1  or percent volume of
toxicant is less  than one,  multiply the dose values by
10 or 100 as logs values less than one are negative.
Then divide the resulting final  values by the same
multiple.  Express the TL values as whole numbers in
the example given.

                         REFERENCES

Leteux, F. and  F. P. Meyer.   The Progressive Fish
     Culturist  34.   1972.   "Mixtures  of Malachite
     Green and  Formalin  for Controlling Ichthyophthirius
     and other  Protozoan Parasites of Fish. "
Woolf, C. M.  1968.  Principles of Biometry.  D  Van
     Nostrand Company.  Princeton, N. J.  359 pp.

-------
                                     Table  3
                TEST ORGANISM SUITABLE FOR THE  STATE  OF HAWAII
                  University of Hawaii at Manoa

                                 Department of Zoology
                             Edmondson Hall • 2538 The Mall
                                 Honolulu, Hawaii 06822
                         Suggested Native Hawaiian Fauna
                               for Aquatic Bioassay

                      (John A. Maciolek - Associate Professor)


      The following  fresh and brackish waters animals are available  on most islands
in Hawaii and generally can be kept without undue difficulty in aquaria   and holding
tanks.

A.  Freshwater species.

    1.  Shrimp:  Atya  bisulcata - opae kalaole, "mountain opae".   Occurs  in fast-
        flowing  streams to about 3,000' elevation.  Very abundant  in pristine streams,
        but on Oahu, it is common only at higher elevations.  Filter-feeds on stream
        seston and epilithic algae.  Normally completes its  life cycle in freshwater
        but larvae can tolerate salinity.  Size:  to about 2".

    2.  Fish: Awaous  stamineus » o'opu nakea and Sicydium stimpsoni » o'opu nopili.
        Both species are abundent in the lower to middle reaches of  perennial streams
        on neighbor  islands; much less common on Oahu.   Larvae  develop in ocean and
        migrate  upstream as post-larvae (hinana), often in great numbers, during
        several  months of the year.  Juveniles and adults do not tolerate saline
        water.  Feed on benthic algae (especially nopili) and small  invertebrates.
        Size: hinana  about 1"; nakea adult to 12"; nopili adult to  7".


B.  Brackish water species:  the following shrimp and fishes are broadly  euryhaline
    (freshwater  to seawater).

    1.  Shrimp:  Palaemon debilis = opae huna, "glass shrimp".   Most common in estu-
        aries and brackish shoreline ponds, but is also found in most protected
        inshore  marine areas.  Omnivorous, feeds on plant materials, detritus, etc.
        Can complete its life cycle in brackish water.   Size:  to  1.5".

    2.  Fish: Kuhlia  sandvicensis = ahole, aholehole.   Occur in estuaries and inshore
        marine areas.  Juveniles (to 3") invade lower reaches of streams.  Carnivorous;
        predaceous on  invertebrates (shrimps, worms) and small  fishes.  Size:  to 12".

    3.  Fish: Mugil cephalus = amaama, grey mullet.  Habitat similar to  Kuhlia, but
        is herbivorous—feeding on phytoplankton, bottom sediments,  etc.  Fry and
        small juveniles common in estuaries.  Size:  to at least 2 feet.

-------
                      Holes increase in size from middle  to  end

^PVC pipe 3/4" diam'^'^      	^


\ 	 • w.
\ 	 1*

Sand
Filter
L
2"
Spreader
Board at an
••*».
*
r-r V
1 	 . 	 1 I
                                                                 o   O  Q  D T
                                                                             -cap
      Hose 3/4" diam.
                Pump
                                                    PVC Pipe  (bottom view)
                                   Board at angle   Water goes through holes in PVC
                                           "~~~~"      pipe onto spreader board.
                                                          nergency overflow
                                                         (in event of sand clogging)
                                " diam.  PVC
      1 1/4" diam     Valve
     g.	PVC      	
                        Fish Tanks with  Filtering  System
       8" depth sand
   (No. 12, White Monterey)
    6" depth pea gravel
  1?" depth rock (l"-3" in size)

 row of PVC collector pipe
                                                                  3/4" diam.
  Sand Filter. Details
  (side view)

Surface of sand in square feet
should be approximately equal to
the flow in gallons per minute

. /


^2" diam.

^outlet (2
1 — 	 1
                                                                            cap
                       PVC Collector Pipes, Details
                       (viewed from above)

                       Each pipe has holes of 3/8" - 1/2" diam.
                       spaced regularly at 2" intervals along
                       length.
          Figure 1.  Diagram of Fish Tanks with  Filtering System
                     (Details of Sand Filter  and Collection Pipes Included)

-------
                           GLASS TUBING
                              FLEXIBLE TUBING
                                        PINCH CLAMP
                                           BUCKET
Figure  2  .  The siphon is first filled with distilled water.



After putting the glass tubing into the test water, the pinch




clamp can be released and enough water siphoned into the



bucket to displace the distilled water by test water.  Then




the end of the tube is put into the BOD bottle all the way to



the bottom.  After overflowing the bottle about twice, slowly




withdraw the tubing, allowing the water to flow until the tube




is out of the bottle.  Start with the control water and




proceed from the lower toxicant additions through the more



concentrated ones.  Then the siphon can be utilized without




rinsing it with distilled water.  Stopper the BOD bottles and




measure the dissolved oxygen by the routine Winkler method.

-------
    Figure 3 - Data Recording Sheet
Source



Number and Kinds of Individuals
Collection Date
Bioassay Date




Temperature Range
1
Time
>
i
[
0
hour j
i

24
hour
48
hour
72
hour
96
hour
Parameter
DO, mg/1
PH
EC , jumhos/cm
EDTA, as mg/1
CaC00
JTU initial
1 hr
Survival
DO, mg/1

Survival
DO, mg/1

Survival
DO, mg/1

Survival
DO, mg/1
PH
EC , /omhos/cm
EDTA, as mg/1
CaCO-j
JTU

Control
Holding






















Dilution






















Waste Concentrations



















































































































































/





	 	 _

























-------
                                           \-
                        Inset  indicates determination  of
                           from percent survival  and concen-
                       trations given  in Table  2.   Plot 30%
                       1 survival at  75% concentration  and
                       •  60% survival  at 65% concentration
                           Draw straight line between  points.
                             Line intercepts 50%  survival at
                       12      point  of 68% concentration.
              Survival of Fish in
      Regular Arithmetic Increments of the Log Scale
Figure 4.  Guide to Fish Bioassay Concentration Selection

-------
             1 — 3  -   Completed Data Form

Source NOf»7Vv u)*4"f>"  STP        Collection Date

Number and  Kinds  of  Individuals  IQ ST» CKit t>*cK / I1Vr\$
Bioassay D
Temperature Range
    )£. S -170  °C
Time
0
hour

24
hour
48
hour
72
hour
96
hour
Parameter
DO, mg/1
PH
EC , jo.Tah.os/ cm
EDTA, as mg/1
CaCO.,
JTU initial
1 hr
Survival
DO, mg/1

Survival
DO, mg/1

Survival
DO, mg/1

Survival
DO, mg/1
pli
EC , jumhos/cm
EDTA, as mg/1
CaCO^
JTU
Control
Holding
^-H
"7- »
3£oo_o
-702,0
<*

)t>
1.2-

) 0
<1. 2-

10
T, \

10
-?. o




Dilution
<^.l
-7. 6
a^ooa
•70*40
-<-ff

10
<*. 0

1 o
9- i

ID
«j. j

JO
q.z-




p TT»y ? * ' ^
V7aste Concentrations /*
;s
^•H
i. S 13 loo
90
fi-6
i3oo«
3o%o
M-C>

J 0
e.o

<*
9. I


-------
                               Figure 6
        CALCULATIONS FOR FISH BIOASSAY STATISTICAL CONFIDENCE  LIMITS
Dose
18%
32
42
56
65
75
100
Log of
Dose
1.2553
1.5052
1.6232
1.7482
1.8129
1.8751
2.0000
No.
10
10
10
10
10
10
10
Num
Dead
0
0
3
10
10
10
10
per
Alive
10
10
7
0
0
0
0
Accumu.
Dead1
0
0
3
13
23
33
43
ated
Alive1
27
17
7
0
0
0
0
Total
27
17
10
13
23
33
43
Cumulated
% Mort.2
0
0
30
100
100
100
100
 (S.E.) Standard error =  / 0.79 hR
                        v    n

h = interval between doses

R = interquartile  range which is TLys - TL25-  If either of  these
    values are not found/ use either 2(TLso - TL25) or 2(TLys -
    as the R value.

0.79 is a constant

n = number of organisms in each concentration (use mean number if variable)

h = 0.2499 + 0.1180 + 0.1250 + 0.647 + 0.0622 + 0.1249
h = 0.1241
TL2s = 1.5052 + 25S(0.1180) = 1.6035 = 40.1%
                3(5"

TL50 = 1.6232 + 20 (0.1250) = 1.6589 = 45.6%
TL75 = 1.6232 + 45 (0.1250) = 1.7036 = 50.5%
                Tff
R = 1.7036 - 1.6035 = .1001

SE =
      /0.79 x O.i;
     y         TO~
1241 x .1001
.0313
95% confidence limits equal:

TLso ± 1.96  (SE)

1.6589-1.96  (.0313) = 1.5976 =  39.5%

1.6589+1.96  (.0313) = 1.7202 =  52.5%

  95% CL = 40% - 52%
1.  Accumulative dead are derived from adding downwards in the  numbers
    dead column and those alive by starting at the bottom of  the  numbers
    alive column and adding upwards.

2.  Cummulative % Mortality = Accumulative dead -1 total       x  100

3.  Interpolation to determine log value between 0 and  30% mortality.

-------
                                                             1.
VI.   Use of Random Numbers

     A.   A table of random numbers is given in Table  1.  This
          listing can be used in randomization processes needed
          for sample collection or experimental design.

          For sample collection, the numbers selected  would
          be used to pick the locations to be sampled.  It
          would be essentially a process of limiting the
          number of sample points, all points having an equal
          probability of being selected.

          In experiments the random numbers are used to
          assign positions of flasks, sequence of inoculation,
          etc.  The uses of random tables for the two  purposes
          will be explained below.

          First it will be necessary to select the numbers
          from the table.  This consists of  (1) selecting
          the starting point and  (2)'listing sequentially
          a sufficient amount of numbers.

          1.   Selecting the starting point

                   Table 1 has the columns and rows each
               numbered 0-49.  Without looking, put your eraser
               or finger-tip on any location in the table.
               Assume the point is at the intersection of
               column 20 and row 30.  The numbers there are
               4113.  Then we can start using numbers  at
               column 41 row 13.  If the number selected is
               too high, just move along the row until a number
               under 50 is encountered or find another starting
               location.

          2.   Listing the numbers

                   Using the above location, write down the
               numbers.  When getting to the end of the row start
               back in the reverse direction in the next lower

-------
                                                       2.
B.
     row.  Group the numbers singly or in pairs
     depending on whether more or less than 10
     samples have to be randomized.

         Assume that we have 15 samples to put
     in random order, then starting at our above
     location we would have:  93  15  11  80  45  81
     51  41  80  16  57  42  87  53  95  65  36  etc.,
     continuing until we have encountered numbers
     1-15.  In actual practice we would not write
     down the numbers until one 15 or under was
     encountered.

Use of the tables in experiments

Fish bioassays

     In these experiments, we would like at least
to randomize the position of the jugs on the bench,
and add 2 fish per jug in the randomized order.  For
example if we had the following jugs:
              Control
                   1%  10%  25%  50%  75%  100%
     Given
     Numbers      1      234567

          Utilizing the single sequence of numbers above,
     we would have on the bench the jugs so:
     10%
      Control
50%
25%
75%
100%
1%
          We could re-number the jugs as they appear now
     on the bench 1,  2,  3 	 and utilize a new selected
     sequence of numbers for adding 2 more fish per jug.
     However, time considerations would probably preclude
     this approach, although its advantage should not be
     overlooked in a completely randomized experiment.

-------
                                                       3.
          Variations to the above approach, will probably
     be obvious.

C.   Algal Bioassay Flasks

          Randomization is possible in the sequence of
     adding algae, placement of flasks on shelves, mass
     measurement order, etc.  The importance of randomi-
     zation in bioassays probably should be secondary to
     an orderly sequence which would minimize errors.

-------
Table  1
TEN THOUSAND RANDOM DIGITS

00
01
02
03
04
05
06
07
08
09
10
11
12
13
H
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
411
•14
•15
•Id
47
'It!
•Ill
00-04
88758
35661
2(1335
60826
95044
83746
27998
02635
18386
21717
18446
66027
51420
27045
13094
92382
16215
09342
38148
23689
25407
25349
02322
15072
27002
66181
09779
10791
74833
17583
45601
60683
29956
91713
85704
17921
13929
03248
50583
10636
431!%
76714
:''.'.:i93
70'H2
9'jon
(16456
9i>292
196(10
67IM7
JlfilitlJI

05-09
.66605
42832
03771
74718
99896
47694
42562
32323
13862
13141
83052
75177
96779
62626
17725
62518
50809
14528
79001
19997
37726
69456
77491
33261
31036
83316
01822
07706
55767
24038
46977
33112
81169
84235
86588
26111
71341
18880
17972
•16975
41278
110963
•1(1719
920-12
(10326
00126
443 l!l
07 Mil
511-12
10-14
33843
16240
46115
56527
13763
06143
63402
74625
10988
22707
31842
47398
54309
73159
14103
17752
49326
64727
03509
72382
73099
19693
56095
99219
85278
40386
45537
87481
31312
83701
39325
65995
18877
75296
82837
35373
80488
21667
12690
09449
42205
74907
15-19
43623
77410
88133
29508
31764
42741
10056
14510
04197
68165
08634
66423
87-156
91149
00067
53163
77232
71403
79424
15247
51057
85568
03055
43307
74547
54316
13128
26107
76611
28570
09286
64203
15296
69875
67822
86494
89827
01311
00452
45986
10425
16890
02083 62-1-28
20-24
62774
20686
40721
91975
93970
38338
81668
85927
18770
58440
11887
70160
78967
96509
68843
63852
90155
84156
39625
80205
68733
93876
37738
39239
84809
29505
51128
24857
67389
63561
41133
18070
94368
82414
95963
48266
•18277
61806
93766
34672
66560
15492
45177
22776 -17761 j 13503
863-16
45685
26738 . 019H3
67607 70796
25-29 30-34
25517
26656
06787
13695
60987
97694
48744
28017
72757
19187
86070
16232
79638
44204
63565
44840
69955
34083
73315
58090
75768
18661
18216
79712
36252
86032
82703
27805
04691
00098
34031
65437
16317
05197
83021
01888
07229
80201
16414
46916
59967
27489
57562
09560
59698
95962
25215
14692
69300
08400
80588
71418
08421
08464
67343
68869
92237
93578
02592
93892
35613
18811
43804
77991
69018
81781
94753
09373
34563
75350
42710
39687
60784
94867
13624
34239
66596
90732
65735
71953
47889
01212
63881
90139
06067
49243
16037 j 30875
04186 41388
04889 ; 98128
208' W 02227 76512 ' 53185 1)3057
53051 1()')35 W133 76233 13706
215311 I>OI51 054911 64678 87569
59255 ()l>898 '.'9137
'
50871 81265 j 42223
35-39 40^4
41880
86241
60841
72237
71039
99864
83124
14756
81133
23872
20565
36205
49062
29969
24756
88572
70445
35670
86230
94548
72641
10332
32245
41450
69471
93204
25179
63471
13596
76098
11849
90896
03643
13083
32661
05315
16128
83052
27964
83117
73563
22287
31748
80754
03848
13599
61375
20502
65066
83303
1 1
85126
13152
91788
06337
34165
19641
19896
54937
69503
03036
74390
50036
02196
49315
10814
03107
00906
10549
99682
82693
95386
83137
84081
30944
15606
72973
86104
08804
88730
84217
75171
80945
66081
46278
64751
79328
65074
31029
02766
53947
29875
19760
64278
47491
78354
93710
10760
60405
17790
48694

45-49
60755
49187
86386
73439
21297
15083
18805
76379
44037
34208
36541
59411
55109
11804
15185
90169
57002
07468
82896
22799
70138
88257
18436
53912
77209
90760
40638
23455
86850
34997
57682
71987
12242
73498
83903
13367
28782
06023
28786
95218
79033
13056
05731
96012
14964
23974
26889
09745
55413
81953


-------
VII.  APPENDIX



     Contents                                            Page

Equivalent Values                                         1

Physical Constants                                        4

Specific Conductance Conversion Figure                    4

Mathematical Formulae                                     5

Oxygen Solubility and Nomograph                           6

Interconversion Tables
   Centegrade to Fahrenheit                               7
   Meters to Feet                                         8

Plankton Neeting Aperture Size and Grades                 9

Sediment Size Classification                              9

Sieve Scales - Wentworth, Tyler, and U.S.
  Sieve Series                                           10

Formula Weights                                          11

Atomic Weights                                           13

Relative Humidity                                        14

Stock Solutions                                          15

Composition of Commercial Acids and Bases                15

Exponential Arithmetic                                   16

Significant Figures                                      17

Use of Logarithms and Exponents                          18

-------
                            -1-
                             EQUIVALENT VALUES
Depth
•  1 fathom = 6 feet
           = 1.8 29 meters

Area
  1 square inch = 6.42 square centimeters
  1 square foot = 929.03 square centimeters
  1 square yard = 0.836 square meter
  1 acre = 43,560 square feet
        = 4840 square yards
        = 160 square rods
        = 10 square chains (Gunter's)
        = 0.4047 hectare       i
  1 section = 640 acres
           = 1 square mile
  1 square mile = 640 acres
               = 2 59 hectares
               — 2.59 square kilometers
  1 square millimeter = 0.0015 square inch
  1 square meter — 10.758 square feet
  1 hectare = 10,000 square meters
           = 2.5 acres (approximately)

Volume
  1 cubic inch =  16.386 cubic centimeters
  1 cubic foot = 28,316 cubic centimeters
              = 7.48 gallons
              = 0.0283 cubic meter
  1 cubic yard = 0.7646 cubic meter
  1 acre-foot = 325,850 gallons
  1,000,000 cubic feet = 22.95 acre-feet
  1 cubic centimeter = 0.061  cubic inch
  1 cubic meter -= 35.314 cubic feet
               = 1.308 cubic yards
Length
  1 inch = 25.40 millimeters
        =  2.54 centimeters
  1 foot = 0.305 meter
        = 30.5 centimeters
  1 yard = 3 feet
        = 0.914 meter
  lrod = 16.5 feet
        =  5.5 yards
  1 mile (statute) = 63,360 inches
                 = 5280 feet
                 = 1760 yards
                 = 320 rods
                 = 1609 meters
                 = 1.609 kilometers
                 = 0.867 geographic mile
  1 millimeter = 0.0393 inch
  1 centimeter = 0.393 inch
  1 meter = 39.37 inches
          = 3.281 feet
          = 1.0936 yards
          = 0.000621 mile
  1 kilometer = 3281 feet
             = 1000 meters
  1 chain (Gunter's) = 792 inches
                    = 66 feet
                    = 4 rods
                    = 0.0125 mile
  1 link (Gunter's) = 7.92 inches
                   = 0.04 rod
  1 chain (engineer's) = 100 feet
  1 link (engineer's) = 1 foot

-------
                   -2-
                                   U-.N r VALLT.S

        Capacity
           I U.S. pint = 473.IS cubic centimeters
           1 U.S. quart  = 2 pints
                        = 946 cubic centimeters
                        = 0.946 liter
           1 U.S. gallon = 231 cubic inches
                        = 4 quarts
                        = 3784 cubic centimeters
                        = 3.7K4 liters
           1,000,000 gallons = 3.07 acre-feet
           1 liter = 61.027 cubic inches
                 = 2.11 pints
                 = 1.0567 quarts
                 = 1000 cubic centimeters

        Miscellaneous
           1 atmosphere pressure — about 15 pounds per square inch
                                 -- about 1 ton per square foot
                                 = about 1 kilo per square centimeter

        Angles
           1 circumference = 360 degrees
           1 degree = 60 minutes
           1 minute = 60 seconds
          METRIC SYSTEM                            ENGLISH  SYSTEM

                                Units of Length
 Meter (in.) = 39.37 inches (in.)              Yard  = 0.914-1 m.
 Centimeter (cm.)  = 0.01 in.                  Inch (U.S.) = 2.5t cm. (Fig. 1-5)
 Millimeter (mm.)  = 0.001 m.
 Kilometer (km.) = 1000 in.                  Mile (U.S.) = 1.609km.
 Angstrom unit (A.lJ. or A) = 10~s cm."

                                Units of Volume
 Liter  (1.) = volume of 1 kg. of water          Liquid quart (U.S.) = 0.9463 1.
 Millililcr (nil.) = 0.001 I.                    Cubic foot (U.S.) = 28.316 1.

                                Units of Weight
 Grmn (n.) = weight of 1  ml.  of water          Ounce (oz.)(avoirdupois)  = 28.35 g.

 Milligram (mg.) = 0.001 g.                   Pound (Ib.) (avoirdupois) = 0.4536 kg.
 Kilogram (kg.) = 1000 g.                    Ton (short.) = 907.185 kg.
Ton (metric) = 1000 kg.  = 2204.62 Ib.        Ton (long) = 2210lb.  = 1.016 metric tons

-------
                      -3-
                      NUMERICAL EQUIVALENTS
             LENGTH
          1 in. = 2.540 cm
           1 ft = 30.48 cm
          1 mi = 1.609. km
          1 cm = 0.3937 in.
           1 m = 39.37 in.
          1 km = 0.6214 mi
           1 m = 3.28 ft

              SPEED
      15 mi/hr = 22 ft/sec
       1 mi/hr = 1.467 ft/sec
       1 mi/hr = 44.7 cm/sec
       1 km/hr = 27.78 cm/sec

              FORCE
         1 g-wt = 980 dynes
       1 kg-wt = 2.205 Ih
          1 oz = 28.35 g-wt
           1 Ib = 453.6 g-wt
          " 111) = 4.448 X 10s dynes
           1 11)= 4.448 newtons
      1 newton = 10s dynes
      1 newton = 3.60 oz

            PRESSURE
1 in. of mercury
1 cm of mercury
1 cm of mercury
   1 ft of water
  1 in. of water
  1 cm of water
  1 cm of water
       1 lb/in.2
          1 bar =
          fbar
  1 atmosphere
  1 atmosphere
  0.491 Ib/in.2
  0.1934 lb/in.2
  0.0133 bar
  0.433 lb/in.2
  0.0361 lb/in.2
  0.0142 lb/in.2
  0.980 millibar
  0.0690 bar
= 10° dynes/cm2
  14.5lb/in'.2
  1.0132 bars
  14.7 lb/in.2
1 atmosphere = 1.058 tons/ft2
1 atmosphere = 76 cm of mercury

       WORK AND ENERGY
      1 joule = 107 ergs
      1 joule = 0.738 ft-lb
      1 joule = 0.000000278 kw-hr
      1 joule = 0.000000373 hp-hr
      1 joule = 0.239 cal
       1 ft-lb = 1.35 joules
       1 ft-lb =1.35  X 107crgs
       1 ft-lb = 0.324 cal
       1 ft-lb = 0.001286 Btu
        1 cal = 4.18 joules
        1 cal = 3.086 ft-lb
       1 Btu = 252 cal
       1 Btu = 778 ft-lb
       1 Btu= 1055 joules
     1 kw-hr = 3.6 X 106 joules
     1 kw-hr = 2.655 X 10° ft-lb
     1 kw-hr = 1.341 hp-hr
     1 hp-hr = 1.98X 10fi ft-lb
     1 hp-hr = 2.68  X 106 joules
     1 hp-hr = 0.746 kw-hr

             POWER
        1 hp = 746 watts
        1 hp=178cal/sec
    1 Btu/hr= 0.293 watts
        1 kw=1.34hp
      1 watt = 0.239 cal/sec

     ELECTRICAL QUANTITIES
     10 amp = 1 em unit
 10 coulombs = 1 em unit
   1 coulomb = 3 X 109 es units
    300 volts = 1 es unit
 1 microfarad = 9 X 105 es units
 1 millihenry = 10° em units

-------
                         -4-
               ACCEPTED VALUES OF CERTAIN QUANTITIES
Velocity of light in vacuo
Gravitation constant
Electronic charge
Electronic charge
Number of molecules  at 0° C atmospheric pressure
Number of molecules in 1 gram-molecular weight at
  0° C  atmospheric pressure (Avogadro's number)
Mass of hydrogen atom
Mass of electron
Mass of electron  in atomic mass units
Mass of proton
Unit of atomic mass
Unit of atomic mass equivalent to
1 electron-volt
Planck's constant (h)
                       299,776 km/sec
                       6.670 X 10~8cgs unit
                        4.80 X 10~10esunit
                        1.60X 10-'9coul
                        2.69 X 1019 per cm2

                      6.0233 X 1023
                        1.67 X 10~24g
                        9.11 X 10-28g
                       5.486 X 10~4amu
                        1.67 X 10"24g
                       1.660X 10-24g
                       0.00146 erg
                        1.60 X 10~12erg
                       6.624 X 10~27 erg-sec
                     SOME GEOMETRICAL RELATIONS

                     TT — 3.1416, or 3f approximately
                     Circumference of a  circle = 2 jrr
                     Area of a circle          = Trr2
                     Area of a sphere         = 4 vrr2
                     Volume of a sphere      = g Trr3
                     SOME TRIGONOMETRIC RELATIONS
            sin 6 = -7- • or y = R sin 6.
                   K

            cos 6 = — > or x = R cos 6.
                   K
               a   y   sin 6              .
            tan 0 = - =	^, or y = x tan 6.
                        cos 0
               a   X   COS
            cot 6 —- =
                   y   sin
                        in 6
or x — y cot 6.
1.60
1.50
1.40
"• 1 30
1
S 1 20
'£
3
2
1.10
1.00
090
n an
\









\
\









\
\








\
\









\









\

                                                   Factors for converting specific
                                                   conductance of water to equiva-
                                                   lent values at 25 C  (based on
                                                   0.01M KC1 solution).
                    5    10    15    20   25    30
                      Temperature of Sample-*C

-------
                    — 5 —
                     MATHEMATICAL FORMULAS
          Given
                                 Sought
                                                     Formula
Triangle
   1.  Base  (b)  and
      altitude (a")
   2.  Area (a) and base (b)
      or altitude (a')
   3.  Three sides (d, d', d")
   4.  Base  (b) and perpen-
      dicular (p) of right-
      angle triangle
   5.  Base  (b) or perpen-
      dicular (p)  and hy-
      potenuse (b) of right-
      angle triangle
Trapezoid
   6.  Sides  (s and /) and
      altitude (a')

Trapezium
   7.  Diagonal (d) and per-
      pendiculars  (p  and
      p') to diagonal drawn
      from vertices of op-
      posite angles
Circle
   8.  Radius (r)

   9.  Circumference (c)

  10.  Radius (r)
Sphere
  11.  Radius (r)

  12.  Radius (r)
                           Area (/i)

                           Base (b), or
                             altitude  (tf)
                           Area (a)
                           Hypotenuse (h)
                           Base  (b),  or per-
                             pendicular (p)
                           Area
                           Area (a)
                        2a      .   2a
                   b =  — , or a = —-
                        cf          b
                   Let  s = sum of three
                   sides, then
 Cylinder
   13. Radius (r) and
      altitude (a')
   14. Radius (r) and
      altitude (a')
   15. Radius (r) and
      slant height (/.')

Fnistnnn of Cone
   16. Areas  of both  bases
      (b  and b') and  alti-
      tude (a')
   17. Circumferences    (c
      and  
-------
                  -b-
         Solubility of oxygen, from a wet atmosphere at a pressure of
760 mm. Hg, in mg. per liter, at temperatures from 0° to 35° C.






































Temp.
0
1
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
30
31
32
33
34
35

0.0
14.16
13.77
13.40
13.05
12.70
12.37
12.06
11.76
11.47
11.19
10.92
10.67
10.43
10.20
9.98
9.76
9.56
9.37
9.18
9.01
8.84
8.68
8.53
8.38
8.25
8.11
7.99
7.86
7.75
7.64
7.53
7.42
7.32
7.22
7.13
7.04

COfimlwn FacloM Iw O.ygen
0.1
14.12
13.74
13.37
13.01
12.67
12.34
12.03
11.73
11.44
11.16
10.90
10.65
10.40
10.17
9.95
9.74
9.54
9.35
9.17
8.99
8.83
8.67
8.52
8.37
8.23
8.10
7.97
7.85
7.74
7.62
7.52
7.41
7.31
7.21
7.12
7.03
o
i .
Situation *l Vanout Altitude*
Altitude
F«>?l MMrev
0 0
1JO 100
(j!>!» i'OO
4KO JOO
1110 100
IMO " SOO
1470 (XX)
.MOO 700
?MO rtoo
WO 'KX)
3?80 HXX)
IMO MiXl
t'MO 1AM)
4:vn i KX)
4NX) MOO
 I
PfflMjfd
mm factor
760 00
?bf) 01
Ml OJ
7.1? 04
7?( OS
714 06
70'> 1W
61h 0*)
b8J 11
679 1?
671 IJ
(ifi.l 15
br»5 lt>
MJ 17
6J<» \1
631 ?n
1>?1
6t:> :••!
WH .''ti
^O4 .'H
SflO 11
571 53
MO to




0.2
14.08
13.70
13.33
12.98
12.64
12.31
12.00
11.70
11.41
11.14
10.87
10.62
10.38
10.15
9.93
9.72
9.52
9.33
9.15
8.98
8.81
8.65
8.50
8.36
8.22
8.09
7.96
7.84
7.72
7.61
7.51
7.40
7.30
7.20
7.11
7.02
f
i , 1



0.3
14.04
13.66
13.30
12.94
12.60
12.28
11.97
11.67
11.38
11.11
10.85
10.60
10.36
10.13
9.91
9.70
9.50
9.31
9.13
8.96
8.79
8.64
8.49
8.34
8.21
8.07
7.95
7.83
7.71
7.60
7.50
7.39
7.29
7.20
7.10
7.01





0.4
14.00
13.63
13.26
12.91
12.57
12.25
11.94
11.64
11.36
11.08
10.82
10.57
10.34
10.11
9.89
9.68
9.48
9.30
9.12
8.94
8.78
8.62
8.47
8.33
8.19
8.06
7.94
7.82
7.70
7.59
7.48
7.38
7.28
7.19
7.09
7.00
10
.1,1



WATER TEMPERATURES













































0.5
13.97
13.59
13.22
12.87
12.54
12.22
11.91
11.61
11.33
11.06
10.80
10.55
10.31
10.09
9.87
9.66
9.46
9.28
9.10
8.93
8.76
8.61
8.46
8.32
8.18
8.05
7.92
7.81
7.69
7.58
7.47
7.37
7.27
7.18
7.08
6.99
15
, , 1 . ,



•CENT









0.6
13.93
13.55
13.19
12.84
12.51
12.18
11.88
11.58
11.30
11.03
10.77
10.53
10.29
10.06
9.85
9.64
9.45
9.26
9.08
8.91
8.75
8.59
8.44
8.30
8.17
8.04
7.91
7.79
7.68
7.57
7.46
7.36
7.26
7.17
7.07
6.98
30
i i 1 i i













0.7
13.89
13.51
13.15
12.81
12.47
12.15
11.85
11.55
11.27
11.00
10.75
10.50
10.27
10.04
9.83
9.62
9.43
9.24
9.06
8.89
8.73
8.58
8.43
8.29
8.15
8.02
7.90
7.78
7.67
7.56
7.45
7.35
7.25
7.16
7.06
6.97
23 JO
i iliniJ













0.8
13.85
13.48
13.12
12.77
12.44
12.12
11.82
11.52
11.25
10.98
10.72
10.48
10.24
10.02
9.81
9.60
9.41
9.22
9.04
8.88
8.71
8.56
8.41
8.27
8.14
8.01
7.89
7.77
7.66
7.55
7.44
7.34
7.24
7.15
7.05
6.96















0.9
13.81
13.44
13.08
12.74
12.41
12.09
11.79
11.50
11.22
10.95
10.70
10.45
10.22
10.00
9.78
9.58
9.39
9.20
9.03
8.86
8.70
8.55
8.40
8.26
8.13
8.00
7.88
7.76
7.65
7.54
7.43
7.33
7.23
7.14
7.05
6.95



























0










»o
^








6°
I0 ^
^>^









>•
^








6° O
v>^
>^






\1*

\3^*
-V^






Q
\
~> v v>
1>^







**

-^
>^
































      for determining Oj saturation at different t<-mr>fratiirw and akil

-------
                                    TEMPERATURES—CENTIGRADE TO FAHRENHEIT*
Temp. ° C.
0
10
20
30
40
50
0
32.0
50.0
68.0
86.0
104.0
122.0
1
33.8
51.8
69.8
87.8
105.8
123.8
2
35.6
53.6
71.6
89.6
107.6
125.6
3
37.4
55.4
73.4
91.4
109.4
127.4
4
39.2
57.2
75.2
93.2
111.2
129.2
5
41.0
59.0
77.0
95.0
113.0
131.0
6
42.8
60.8
78.8
96.8
114.8
132.8
7
44.6
62.6
80.6
98. 6
116.6
134.6
8
46.4
64.4
82.4
100.4
118.4
136.4
9
48.2
66.2
84.2
102.2
120.2
138.2
   •Temperatures in degrees Centigrade expressed in left vertical column and  in top horizontal  row;  corresponding temperatures  in
degrees Fahrenheit in body of table.
                                    TEMPERATURES—FAHRENHEIT TO CENTIGRADE*
Temp. ° F.
30
40
50
60
70
80
90
100
0
-1.11
4.44
10.00
15.56
21.11
26.67
32.22
37.78
;
-0.56
5.00
10.56
16.11
21.67
27.22
32.78
38.33
2
0.00
5.56
11.11
16.67
22.22
27.78
33.33
38.89
3
0.56
6.11
11.67
17.22
22.78
28.33
33.89
39.44
4
1.11
6.67
12.22
17.78
23.33
28.89
34.44
40.00
5
1.67
7.72
12.78
18.33
23.89
29.44
35.00
40.56
6
2.22
7.78
13.33
18.89
24.44
30.00
35.56
41.11
•T
2.78
8.33
13.89
19.44
25.00
30.56
36.11
41.67
8
3.33
8.89
14.44
20.00
25.56
31.11
36.67
42.22
9
3.89
9.44
15.00
20.56
26.11
31.67
37.22
42.78
   'Temperatures in degrees Fahrenheit expressed in left vertical column  and in  top horizontal row; corresponding temperatures in
degrees Centigrade in body of table.

-------
                                                 METERS TO  FEET*
Meters
0
10
20
30
40
50
60
70
80
90
100
0
0.00
32. SI
65.62
98.45
131.24
164.04
196.85
229.66
262.47
295. 2S
328.09
;
3.28
36.09
68.90
101.71
134.52
167.33
200.13
232.94
265.75
298.56
331.37
2
6.56
39.37
72.18
104.99
137.80
170.61
203.42
236.22
269.03
391.84
334.65
3
9.84
42.65
75.46
108.27
141.08
173.89
206.70
239.51
272.31
305.12
337.93
4
13.12
45.93
78.74
111.55
144.36
177.17
209.98
242.79
275.60
308.40
341.21
5
16.40
49.21
82.02
1 14.83
147.64
180.45
213.26
246.07
278.88
311.69
344.49
6
19.69
52.49
85.30
118.11
150.92
183.73
216.54
249.35
282.16
314.97
347.78
7
22.97
55.78
88.58
121.39
154.20
187.01
219.82
252.63
285.44
318.25
351.06
S
26.25
59.06
91.87
124.67
157.48
190.29
223.10
255.91
288.72
321.53
354.34
9
29.53
62.34
95.15
127.96
160.76
193.57
226.38
259.19
292.00
324.81
357.62
"Length in meters expressed  in left vertical column and in top horizontal row; corresponding lengths in  feet in body
of table.
                                                                                                                                          I
                                                                                                                                          CD
                                                                                                                                          I
                                                 FEET  TO METERS*
Feet
0
10
20
30
40
50
60
70
80
90
100
0
0.000
3.048
6.036
9.144
12.192
15.239
18.287
21.335
24.383
27.431
30.479
;
0.305
3.353
6.401
9.449
12.496
15.544
18.592
21.640
24.688
27.736
30.784
2
0.610
3.658
6.706
9.753
12.801
15.S49
18.897
2 1 .945
24.993
28.041
31.089
3
0.914
3.962
7.010
10.058
13.106
16.154
19.202
22.250
25.298
28.346
31.394
4
1.219
4.267
7.315
10.363
13.41 1
16.459
19.507
22.555
25.602
28.651
3 1 .698
5
1.524
4.572
7.620
10.668
13.716
16.763
19.811
22.859
25.907
28.955
32.003
6
1.829
4.877
7.925
10.972
14.020
17.068
20.116
23.164
26.212
29.260
32.308
7
2.134
5.182
8.229
11.277
14.325
17.373
20.421
23.469
26.517
29.565
32.613
8
2.438
5.486
8.534
11.582
14.630
17.678
20.726
23.774
26.822
29.870
32.918
9
2.743
5.791
8.839
11.8H7
14.935
17.983
21.031
24.079
27.126
30.174
33.222
'Length in feet expressed in left vertical column and in top horizontal row; corresponding lengths in meters in body of table.

-------
                   -99
  AVERAGE APERTURE SI/E OF STANDARD GRADE DEVOUR BOLTING SII.K
Silk No.
0000
000
00
0
1
2
3
4. •
5
6
7
8
9
Meshes
per
Inch
18
23
29
38
48
54
58
62
66
74
82
86
97
She of
Aperture
(van.)
1.364
1.024
0.752
0.569
0.417
0.366
0.333
0.318
0.282
0.239
0.224
0.203
0.168
Silk No.
10
11
12
13
14
15
16
17
18
20
21
25

Meshes
per
Inch
109
116
125
129
139
150
157
163
166
173
178
200

Size of
Aperture
(mm.)
0.158
0.145
0.119
0.112
0.099
0.094
0.08VS
0.081
0.079
0.076
0.069
0.064

           GRADES AND SI/E RANGES OF SILK BOLTING CLOTH
             Grade
            Standard
           X quality
           XX quality
          XXX quality
           Grit gauze
        XXX Grit gauze
Range of Sizes
 Nos. 0000-25
 Nos.    6-17
 Nos. 0000-16
 Nos.    6-18
 Nos.   14-72
 Nos.   14-72
WENTWORTH'S  CLASSIFICATION OF COARSER SEDIMENTS BASED UPON SIZE
                          OF PARTICLES
Diameter of Panicle
in mm.
More than 256
256-64
64-4
4-2
2-1
1-0.5
0.5-0.25
0.25-0.125
0.125-0.062
0.062-0.004
Less than 0.004
Name Applied to
Particle
Boulder
Cobble
Pebble
Granule
Very coarse sand
Coarse sand
Medium sand
Fine sand
Very fine sand
Silt
Clay

-------
                  -10-
WENTVVORTH GRADE SCALK., \,/2 SCAI K. ^2 Sc\f.F., CORRESPONDING TYLER
  SIEVE OPENINGS AND MESH, AND COKF.F.SPONDING MESH OF U.S.  SIEVE
                             SERIES

Wentwonb Grade
Scjle
(iinn.)


4
Granule


2
Very coarse sand


1
Coarse sand


0.500 (%)
Medium sand


0.250 ('/,)
Fine sand


0.125 (Is)
Very fine sand


0.062 (>•!„)
Silt
V'/.Y> Openings
Increase in the
Ratio of
.—

1 A 14 mm.
4.00

2.83

2.00

1.41

1.00

0.707

0.500

0.354

0.250

0.177

0.125

O.OSS

0.062

..—

1.189mm.
4.00
3.36
2.83
2.38
2.00
1.68
1.41
1.19
1.00
0.840
0.707
0.595
0.500
0.420
0.354
0.297
0.250
0.210
0.177
0.149
0.125
0.105
0.088
0.074
0.062


Tyler Screens


Mm.

3.96
3.33
2.79
2.36
1.98
1.65
1.40
1.17
0.991
0.833
0.701
0.589
0.495
0.417
0.351
0.295
0.246
0.208
0.175
0.147
0.124
0.104
O.OSS
0.074
0.061


Mesh

5
6
7
8
9
10
12
14
16
20
24
28
32
35
42
48
60
65
80
100
115
150
170
200
250


US. Sieve
Series,
Mesh


5
6
7
8
10
12
14
16
18
20
25
30
35
40
45
50
60
70
80
100
120
140
170
200
230


-------
—IT —

AgBr
AgBrOj
AgCNS
AgCI
Ag2CrO4
Agl 	 	
AglO,
AgNO,
A9J0
Ag,P04
Ag,S
.MO, . „ 	 . 	
AI(OH),
AI,(S04),
.AfcOj .... . _._
As205
As2S,
BaCOj 	 	 	 	 	 	 -
Ba(CNS)2
BaCI2
Ba(CIO«),
BaCrO4
BaO
BaO2 -
Ba(OH)2
Ba,(P04)2
BaSO4 -
Bi2S,
Ca,(AsO4)i
CaBr2
CaCO,
CaC2O4
CaF2
Ca(IO,)2
CaO
Ca(OH)2
Ca,(P04)2
CaS04
CeO2
Ce(S04),
H4Ce(S04)4
(NH,),Ce(NO,)t
(NH4)2Ce(S04),-2H20
C02
CO(NH2)2 (urea)
0,0,
CuCO,
Cul
CuO
Cu2O
CuSO4-5H2O
....
187.80
235.80
165.96 ;
143.34
231.77
234.79.
282.79
169.89
231.76
418.62
247.83
101.96
78.00
342.16
197.82
229.82
246.02
197.37
253.53
208.27
336.27
253.37
153.36
169.36
171.38
602.03
233.43
514.20
398.06
199,91
100.09
128.10
78.08
389.90
56.08
74.10
310.19 |
136.15
172.13 F
332.26
528.42 |
548.26
500.44 .
44.01 i
60.06 i
152.02
123.55
190.45 i
79.54 I
143.08
249.69 j

CuS
Cu2S
FeC03
Fe(CrO,j,
FeO
FejO, 	 	 ._ „ . ..
Fe,04
Fe(OH)2
Fe(OH),.__, 	 „.. 	
FeS2
FeS04-7H20
FeSO4-(NH4)2SO4-6H2O.
Fe2(S04),
HBr
H2C204-.2H2O (oxalic) 	 	
HC2H3O2 (acetic)
HC7H5O2 (benzoic)
HCI 	 	 	
HCI04
HNO3
HNH2SO3 (sulfamk) 	
H202
H,P04
H2S.. 	 	 . _. 	
H2SO,
H2S04
Hg(NO,), 	
HgO
HgS
Hg2Br2 ..._ 	 	
Hg2CI2
Hg2l2
KBr
KBrO,
KCN
KCNS
K2C03
KC!
KCIO,
KC!O4
K;CrO4
K2Cr2O7
K,Fe(CN)4
K4Fe(CN)4
KHC2O4
KHC204-H2C204-2H20
KHC4H4Ot (tartrate)
KhiC8H4O4 (phthalate)
KH(IO,)i
KH;PO4
K2HPO4 ;

9561
159 15
11584
223 57
71.85
159.70
231.55
89.87
.10687
119.93
278.03
392.16
399.90
80.92
,126.07
60.05
122.12
36.46
100.46
63.02
97.10
34.02
98.00
34.08
82.08
98.08
324.63
21661
232.68
561.05
472.13
655.04
119.02
167.02
65.12
97.19
138.21
74.56
122.56
138.56
194.21
29422
329.26
363.36
128.13
254.20
188. IS
204.7.'
389.93
137.09
17513

-------
-12-
Kl
•ciO,
MO, 	 	 	
M.'.nO,
>'NO;
rNO,
K O
KOH
K.PtCI,
K SO,
I. COi
I'CI
Li ,50,
MgCO,
Mg CIO,),
MqNH,PO4
MgO
Mq'OH),
Mg:P,O,
MgSO4
MnO,
MnjO,
Mn,O4
Mn.'OH);
MrvP.O,
No.AsO,
No:B,O,
NoBr
NoBrO,
NaC,H,O,
NoCN
NoCNS
Na.CO,
N0.C;O4
NoCI
NoCIO
NoCIO,
NoHCO,
Nal
NaNO,
No O
No.Oj
NnOH
No.PO.
No.S
No. SO,
Nn;SO,
'o.S.O, 5H,O
NH,
N,H.
(NH.^CiO,
166.01
214.01
230.01
158.03
85.11
101.il
94.20
56.11
486.03
174.27
73.89
42.40
109.95
84.33
223.23
137.34
40.32
58.34
222.59
120.39
86.94
157.88
228.82
88.96
283.83
191.88
201.26
102.91
150.91
82.03
49.01
81.08
106.00
134.01
58.45
74.45
90.45
84.02
149.90
85.00
61.98
77.98
40.00
163.95
78.05
126.05
142.05
248.19
17.03
32.05
124.10

















































!

NH4CI
NH4NOj
NH4OH
(NH4))PO4-12MoO3
(NH4)2PtCI6
(NH4)2S04
P2O5
PbCO,
PbC204
PbCrO4
Pbl2
Pb(IO,)2
PbMoO4
Pb(NO,)2
PbO
PbO2
Pb,O4
Pb;(P04)2
PbSO4
Sb;Oj
Sb2O4
Sb206
Sb2S3
SiO2
SnCI2
SnO2
SO2
SO,
SrCO,
SrC2O4
SrO
Sr,(P04)2
SrSO4
TiO2
UFt
UO,
U,08
V205
ZnBr2
ZnO
Zn2P2O7
ZnS
ZnSO4
Water for Hydrates:
1 H2O
2 H2O
3 H2O
4 H20
5 H2O
6 H2O
7 H2O
53.50
80.05
35.05
1876.50
443.91
132.15
141.95
267.22
295.23
323.22
461.03
557.03
367.16
331.23
223.21
239.21
685.63
811.58
303.27
291.52
307.52
323.52
339.72
60.09
189.61
150.70
64.07
80.07
147.64
175.65
103.63
452.84
183.70
79.90
352.07
286.07
842.21
181.90
225.21
81.38
304.71
97.45
161.45

18.02
36.03
54.04
72.06
90.08
108.10
126.11

-------
                    -13-
Element
   •  Actinium
   :  Aluminum
     Americium
     Antimony
   j  Argon
   !  Arsenic
     Astatine
     Barium
   i  Berkelium
     Beryllium
     Bismuth
     Boron
     Bromine
     Cadmium
     Calcium
     Californium
     Carbon
     Cerium
     Cesium
     Chlorine
     Chromium
     Cobalt
     Columbium (see
     Copper
     Curium
     Dysprosium
     Einsteinium
     Erbium
     Europium
     Fermium
     Fluorine
     Francium
     Gadolinium
     Gallium
     Germanium
     Gold
     Hafnium
     Helium
     Holmium
     Hydrogen
     Indium
     Iodine
     Iridium
     Iron
     Krypton
     Lanthanum
     Lead
     Lithium
     Lutetium
     Magnesium
     Manganese
  Svmbol
   "
     Al
     Am
     Sb
     A
     As
     At
     Ba
     Bk
     Be
     Bi
     B
     Br
     Cd
     Ca
     Cf
     C
     Ce
     Cs
     Cl
     Cr
     Co
Niobium)
     Cu
     Cm
     Dy
     E
     Er
     Eu
     Fm
     F
     Fr
     Gd
     Ga
     Ge
     Au
     Hf
     He
     Ho
     H
     In
     I
     Ir
     Fe
     Kr
     La
     Pb
     Li
     Lu
     My
     Mn
At.
  /(89
  13
  95
  51
  18
  33
  85
  56
  97
   4
  83
   5
  35
  48
  20
  98
   6
  58
  55
  17
  24
  27

  29
  96
  66
  99
  68
  63
 100
   9
  87
  64
  31
  32
  79
  72
   2
  67
   1
  49
  53
  77
  26
  36
  57
  82
  3
  71
  12
  25
                                      No
db> W
1 f ' '
\ t. A
At.Wt.
227
26.98
[243|
121.76
39.944
74.91
[210|
137.36
[245|
9.013
209.00
10.82
79.916
112.41
40.08
[248|
12.011
140.13
132.91
35.457
52.01
58.94

63.54
[245]
162.51
[254]
167.27
152.0
[252]
19.00
(233)
157.26
69.72
72.60
197.0
178.50
4.003
164.94
J.0080
1U.82
126.91
1922
5585
83.80
13392
207.21
6940
17499
2432
54.94
- JL,. ,. ,,. r^
it •! • 4^.'d ^ rt •Jf ^

Element
...-•rWevium
•,.,-rcury
Molybdenum
s>odyiTiium
NVon
•jcptunium
s',
-------
RELATIVE HUMIDITY
Dry-Bulb Ther-
mometer: Degrees,
Fahrenheit
50
52
54
56
58
60
62
64
66
68
70
72
74
76
78
80
82
84
86
88
90
92
94
96
Difference between Dry-Bulb and Wet-Bulb Thermometers
1°
93
94
94
94
94
94
95
95
95
95
95
95
95
95
96
96
96
96
96
96
96
96
96
96
2°
87
87
88
88
89
89
89
90
90
90
90
91
91
91
91
92
92
92
92
92
92
92
93
93
3°
80
81
82
82
83
84
84
85
85
85
86
86
86
87
87
87
88
88
88
88
88
89
89
89
4°
74
75
76
77
78
78
79
79
80
81
81
82
82
82
83
83
84
84
84
85
85
85
86
86
5°
67
69
70
71
72
73
74
74
75
76
77
77
78
78
79
79
80
80
81
81
81
82
82
82
6°
61
63
64
65
67
68
69
70
71
71
72
73
74
74
75
75
76
77
77
77
78
78
79
79
7°
55
57
59
60
61
63
64
65
66
67
68
69
70
70
71
72
72
73
73
74
75
75
75
76
8°
50
51
53
55
56
58
59
60
61
63
64
65
66
66
67
68
69
69
70
71
71
72
72
73
9°
44
46
48
50
51
53
54
56
57
58
60
61
62
63
63
64
65
66
67
67
68
69
69
70
10°
38
40
43
44
46
48
50
51
53
54
55
57
58
59
60
61
62
63
63
64
65
65
66
67
11°
33
35
38
40
42
44
45
47
49
50
52
53
54
55
56
57
58
59
60
61
62
62
63
64
12°
27
30
32
35
37
39
41
43
45
46
48
49
50
52
53
54
55
56
57
58
59
59
60
61
13°
22
24
28
30
33
34
37
38
40
42
44
45
47
48
49
51
52
53
54
55
56
56
57
58
14°
16
20
23
25
28
30
32
34
36
38
40
42
43
45
46
47
48
49
51
52
53
54
54
55
15°
11
15
18
21
24
26
28
30
32
34
36
38
40
41
43
44
45
46
48
49
50
51
52
53
16°
6
10
13
16
19
22
24
27
29
31
33
35
36
38
39
41
42
44
45
46
47
48
49
50
17°
1
5
8
12
15
18
20
23
25
27
29
31
33
35
36
38
39
41
42
43
44
45
46
47
18°
0
0
4
8
11
14
16
19
22
24
26
28
30
31
33
35
36
38
39
40
41
43
44
45

-------
          -15-
Slock Solution! of Cations (50
Group Ion
I Ag+
Pb++
Hg»++
II Pb++
Bi+++
Cu++
Cd++
llg++
AB+++

Sb+++

Sa++


Sn++++
III Co++
Ni++
Mn++
Fe'l"l"f
AJ+++
Cr+++
Zn"1"1-
IV Ba++
Sr++
Ca++
VI f 11
Mg++
NH4+
Na+
K+
mg. of cation per ml.)
formula of Salt
AgNO,
Pb(NO,)2
Hg,(NO,),
Pl>(NOa),
Bi(NO3)3-5 H20
Cu(NO,)2-3 H20
Cd(NO,)2-4II,O
HgCI,
As,O,

SbCl,

SnCU- 2 FI20


SnCl4-3II20
Co(NO,)2-6II2O
Ni(NO,)2-6 I12O
Mn(NO5)2-6 H20
Fc(NO,),-9 II20
AI(NO,),-9 II20
Cr(NOs),
Zn(NOa),
BaCl,-2 H,0
Sr(NO,)2
Ca(NOs)2-4 HiO
Mg(NO,)2-6 II2O
N1I4NO,
NaNO,
KNO,

Grams per iOO ml. of Solution
8.0
8.0
7.0 (dissolve in 0.6 M UNO,)
8.0
11.5 (dissolve in 3 M UNO,)
19.0
13.8
6.8
3.3 (heat in 50 ml. of 12 M IICI, then
add 50 ml. of water)
9.5 (dissolve in 6 M IICI, and dilute
with 2 M IICI)
9.5 (dissolve in 50 ml. of 12 M IICI.
Dilute to 100 ml. with water.
Add a piece of tin metal)
13T3 (dissolve in 6 M IICI)
24.7
24.8
26.2
36.2
69.5
23.0
14.5
8.9
12.0
29.5
52.8
22 2
18.5
13.0
Composition of Commercial Acids and Bases
Acid or Jiase
Hydrochloric
Nitric
Sulfuric
Acetic
Aqueous ammonia
Specific
Gravity
1.19
1.42
1.84
1.05
0.90
Percentage
by Weight
38
70
95
99
28
Molar ily
12.4
15.8
17.8
17.3
14.8
Normality
12.4
15.8
35.6
17.3
14.8

-------
      Exponential Arithmetic
                                  -10-
   In chemistry we use the exponential metliod of expressing very large nnd very
 .small numbers.  These numbers are expressed as a product of two numbers.  Tin-
 first number of the product is called the digit term.  This term is usually a number
 not less than 1 and not greater than 10.  The second number of the  product is
 called the exponential term and is written as 10 with an exponent.  Some examplo
 of the exponential method of expressing numbers are given below.

                       1000 = 1 X 103
                        100 = 1 X 102
                         10 = 1 X 10l
                          1 = 1 X 10°
                         0.1 = 1 X 10-1
                       0.01 = 1 X 10-2
                      0.001 = 1 X 10-3
                       2386 = 2.386 X 1000 = 2.386 X 10s
                      0.123 = 1.23 X .1  = 1.23 X 1Q-1
   The power (exponent) ofJO is equal to the number of places the decimal is shifted
 to  give  the  digit  number.  The exponential  method  is particularly useful as n
 shorthand  for  big numbers.  For  example,  1,230,000,000 = 1.23 X 109;  and
 0.000,000,000,36 = 3.6 X IQ-'0.
   1.  Addition of Exponentials.  Convert  all the  numbers to the same power of
 10 and  add the digit terms of  the number.
   EXAMPLE.  Add 5 X  10-6 and 3 X 10~3
   SOLUTION.  3 X 10~3  = 300 X  10~6
              (5 X 10-6) + (300 X  10-6) = 305 X lO"6 = 3.05 X 1Q-'
   2.  Subtraction of Exponentials.   Convert all the numbers to the same power of
 10 and  take the difference of the digit terms.
   EXAMPLE.  Subtract 4 X 10~7 from 5 X  10"8
   SOLUTION.  4 X 10~7 =  0.4 X 10~s
              (5 X 10-6) - (0.4 X  10 6) =  4.6 X Ifl-6
   3.  Multiplication of Exponentials.   .Multiply the digit terms in  the  usual way
 nnd add algebraically the exponents of the exponential terms.
   EXAMPLE.  Multiply 4.2 X 10~a by 2 X 103
   SOLUTION.  4.2  X 10~8
               2X 10*
              8.4  X 10~*
  4. Division of Exponentials.   Divide  the digit  term of the  numerator by the
 digit  term of the  denominator and subtract algebraically  the exponents  of the
 exponential terms.
  EXAMPLE.  Divide 3.6 X lO"6 by 6 X 10-J
              •} f. v If)-6
  SOLUTION,  -y^pr = °-6 x ™~l = 6 X 10^2

  5. The Squaring of Exponentials. Square the digit term in the usual way and
 multiply the  exponent of the exponential term by 2.
  EXAMPLE.  Square the number 4 X 10~6
  SOLUTION.  (4 X 10-6)- = 16  X 10-'= =  1.6 X JO"11
  6. The Cubing of Exponentials.  Cube  the digit  term in the usual  way  arid
multiply the exponent of (lie exponential term by 3.
  EXAMPLE.  Cube the number 2 X 103
  SOLUTION.  (2 X 103)3 = 2 X  2 X 2 X H)9 = 8 X 109
  7. Extraction of Square  Knots of  Exponentials.   Decrease or increase  the expo-
nential term so that the power of ten is evenly divisible by 2.  Extract the square
root of the digit  term  by inspection or by logarithms and divide the exponential
term by  2.
  EXAMPLE.  Extract the square root  of 1.6 X 10~7
  SOLUTION.   1.6 X 10~7  = 16 X 10-"
                  X lO"8 = VT6 x VlcF1 = 4 X 10-4

-------
                             -17-
     Signiflcant Figures

   A bee keeper reports that he has 525,311 bees.  The last three figures of the nutn-
 bcr  are obviously inaccurate, for during the time the keeper was  counting the
 bees, some of them would have died and others would have hatched; this would
 have made the exact number of bees quite difficult to determine.  It would  have
 been more accurate if  he had  reported the number 525,000.  In other words, the
 last three figures are not significant, except to set the position of the decimal point.
 Their exact values have no meaning.
   In reporting any  information in terms of numbers,  only  as many  significant
 figures should be used as are warranted by the accuracy of the measurement.  The
 accuracy  of  measurements is dependent  upon  the sensitivity  of the  measuring
 instruments used.   For example, if the weight of an object has been reported as
 2.13 g., it is assumed that the last figure (3) has been estimated and that  the weight
 lies  between  2.125 g. and  2.135 g.  The quantity 2.13  g. represents three signifi-
 cant figures.  The weight of this same object as determined by a more sensitive
 balance may have been reported  as 2.134 g.  In this case one  would assume the
 correct weight  to be between  2.1335 g.  and 2.1315 g.,  and the quantity 2.134 g.
 represents 4 significant figures. Note that the last figure is estimated and is also
 considered as a significant figure.
   A zero  in a number may or may not be significant, depending upon the manner
 in which it ia used.  When one or more zeros are used in locating a decimal point,
 they are  not significant.  For example, the  numbers 0.063, 0.0063,  and 0.00063,
 each have two significant figures.  When zeros appear between digits in a number
 they arc significant.  For example, 1.008 g. has four significant figures.   Likewise,
 the  zero in 12.50 is significant.  However, the quantity 1370 cm. has four signifi-
 cant figures provided the accuracy of the  measurement includes the zero as  a sig-
 nificant digit; if the digit 7 is estimated, then the number has only three significant
 figures.
   The importance of significant figures lies in their application to fundamental
 computation.   When adding or subtracting, the last digit that is retained in the
 sum or difference should correspond to  the first doubtful decimal place (as  indi-
 cated by underscoring).
   EXAMPLE.  Add 4.383 g. and 0.0023 g.
   SOLUTION.  4.383 g.
             0.0023
             4.385 g.

 When multiplying or dividing, the product or  quotient should contain no  more
 digits than the  least number of significant figures in the numbers involved in the
computation.
  KXAMPLI:.  Multiply 0.6238J)y 6.6
  SOLUTION.  O.d2:$8 X 6.6 = 4.1_   ~
   In rounding off numbers, increase the last digit retained by one if the following
digit is five or more.  Thus 26.5 becomes 27, and 26.4 becomes 26 in the rounding-
ofl process.

-------
                              -18-
     The Use of Logarithms and Exponential Numbers

  Tlie common logarithm of a number is the power to which the number 10 must
be raised to equal that number.  For example, the logarithm of 100 is 2 because the
number 10 must be raised to the second power to be equal to  100.  Additional
examples are as follows:
Number
10
1






,000
,000
10
1
0.1
0.01
0.001
0.0001
Ntitnlier Expressed
ttsponenlially
10*
10'
10'
10°
!()->
lir2
10-'
l<)"«
Logarithm
4
3
1
0
-1
	 2
-3
-I
  What is the logarithm of 60?  Because 60 lies between 10 and 100, which have
logarithms of 1 and 2, respectively, the logarithm of 60 must lie between 1 and 2.
The logarithm of 60 is 1.7782, i.e.,  60 = 10'm".
  Every logarithm is made up of two parts, called the characteristic and the man-
tissa. The characteristic is that part of the logarithm which lies to the left of the
decimal point;  thus the characteristic of  the logarithm of 60 is 1.  The mantissa
is that part of the logarithm which lies 1o the right of  the decimal point;  thus
the mantissa of the logarithm of 60 is .7782.  The characteristic of the logarithm
of a number greater than 1 is one less than the number of digits to the left of the
decimal point in the number.
Number
60
600
6000
52840
Cliaracteristic
1
2
3
4
Number
2.310
23.40
234.0
2340.0
Characteristic
0
1
2
3
The mantissa of (he logarithm of a number is found in the logarithm table (see
Appendix B), and its value is independent of the position of the decimal point.
Thus  2.340, 23.4-0, 234.0, and 2340.0 all have the same mantissa.  The logarithm
of 2.340 is 0.3692, that of 23.40 is 1.3692, that of 231,0 is 2.3692, and that of 2340.0
is 3.3692.
  The meaning of the mantissa and characteristic can be better understood from
a consideration of their relationship to exponential numbers.   For example, 2340
may be  written 2.31 X 103.  The logarithm of  (2.34 X 103)  = the  logarithm  of
2.34 + the logarithm  of 103.  The logarithm of 2.34 is .3692  (mantissa) and  the
logarithm of 10' is 3 (characteristic).  Thus the logarithm of 2340 = 3 + .3692,
or 3.3692.

-------
                          -19-
  The logarithm of a number less than 1 lias a negative value, and a convenient
method of obtaining the logarithm of such a number is given below.  For example,
we may obtain the logarithm of .00231 its follows:  When expressed exponentially,
.00234 = 2.34 X 10-3.  The logarithm of 2.31  X  10  ' = the logarithm of 2.31 + the
logarithm  of J0~3.  The logarithm of 2.31  is .3692 (mantissa) and the logarithm of
10-3 is -3  (characteristic).  Thus the logarithm of .002:51 = .3692 + (-3) = .3602 -
3 = -2.6208.   The abbreviated form for the expression  (.3602 - 3) is 3.3602.
Note thai only the characteristic has a  negative value in  the  logarithm 3.3602,
and that the mantissa is positive.  The logarithm 3.3692 may also be written as
7.3692-10.
  To multiply two numbers we  add the logarithms of the numbers.  For example,
suppose we multiply 412 by 353.
                       Logarithm of 112    =2.6149
                       Logarithm of 353    = 2.5478
                       Logarithm of product = 5.1627

The number which corresponds to the logarithm  5.1627  is  145400 or 1.454 X 10s.
Thus 1.45 X 10s is the product of 412 and 353.
  To divide two  numbers we subtract the logarithms of the numbers.  Suppose
we divide 412 by  353.
                       Logarithm of 412     = 2.6149
                       Logarithm of 353     = 2.5478
                       Logarithm of quotient = 0.0671

The number which corresponds  to the logarithm 0.0671 is 1.17.  Thus 412 divided
by 353 is 1.17.
   Suppose we multiply 5432 by 0.3124.  Add  the logarithm of 0.312* to that of
5432.
                      Logarithm of 5432      = 3.7350
                      Logarithm of 0.3124     = 1.4918
                      Logarithm of the product = 3.2298

The number which corresponds  lo the logarithm 3.2208 is 1697 or 1.697 X 10s.
   Let us  divide 5432 by 0.3121.  Subtract the logarithm of 0.3124 from that of
5432.
                      Logarithm of 5132        = 3.7350
                      Logarithm of 0.3124      = Xl?48
                      Logarithm of I hi:  quotient = 4.2102

The number which corresponds to the logarithm  4.2102 is  17390 or I.739X 104.
   The extraction  of roots of numbers by means  of logarithms is a simple  pro-
cedure.   For example, suppose  we extract I lie  cube root of 7235.  The logarithm
of  ^7235 or (7235)* is eqiii.l to >- of (he.  logarithm of 7235.

                         Logarithm of  7235 = 3.8594
                                & of 3.8391 = 1.2865

The number which corresponds to  the  logarithm 1.2865 is  19.34.  Thus,  19.31
is the cube root of 7235.

-------
      ELEMENTS OF A QUALITY ASSURANCE PROGRAM
                         By

                  Kathleen Shimmin
                  EPA, Region IX
                  San Francisco CA
Presented at the Workshop on Sampling, Monitoring and Analysis
  of Water and Wastewater, March 6-12, 1974, Honolulu HI.

-------
(These procedures are minimum efforts.  Depending upon the purpose  of the
analysis, even more rigorous procedures may be warranted.)
                  ELEMENTS OF A QUALITY ASSURANCE PROGRAM
A.  Procedures for any given laboratory - CHEMISTRY
     I.  Intralaboratory procedures
         1.  Use established procedures (St.Meth., ASTM,  EPA - choice depends  upon  need)
            a.  Choose proper procedure for given sample  - taking note of  interferences .
                                                                                 etc.
            b.  Choose procedure with sensitivity appropriate for need.
                Note  sensitivity.
            c.  Have written laboratory manual and use it.
            d0  Note procedure used when data reported (if a choice exists
                in the laboratory manual).
         2.  Demonstrate that analyst is capable of analysis
            a.  Give proper training if necessary
            b.  Routinely run standard curves, unknowns,   blanks.
            c.  Periodically run reference samples (preferably prepared by  an
                independent laboratory)
            d.  Prepare quality control charts for each analyst and each analysis
                i)  Precision
                ii) Accuracy
            e.  Have analysts crosscheck each other's calculations  and technique
         3.  Have established procedures for quality assurance for  the data
             being produced
            a.  Check precision by analyzing duplicates,  at least one per  ten  samples
            b0  Check accuracy by analyzing spikes, at least one per ten samples
         4.  Take appropriate steps to redo samples when  quality control chart
             limits are exceeded
         5.  Maintain permanent records in bound volumes
         6.  Have established safety precautions and adhere to them
         7.  Have established procedures to assure the quality of equipment,
             reagents, glassware
            a.  Regular servicing
            b.  Conductivity checks on deionized, distilled waters
            c.  Date reagents,  chemicals, solutions.  Store properly
         8.  Prepare and follow written  sampling and handling procedures.
             Procedures should  be in accordance with EPA  guidelines.
     II.  Interlaboratory procedures
         1.  Periodically analyze samples split with another lab.
         2.  Participate in round-robin test evaluations
         3.  Maintain  contact  with other laboratories
        4.   Participate in Laboratory  evaluation programs

-------
 ELEMENTS, continued                                   page two
B.  Procedures for any given laboratory r BACTERIOLOGY
     I.  Intralaboratory procedures
         1.  Use established procedures specified for a given sample (St. Meth. or EPA)
            a.  Choose proper procedure for a given sample
            b.  When required to quantify bacterial levels, choose procedure
                with appropriate sensitivity
            c.  Have written laboratory manual
            d.  Note procedure used when data reported
         2,  Demonstrate that analyst is capable of analysis
            a.  Give proper training
            b.  Split samples with other analysts routinely
            c.  Have analysts crosscheck each other's calculations and procedures
         3.  Have established procedures for quality assurance for data being produced
            (Example - EPA)
            a.  Membrane filters - run in duplicate, at least 4 dilutions per
                medium per sample; periodically confirm selected colonies
                with MPN procedures
            b.  MPN - use 5 tubes per dilution, at least 3 dilutions
                For a selected percentage of tests go through to Completed Test
            c.  Standard Plate Count - run in duplicate
            d.  Analyze controls (blanks, known spikes)
            e.  Report data which falls within statistically significant
                confidence range for given test
                i.  MF, total coliforms  20 - 80  colonies per plate
                        Fecal coliforms  20 - 60  colonies per plate
                        Fecal streptococci  20 - 100 colonies per plate
                ii. Plate Count (100 mm diameter)  30 - 300 colonies per plate
                Numbers outside range should be reported "less than" or
                "greater than" and should be redone if necessary
         4.  Have  established procedures to assure quality of media, cultures,
             glassware, equipment, etc.  Maintain records of this.
            a.  Media
                i.    Note dates received, opened
                ii.   Discard outdated material (or use only for screening purposes)
                iiio  Store under proper conditions (temperature, humidity, light)
                iv.   Prepare media properly
                v.    Establish and maintain program to check media periodically
                      (by batch lots) to assure appropriate positive and negative resui
            b_  Stock cultures (if these are maintained in laboratory)
                i.    Transfer at appropriate frequency (e.g., once per month)
                ii.   Routinely check purity of cultures by making streak plates,
                      and repurify if necessary

-------
ELEMENTS, continued                                    page three

            c.  Water supply system (distilled), for media preparation
                i.    Periodically check toxicity of distilled water to a
                      given bacterial culture - usually Enterobacter aerogenes
                      Standard Methods recommends a frequency of at least once per ye,
            d „  Equipment - repair
                i.    Maintain routine service contracts on major equipment
                      such as autoclaves, microscopes, balances, hoods
                ii.   Keep other equipment in good repair - either by contract or
                      through other means
                iii.  Note dates of service by placing labels on the equipment
                      (labels should list date, name, and address of service person)
            e.  Equipment - performance
                i»    Use recording charts for temperature of waterbath, autoclave
                      Store these as lab records
                ii.   Record temperatures periodically for incubators - frequency
                      depends upon usage
                      As an alternative, use a max-min thermometer and record results
                iii.  Test accuracy of automatic pipetting machine before
                      and during use
                iv„   Test accuracy of thermometers against NBS-registered
                      thermometer (with chart), appropriate for the desired
                      temperature range
         5.  Maintain permanent records in bound volumes
         6.  Establish safety precautions and adhere to them
            a.  Sterilize all contaminated material before washing or discarding it
            b„  Use aseptic technique
            c0  Properly train all individuals before allowing them to work
                with potentially-contaminated material
            d.  Immunize lab workers against tetanus (and possibly typhoid or
                other disease as appropriate)
            e.  Etc.
         7.  Follow established sample handling procedures, which are in agreement
             with EPA guidelines
            a.  Adhere to temperature-of-storage conditions
            b.  Do not exceed maximum allowable period for time elapsing
                between collection and processing of sample
            c.  Establish chain-of-custody routine for possible enforcement samples
     II.  Interlaboratory procedures
         1.  Periodically analyze samples split with another lab
         2.  Participate in round-robin test evaluations
         3.  Maintain contact with other laboratories
         4.  Participate in laboratory evaluation programs

-------
ELEMENTS, continued                                    page four
C.  Procedures for any given laboratory - BIOASSAY
     I.   Intralaboratory procedures            ......	 —  -
         1.  Use established procedures
            a.  Choose proper procedure and test animal for given sample
            b.  Hold animal for required time under required conditions prior
                to initiating test
            c.  Choose appropriate dilutions for documentations of standards compliance
            d.  Have written laboratory manual
            e.  Note procedure used when data reported
         2.  Demonstrate that analyst is capable of analysis
            a.  Give proper training
            b.  During learning period split samples with other analysts
         3.  Have established procedures for quality assurance for data
             being produced
            a.  Statistically evaluate results
            b.  Report confidence intervals for data, unless only an estimated
                figure is requested
         4.  Maintain permanent records in bound volumes
         5.  Have established safety precautions and adhere to them
         6.  Have established procedures to assure quality of equipment,
             reagents, glassware
         7.  Follow established sample handling procedures in accordance with
             EPA guidelines
     II.  Interlaboratory procedures
         1.  Periodically analyze samples split with another lab
         2.  Participate in round-robin test evaluations
         3.  Maintain contact with other laboratories
         4.  Participate in laboratory evaluation programs

-------
ENVIRONMENTAL PROTECTION AGENCY




       STANDARD METHODS




            USED BY




           REGION IX




    MICROBIOLOGY LABORATORY

-------
                         Region IX


          Activities of the Microbiology Section


     The Microbiology Laboratory has established tests which
it can perform.  These include:  indicator organisms assays
(total and fecal coliform, fecal streptococci) by multiple
tube dilution and membrane filter techniques; plate counts
at 20° and 35°C; pathogen isolation  (Salmonella), serology,
and fluorescent-antibody scanning.  Methodology is attached.

     Upon request the Section can adapt existing techniques
and develop special ones for recovering such organisms as:
anaerobic bacteria; photosynthetic bacteria; Pseudomonas sp.;
sulfur oxidizers; Klebsiella; and other specific groups.

     Staff from the Section will also offer technical advice
and consultation for review of grants,  permits, standards
and research proposals, etc.  Lectures and training courses
in laboratory and field techniques and sample collection have
been given by the Section in the past and can be developed or
modified upon request.
                             Kathleen G. Shimmin
                             Chief,  Microbiology Section

-------
               TABLE OF CONTENTS


                                                  PAGE

Sample Collection and Chain-of-Custody              1

Determination of Coliform Organisms by
Membrane Filter Procedure
     Total Coliforms                                4
     Fecal Coliforms                                7

Determination of Fecal Streptococcus Organisms
by Membrane Filter Procedure using:

     M-Enterococcus Agar                            9
     KF Streptococcus Agar                         11

Most Probable Number  (MPN) Test for the
Detection and Enumeration of Coliform and
Fecal Coliform Organisms                           12

Most Probable Number  (MPN) Test for the
Detection and Enumeration of Fecal
Streptococcus Organisms                            16

Estimation of Bacterial Density                    18

Gram's Stain Technique                             23

IMViC Procedures                                   24

Procedure for Salmonella Isolation                 26

Fluorescent Antibody  (FA) Salmonella Screening     31

Serological Grouping of Salmonella                 32

Diagram of Slide Agglutination                     34

-------
                       TABLES
                                                      PAGE
*407(1):
*407(2):
 I.
 II.
MPN Index and 95% Confidence Limits
for Various Combinations of Positive
and Negative Results when Five 10-ml
portions are used

MPN Index and 95% Confidence Limits
for Various Combinations of Positive
and Negative Results when Five 10-ml
Portions, Five 1-ml Portions, and Five
0.1-ml Portions are used.

Incubation Time and Colonial
Appearance for Various Organisms in
Selected Media

Salmonelleae - Parameters and
Biochemical Reactions
                                                       20
                                                       21
                                                       28
                                                       29
*Refers to Section Numbers in Standard Methods, 13th Edition.

-------
         STANDARD PROCEDURES USED BY REGION IX
   LABORATORY FOR BACTERIOLOGICAL EXAMINATION
           OF TOTAL AND FECAL COLIFORMS
Sample Collection and Chain-of-Custody

a.   Procedure for collection of samples shall be
     done in accordance with methods as outlined in
      Standard Methods for the Examination of Water
     and Wastewater;13th Edition.

b.   Samples should be properly iced immediately after
     collection  (ice bucket or chest).

c.   Chain-of-custody should be as follows:

     1.   Two people are required; one as sampler,
          one as witness.

     2.   Label on sample container should read:   (See
          Attachment - page 3)

          Sample Number             (Use a self-stickinq
          Sample Description         label which will not
          Time and Date Collected    deteriorate in ice chest)
          Collected by
          Witnessed by

     3.   If samples change hands during transport back
          to laboratory, label or tag should be attached
          to ice bucket or chest, signed by person and
          witness to whom custody was given.  (See Attachment)

     4.   Upon delivery to laboratory the technican should
          make out a receipt stating time and condition
          samples were received.

     5.   Before processing, log in time received, time
          collected, time processed, and processor.  This
          can be done in same log book as results are
          recorded.

Processing

a.   Do at least duplicate replicate plates for each
     dilution.

b.   For unknown water, do at least four dilutions with at  least
     two replicates for each dilution.  (A minimum of eight plates
     per medium ner sample.)

-------
3.    Time lapse between sample collection and processing is
     as follows:

     a.   Seawater                  4 hours for Total Coliforms
                                    2 hours for Fecal Coliforms

     b.   Freshwater                6 hours for Total Coliforms
                                   3-4 hours for Fecal Coliforms

     c.   Shellfish                 6 hours for Total and Fecal
                                   12 hours maximum

     Examination of shellfish for total and fecal coliforms
     should be done according to the procedures recommended
     in most recent edition of American Public Health Association,
     Recommended Procedures for the Examination of Seawater and
     Shellfish.

-------
                  ATTACHMENT
N"
Sample  Description

Date      (c

Sampler

Witness
                                    Time
               SAMPLE  BOTTLE LABEL
ENVIRONMENTAL PROTECTION AGENCY

SAMPLE NO.
SIGNATURE
PRINT NAME AND TITLE f to factor, Analyst or Tacfmfcl«0
SIH. BROKEN »Y
Ul
5
r*»
&
0
U- 
-------
                                        Standard Method used
                                                by
                                        Region IX, Laboratory
       THE DETERMINATION OF COLIFORM ORGANISMS
         BY THE MEMBRANE FILTER PROCEDURE

               M-ENDO - Total Coliforms
I.     Preparation of Medium

      1.   Boil 500 ml.  distilled wate:n

      2.   Add 1.5% Bacto-Agar (7.5 g)  and stir to dissolve.

      3.   Add 24 g. Difco M-Endo medium and 10 ml of 95% ethanol.
           Bring to boil to dissolve (keep stirring).

           DO NOT CONTINUE TO BOIL the medium once boiling point
           reached.

      4.   Cool medium slightly and distribute about 5 ml per
           petri dish (sterile, disposable, 50 x 12 mm).

      5.   Allow the plates to harden.   Pack the plates in an
           inverted position in a basket and cover with brown
           paper.  Place the basket in the refrigerator.

           In the dark and under cool conditions the prepared
           medium can be stored for short periods of time.  The
           prepared plates should be used within a 24 hour period
           Under no circumstances will the plates be stored for
           periods of 72 hours or longer.  Results from such
           plates are questionable.

II.   Testing Procedures

      1.   All filtrations should be carried out according to the
           protocol outlined in Section 408, page 678 of Standard
           Methods, 13th Edition, 1971.

      2.   Sample volumes to be filtered should be chosen so that
           at least one  membrane filter contains between 20-80
           coliform  colonies, and not  to exceed 200 colonies of
           all types on  the filter.

-------
     In the absence of previous bacterial data the
     following are recommended volumes:

     a.   Treated water supplies 	 minimum of 50 ml,
                                      100 ml  recommended.
     b.   Untreated water supplies — 5 - 50 ml.
     c.   Unpolluted surface water -- 1, 4, 15 and 60 ml
                                       (covers range 33-8000).
     d.   Polluted surface water 	 0.02, 0.08, 0.15 and
                                      0.5 ml (covers range
                                      of 4000 - 40,000) .
     e.   Sewage                 	 0.0003, 0.001, 0.003
                                      and 0.01 ml (covers
                                      range of 200,000 -
                                      27,000,000 cells/lOOml)

3.    The plates containing the membranes are placed in
     a 35°C incubator in an inverted position.

4.    After a period of 24 - 2 hours the plates are removed
     from the incubator.  Coliform colonies are dark red
     and have a green metallic surface sheen.  Non-coliform
     colonies range from colorless to pink, however, the
     metallic sheen is absent.  Such type colonies should
     not be included in the coliform count.

5.    The characteristic metallic sheen colonies are counted
     with the aid of a wide-field binocular microscope
     using 10 or 15 x magnification.  For illumination,
     use a light source directly over the membrane filter
     so that an image of the light source is reflected
     off the colony surface into the microscope lens system.
     (Suitable for this purpose is a Stereozoom Microscope
     [A/0 or B&L]  with a fluorescent illuminator).

6.    Select the membranes that have between 20 to 80
     coliform colonies and compute  the density per 100 ml.
     The actual colony counts and the calculated density
     per 100 ml are entered on the data sheet.

     No. of coliform colonies counted x 100 = No.  of colonies
       Sample volume filtered in ml             per -^QQ mj_

7.    The plates are then placed in metal containers and
     sterilized in the autoclave.  At no time will any
     culture medium containing bacteria be disposed of
     first without adequate sterilization.

-------
8.    Calculations

     a.   For routine work,  at least 3 dilutions of the
          sample are made.   However, when testing water
          where no previous  information is available then
          it may be necessary to use as many as 4 or 5
          dilutions.

     b.   Select the membrane that has between 20-80
          coliform colonies  and compute the density
          per 100 ml.

     c.   If several sample  volumes have coliform colonies
          in the range 20-80, then average the counts per
          100 ml.

     d.   If all membranes have counts outside the range
          20-80, the following procedure should be used:

          1.   Low counts (below 20 colonies).  Calculate
               the density  if 20 colonies were to have
               been present.  Report on this calculation
               on the data  sheet preceded by "<" ("less
               than").

          2,   High counts  (above 80 colonies).  Calculate
               the density  if 80 colonies were to have
               been present.  Report this calculation on
               the data sheet preceded by ">"  ("greater
               than").

          It must be realized that data reported with
          "less than" and "greater than" have  limited use
          when strict interpretation of data is required.
          It does provide some idea as to the  relative
          coliform density of the sample.  Most important
          of all, it sh9ws the need for repeat sampling and
          adjustment of sample filtration volume so that
          a membrane with the desired range 20-80 may
          be obtained.

          In order to obtain at least one membrane having
          an acceptable number of colonies, the range of
          filtration volumes should vary by a  factor of
          4 or less.  Different factors apply  to fecal
          coliform and fecal streptococcus.

-------
                         REGION IX



       THE DETERMINATION OF FECAL COLIFORM ORGANISMS

             BY THE MEMBRANE FILTER TECHNIQUE


I.    Medium Preparation

      1.   Rehydrate Bacto-M-FC Broth Base by adding 3.7g in
           100 ml distilled water.

      2.   Add one ml of a rosolic acid solution prepared by
           dissolving one gram rosolic acid in 100 ml 0.2N NaOh.

      3.   Add 1.5% Bacto Agar.

      4.   Dissolve ingredients in a boiling water bath or in
           "Instatherm" apparatus.

      5.   Bring to a boil and pour approximately 5 ml into
           sterile, disposable 50 x 12mm petri dishes.  Final
           reaction of the medium is pH 7.4.

      6.   Allow the medium to harden.

      7.   Pack the plates in baskets in the inverted position
           and place in the refrigerator.

      8.   Prepared medium "shelf life" is 5-7 days if stored in
           the refrigerator away from light.

      9.   The rosolic acid is stable indefinitely in the dry
           state.  In aqueous solution it is stable for two
           weeks under refrigeration.  After this period
           degradation is evidenced by the change of color
           (Red to Brown).

II.    Testing Procedures

      1.   All filtrations  should be carried out according to
           the protocol outlined in Section 408, page 678 of
           Standard Methods, 13th Edition, 1971.

      2.   Sample volumes to be filtered should be chosen so
           that at least one membrane filter contains between
           20-60 fecal coliform colonies.

-------
      3 .    The plates containing the membranes are placed in
           water-proof plastic bags (Whirl-Pak Bags)  and
           submerged in a 44,5 - 0.2°C water bath for 24 hours.
           (Plates should not be held no longer than 20 minutes
           at ambient temperature after filtration.  Immediate
           introduction of plates into the44.5°C water bath
           is recommended) .

      4.    After incubation, colony counts are made using a
           wide-field binocular microscope - lOx magnification.
           Fecal coliform colonies are deep blue in color and
           may vary from one to three mm in diameter.  Non-
           fecal coliform colonies will appear as pink or
           colorless type colonies and should not be included
           in the fecal coliform count.

      5.    The colony counts are entered on the data sheet and
           reported per 100  ml.
No. of fecal coliform colonies counted x ^OQ = NQ.  of colonies
    Sample volume filtered in ml                 *      per
      6.   Calculations and selection of sample filtration
           volumes follow the same theory as that discussed in
           the Method, Total Coliform Determination
           by Membrane Filter Procedure ,   Exceptions are that
           the desired range of fecal coliform colonies on the
           membrane is 20-60, and that fecal coliform counts
           should be based on filtration volumes varying by a
           factor of 3 or less.

      7 .   The plates are than placed in metal containers and
           sterilized in autoclave.   At no time will  any culture
           medium containing bacteria be disposed of  first without
           adequate sterilization.

-------
         THE DETERMINATION OF FECAL STREPTOCOCCI
             BY THE MEMBRANE FILTER PROCEDURE


I.    Preparation of Medium

      1.   Weigh out 21 grams of M-Enterococcus Agar and place
           in one liter flask.

      2.   Add 500 ml cold distilled water and bring solution
           to a boil using "Instatherm" or boiling water bath
           and remove as soon as agar is in solution.
           (Do not overheat.)

      3.   Final pH should be:  7.2

      4.   Pour approximately 5 ml of the medium into sterile,
           disposable, 50 x 12 mm petri dishes.

      5.   Allow the medium to harden.  Pack the plates in an
           inverted position in a basket and cover with brown
           paper.  The prepared plates can be stored in the
           refrigerator up to four weeks.

      6.   The filtration of the sample should be carried out
           according to the methods outlined in the section on
           Filtering Techniques.

      7.   Sample volume to be filtered should be chosen so that
           at least one membrane filter contains between 20-100
           fecal streptococcus colonies.

      8.   The plates containing the membranes are placed in a
           35°C incubator in an inverted position.

      9.   After a period of 48 hours the plates are removed
           from the incubator.  With a wide-field binocular
           microscope, using 10 or 15x magnification, count
           all pink, red and purplish-red colonies.  Colonies
           other than these are not fecal streptococci and should
           not be included in the count.

     10.   The colony counts are entered on the data sheet and
           reported per 100 ml.

\'o.  of fecal streptococci counted x 100 = No.  of fecal streptococci
 Sample volume filtered in ml                            per j^QO ml

-------
                                                      10
11.  Calculation and selection of sample filtration
     volumes follow the same principle as that
     discussed in Method of Coliform Determination By
     Membrane Filter Procedure, except that the desired
     range of colonies on the membrane is between 20-100
     and the counts should be based on filtration volumes
     varying by a factor of 5 or less.

12.  The plates are than placed in metal containers and
     sterilized in the autoclave and discarded.

-------
                                                          11
Standard Methods used by Region IX for the Determination of
Fecal Streptococcus by Membrane Filter using Bacto-KF
Streptococcus Agar


I.   Preparation of Medium

     a.   Weigh out 76.4 grams KF Streptococcus agar and
          place in flask.

     b.   Add 1000 ml cold distilled water and heat to boiling
          to dissolve completely.

     c.   Dispense in 100 ml amounts or multiples thereof
          into flasks and sterilize for 10 minutes at 15 Ib
          pressure  (121°C) .

     d.   Cool to 60°C and add one ml Bacto TTC Solution 1%.
          (Triphenyltetrazolium Chloride 1%) per 100 ml sterile
          medium.

     e.   Mix to obtain uniform distibution of the TTC.
     f.   Final pH 7.2.

     g.   Pour approximately 5 ml of medium into sterile
          disposable, 50x12 mm petri dishes.

     h.   Incubate inoculated plates for 48 hours at 35°c.

     i.   Using a dissecting microscope with a magnification
          of 15 diameters count all colonies showing red or
          pink center as streptococcus.

     j.   Colony counts are entered on data sheet and reported
          per 100 ml.

     k.   Calculation:                         fecal
Number of fecal streptococcus_counted  x 10Q = streptococcus/100m
      Sample volume fxltered in ml


     1.   Desired range of colonies on membrane is 20-100 and
          counts should be based on filtration volumes  varying
          by a factor of 5 or less.

     m.   Plates are then placed in metal containers and
          sterilized in an autoclave.  (120°C, 15 Ib  pressure
          for 1/2 hour).

-------
                                                          12
           THE MOST PROBABLE NUMBER (MPN)  TEST
           FOR THE DETECTION AND ENUMERATION OF
           COLIFORM AND FECAL COLIFORM ORGANISMS
I.     Presumptive Test

      A.    Preparation of medium

           1.    For 1 ml of sample inoculation:   weigh out
                35.6g lauryl tryptose broth and  add to one
                liter of distilled water.   (For  10 ml of
                sample inoculation 53.4g per one liter of
                distilled water).

           2.    Dissolve  ingredients and dispense ten ml of
                mixture 1 above into test tubes.  Dispense
                20 ml of 53.4g per one liter mixture into
                large test tubes.

           3.    Insert one Durham  gas tube, inverted position,
                into each tube containing the broth.  Place auto-
                clavable plastic Kaputs on tubes.  The double-
                strength tubes may be coded by closing them with
                Kaputs of a different color.
           4.    Sterilize in the autoclave for 12 minutes at
                12 Ibs, pressure.

      B.    Procedure

           1.    The five tube, multiple fermentation technique
                will be used.

           2.    Inoculate a series of five tubes in each dilution,
                In all such analyses, at least 3 dilutions must
                be used.  (Region  IX routinely uses 5 dilutions.)

           3.    The portions of the water sample used for
                inoculating the fermentation tubes will vary
                with the type water being tested-  In general,
                decimal multiples  and sub-multiples of one ml
                will be used.

           4.    Incubate the fermentation tubes  at 35.0 - 0.5°C.
                Examine each tube  at the end of  24 ±  2 hours
                and, if no gas had yet been produced, examine
                again at the end of 48  hours ±  three hours.

-------
                                                             13
                Record the presence or absence of gas
                formation at each examination of the tubes.
                The smallest type bubble in the gas tube
                should be recorded as a positive tube, even
                though this may appear as trapped air in the
                tube and not really gas production from the
                fermentation process.
II.  Confirmed  Test

      A.    Preparation of medium

           1.   Weigh out 40.Og of Bacto-Brilliant Green Bile
                2% and add to one liter of distilled water.

           2.   Dissolve ingredients and dispense ten ml into
                each test tube.

           3.   In the inverted position, insert a Durham gas
                vial into each test tube.  Use Kaput closures
                to cover the tubes.

           4.   Sterilize in the autoclave for 12 minutes at
                12 Ibs.  pressure.  Final reaction of the medium
                is pH 7.2.

      B.    Procedure

          *1.   Using a  24 gauge wire loop, with a loop of 3 mm
                in diameter, transfer one loopful of the positive,
                lauryl tryptose broth into a tube of brilliant
                green bile broth.

                (If active fermentation appears in the primary
                fermentation tube before the expiration of the
                24 hour  period of incubation, it is preferable
                to transfer to the confirmatory brilliant green
                bile  broth without waiting for the full 24 hour
                period to elapse.)

           2.    Incubate the inoculated brilliant green lactose
                bile  broth tube for 48 ± 3 hours at 35° ± 0.5°C.

           3.    The formation of gas in any amount in the Durham
                tube,  constitutes a positive Confirmed Test.
                Record all positives and negatives on the data
                sheet.

     *As  an alternative  to transfering with a wire loop, sterilized
     hardwood  applicator sticks (sterilized in dry heat, 1-1/2
     hours, 170°C, stored in glass tubes or syringe-sterilization
     bags)  may  be used.   Each stick is user! once and then dis-
     carded into  a disinfectant-fillQd ^discard container.

-------
                                                             14
III.  Fecal Coliform (MPN)

      A.   Preparation of medium

           1.   Weigh out 37.0g of Bacto E.G. medium and
                add to one liter of cold distilled water.

           2.   Dissolve the ingredients and dispense into
                test tubes in ten ml amounts.

           3.   Insert a Durham gas vial into each tube.
                (In the inverted position.)  Use Kaput closures
                for these tubes.

           4.   Sterilize in the autoclave for 12 minutes at
                12 Ibs. pressure.  Final reaction of the medium
                is pH 6.9.

      B.   Procedure

          *1.   Using a 24 gauge wire loop,  with a loop of 3 mm
                in diameter, transfer one loopful of the positive,
                lauryl tryptose broth into a tube of E.G. medium.

           2.   The inoculated tube must be  put into a 44.5 ±
                0-2°C water bath not later than 20 minutes after
                initial inoculation.

           3.   The formation of any amount  of gas in the vial
                at the end of 24 hours constitutes a positive
                test.  At the end of 24 hours the positive
                and negative results are entered on the data
                sheet.  Readings after 24 hours are invalid.

IV.   Computing of MPN

      1.   The number of positive findings of coliform group
           organisms (Presumptive, Confirmed and Fecal)  resulting
           from multiple-portion, decimal dilution inoculations
           should be computed and recorded in terms of the
           "most probable number" (MPN).

      2.   See MPN and 95%  Confidence Limits for Various
           Combination of Positive Results in Standard Methods,
           13th Edition, 1971.

      *The single-use, sterile, hardwood applicators may be used
       as an alternative to transferring with a wire loop.

-------
                                                                  15
     V.    Schematic Diagram  Illustrating  Steps  in  MPN  Procedures.


                                           Sample

                                  Lauryl  Tryptose Broth

                  Incubate  in  air  incubator at  35°C  + 0.5°C for 24  hours

                       \i-                                       v
               No gas  produced                    Production of gas
               Return  to  incubator           Positive Presumptive  Test
               at 35°C for 24 hours               for Coliform Group
 No gas:                      +gas,  Positive
 coliform                     Presumptive Test
 group absent                 for Coliform Group
                                     v/
     Brilliant Green  Bile  Broth                       E.G.
   Incubate  in 35  ±  0.5°C  in incubator          Incubate in 44.5 ± 0.2°C
            for 24 hours                         water bath  for 24 hours.

 No  gas produced        +Gas                  No gas =        + gas
 return to  incubator    Positive Confirmed   Fecal coliform  Fecal coli-
 for another  24 hrs.    Test  for Coliform    group absent    form group
                          Group                              present
     j                       1-
No g,- s produced    +Gas:
ColiLorm group     Positive  Confirmed
abse \t             Test  for  Coliform
                  Group

-------
                                                               16
      THE MOST PROBABLE NUMBER (MPN)  TEST FOR THE DETECTION
        AND ENUMERATION OF FECAL STREPTOCOCCI ORGANISMS
 I.   Presumptive Test

     A.   Preparation of medium

         1.   For 1 ml of sample inoculation:  weigh out 34.7 grams
             Azide Dextrose Broth and add to one liter (1000 ml)
             of distilled water (for 10 ml of sample inoculation
             prepare double-strength medium).

         2.   Dissolve ingredients and dispense 10 ml of mixture
             into test tubes for 1 ml sample inoculation.  Dis-
             pense 10 ml of double strength medium into large
             (18 x 150 mm)  test tube for a 10 ml sample inoculum.

         3.   Place plastic or metal caps on tubes.

         4.   Sterilize in the autoclave for 15 minutes at 15
             pounds pressure (121°C).  Final reaction of medium
             is pH 7.2 25°C.

     B.   Procedure

         1.   The five-tube, multiple fermentation technique will
             be used.

         2.   Inoculate a series of five tubes in each dilution.
             In all such analyses, at least 3 dilutions must be
             used (EPA, Region IX routinely uses 5  dilutions).

         3.   The dilutions of the water sample used for inoculat-
             ing the fermentation tubes will vary with the type
             of water being analyzed.  Decimal multiples and
             decimal dilutions of one ml are used.

         4.   Incubate the inoculated tubes at 35.0  ± 0.5°C.
             Examine each tube for the presence of  turbidity at
             end of 24 ± two hours.  If no definite turbidity is
             present, reincubate and read again at  end of 48 ±
             three hours.

II.   Confirmed Test

     A.   Preparation of Medium

         1.   Weigh out 35.8 grams EVA Broth (Ethyl  Violet Azide)
             and add to 1 liter (1000 ml)  distilled water.

-------
                                                         17


    2.  Dissolve and dispense 10 ml portions into test tubes,

    3.  Sterilize in autoclave for 15 minutes at 15 pounds
        pressure (121°C).  Final reaction of medium pH 7.0
        at 25°C.

B.  Procedure

    1.  Transfer 3 loopfuls of growth or use a wooden appli-
        cator to transfer growth from each azide dextrose
        broth tube to a tube containing 10 ml ethyl violet
        azide broth.

    2.  Do not discard positive tubes  (presumptive).  Hold
        in the incubator.

    3.  Incubate the inoculated tubes for 24 hours at 35 i
        0.5°C.  The presence of fecal streptococci is indi-
        cated by formation of a purple button at the bottom
        of the tube or by a very dense turbidity.

    4.  Record all positive tubes.  Discard those tubes.

    5.  If no growth  (purple button or heavy tubidity)
        appears in ethyl violet azide in 24 hours, reinocu-
        late the tubes with an additional 3 loopfuls  (or use
        wooden applicator) from the original positive azide
        broth cultures and reincubate for another 24 hours.

    6.  Record results.

C.  Computing and Recording MPN

    Same as with computation of total and fecal coliform
    organisms.

-------
                                                           18


           STANDARD METHODS,  APHA, 13th EDITION
          407 D.  ESTIMATION OF BACTERIAL DENSITY


1.     Precision of Fermentation Tube Test

      It is desirable to bear in mind that unless a large
      number of portions of sample are examined, the precision
      of the fermentation tube test is rather low.  For example,
      even when the sample contains 1 coliform organism per
      milliliter, about 37% of 1-ml tubes may be expected to
      yield negative results because of irregular distribution
      of the bacteria in the sample.  When five tubes, each
      with 1 ml of sample are employed under these conditions,
      a completely negative result may be expected less than
      1% of the time.

      Even when five fermentation tubes are employed, the precision
      of the results obtained is not of a high order.  Consequently,
      great caution must be exercised when interpreting, in
      terms of sanitary significance, the coliform results
      obtained from the use of a few tubes with each dilution
      of sample,  especially when the number of samples from
      a given sampling point is limited.

2.     Computing and Recording of MPN

      The number of positive findings of coliform group organisms
      (either presumptive, confirmed or completed) resulting
      from multiple-portion decimal-dilution plantings should
      be computed as the combination of positives and recorded
      in terms of the Most Probable Number (MPN).  The MPN,
      for a variety of planting series and results, is given
      in Tables 407(1)  through (6).* Included in these tables
      are the 95% confidence limits for each MPN value
      determined.

      The quantities indicated at the heads of the columns
      relate more specifically to finished waters.  The values
      may be used in computing the MPN in larger or smaller
      portion plantings in the following manner:  If, instead
      of portions of 10, 1.0  and 0.1 ml, a combination of
      portions of 100,  10 and 1 ml is used, the MPN is recorded
      as 0.1 times the value  given in the applicable table.


      *Since Region IX  routinely uses the five tube MPN procedure,
       only Table 407(1),  and 407(2)  from Standard Methods 13th Ed.
       are included in  this manual.

-------
                                                     19
If, on the other hand, a combination of corresponding
portions at 1.0, 0.1 and 0.01 ml is planted, record
10 times the value shown in the table; if a combination
of portions of 0.1, 0.01 and 0.001 ml is planted,
record 100 times the value shown in the table; and
so on for other combinations.

When more than three dilutions are employed in a
decimal series of dilutions, the results from only
three of these are used in computing the MPN.   To
select the three dilutions to be employed in determining
the MPN index, taking the system of five tubes of each
dilution as an example, the highest dilution which
gives positive results in all five portions tested
(no lower dilution giving any negative results) and
the next succeeding higher dilutions should be chosen.
The results at these three volumes should then be used
in computing the MPN index.  In the examples given
below, the significant dilution results are shown in
boldface.  The number in the numerator represents
positive tubes; that in the denominator, the total
tubes planted; the combination of positives simply
represents the total number of positive tubes per
dilution:
Example  1 ml    0.1 ml   0.01 ml
                   0.001 ml
                    Combination
                    of positives
(a)
(b)
(c)
5/5
5/5
0/5
5/5
4/5
1/5
2/5
2/5
0/5
0/5
0/5
0/5
5-2-0
5-4-2
0-1-0
In c, the first three dilutions should be taken, so as to
throw the positive result in the middle dilution.

When a case such as shown below in line d arises, where a
positive occurs in a dilution higher than the three chosen
according to the rule, it should be incorporated in the
result for the highest chosen dilution, as in e:
Example  1 ml
0 .1ml
0.01ml
0.001 ml
Combination
of positives
(d)
(e)
5/5
5/5
3/5
3/5
1/5
2/5
1/5 1
0/5 J
5-3-2
When it is desired to summarize with a single MPN value
the results from a series of samples, the geometric mean, the
arithmetic mean, or the median may be used.

-------
                                                          20
    TABLE 407(1):  MPN INDEX AND 95% CONFIDENCE LIMITS FOR
     VARIOUS COMBINATIONS OF POSITIVE AND NEGATIVE RESULTS
               WHEN FIVE 10-ML PORTIONS ARE USED
No. of Tubes
Giving Positive
Reaction out of
5 of 10 ml Each
0
1
2
3
4
5
MPN
Index
per 100 ml
<2.2
2.2
5.1
9.2
16.
>16.
95% Confidence
Limits

Lower
0
0.1
0.5
1.6
3.3
8.0

Upper
6.0
12.6
19.2
29.4
52.9
Infinite
 Formula* for MPN Calculation:

 MPN/100 ml = Number Positive Tubes x 100
              i
              |(ml sample in    x (ml sample
               negative tubes)   in all tubes)
*From Thomas, H.A. Jr.  1942.   Bacterial densities from
fermentation tube tests.   JAWWA 3^:572.

-------
I
                                                                 21
BLE 407(2):  MPN INDEX AND  95%  CONFIDENCE LIMITS FOR VARIOUS COMBINATION;
 POSITIVE AND NEGATIVE RESULTS  WHEN FIVE 10-ML PORTIONS, FIVE 1-ML
                PORTIONS AND  FIVE  0.1-ML PORTION ARE USED
No. of Tubes Giving
Positive Reaction out of
5 of 10
ml Each
0
0
0
0
1
1
1
1
1
2
2
2
2
2
2
3
3
3
3
3
3
3
4
4
1
• t
4
4
•I
5 of 1
ml Each
0
0
1
2
0
0
1
1
2
0
0
1
1
2
3
0
0
1
1
2
2
3
0
0
1
1
1
2
5 of 0.1
ml Each
0
MPN
Index
per
100 ml
< 2
1 i 2
0 2
0
0
1
0
1
0
0
1
0
1
0
0
0
1
0
1
0
1
0
0
1
0
1
2
0
4
2
4
4
6
6
5
7
7
9
9
12
8
95% Confidence
Limits
Lower

< 0.5
< 0.5
< 0.5
< 0.5
< 0.5
< 0.5
< 0.5
< 0.5
< 0.5
1
1
2
2
3
1
11 i 2
11 2
14 ! 4
14 4
17 5
17
13
17
17
21
26
22
5
3
5
5
7
9
Upper

7
7
11
7
11
11
15
15
13
17
17
21
21
28
19
25
25
34
34
46
46
31
46
46
63
78
7 67
      . d  on  page 22

-------
                                                                  22
 (Cont'd.  from  page  21)

TABLE 407(2)i  MPN INDEX AND 95% CONFIDENCE LIMITS FOR VARIOUS  COMBINATION
OF POSITIVE AND NEGATIVE RESULTS WHEN FIVE 10-ML PORTIONS,  FIVE 1-ML
                  PORTIONS AND FIVE 0.1-ML PORTION ARE USED
No. of Tubes Giving
Positive Reaction out of
5 of 10
ml Each
4
4
4
4
5
5
5
5
5
5
5
5
5
5
5
5

5
5
5
5
5
5
5
5
5
5
5
5
5 of 1
ml Each
2
3
3
4
0
0
0
1
5 of 0.1
ml Each
1
0
1
0
0
1
MPN
Index
per
100 ml
26
27
33
34
23
31
2 43
0
1 ' 1
1
2
2
0
2 1
2 2
3 0
3 1
3 2
i
3 ; 3
4 ! 0
4 1
4 1 2
4 i 3
4
5
5
5
5
5
5
4
33
46
63
49
70
94
79
110
140

180
130
170
220
280
350
0 240
1 350
2 ! 540
3 920
4
5
1600
>2400
95% Confidence
Limits
Lower
9
9
11
12
7
11
15
11
16
21
17
23
28
25
31
37

Upper
78
80
93
93
70
89
110
93
120
150
130
170
220
190
250
340

44 i 500
!
35
43
57
90
120
68
300
490
700
850
1,000
750
120 i 1,000
180 1,400
300 3.200
640

5,800


-------
                                                          23
GRAMS STAIN

1.    Using a clean slide, make a thin smear of the culture
      on the slide.

2.    Air dry

3.    Heat fix by passing slide briefly through flame.

4.    Add gentian violet dye - let stand one minute.   Rinse
      with tap water.   Rinse with Gram's iodine.

5.    Add Gram's iodine - let stand one minute.  Drain slide

6.    Add decolorizer  - let stand 10-15 seconds.
      Rinse with water.  Rinse with Safranin.

7.    Add Safranin dye to counterstain - one minute.   Rinse
      with tap water.   Blot dry with paper towel.

      View slide under microscope.

      Gram-positive organisms appear blue-violet;  gram-
      negative bacteria retain only the counterstain,
      appearing red when Safranin is counterstain.

-------
                                                           24


                    IMViC PROCEDURES
                (Standard Methods, 13th Ed.)


Be sure to use a pure culture for all IMViC inoculations.

Indole production from tryptophane broth

1.    Prepare Medium:

      Add 10.0 g.  tryptone (or trypticase) to 1 liter distilled
      water.  Distribute in 5 ml portions into screw-capped
      test tubes.   Autoclave at 120°C for 15 minutes.

2 .    Prepare reagent:

      Dissolve 5 g. paradimethylaminobenzaldehyde in 75 ml
      isoamyl (or normal amyl) alcohol, ACS grade.  Add 25 ml
      concentrated hydrochloric acid (perform this operation
      under a hood if possible or else in a room with good
      ventilation).  The reagent should turn yellow.  Final
      pH should be less than 6.0.

3.    Procedure:

      Inoculate 5 ml medium.   Incubate at 35 ± 0.5°C for
      24 ± 2 hours.  Add 0.2-0.3 ml reagent and shake tube.
      Let stand for 10 minutes.

4.    Results:

      Dark red color in surface layer = (+)  for indole.
      Yellow  (Color of reagent) color in surface layer =
      (-) for indole.  Orange color in surface layer =
      (±) reaction.

Methyl Red Test

1.    Prepare Medium:

      Add 17 g.  MR-VP Broth Base (or equivalent)  to 1 liter
      distilled water.   Heat  slightly to dissolve.   Distribute
      10-ml portions into screw-capped test tubes.  Autoclave
      at 121°C for 12-15 minutes.

2.    Prepare reagent:

      Dissolve 0.1 g. methyl  red dye in 300 ml 95% ethyl alcohol
      Dilute to  500 ml  total  volume with distilled water.

3.    Procedure:

      Inoculate  10-ml medium.   Incubate at 35°C for 5 days.

-------
                                                               25
After incubation, pipette 5 ml culture into a clean test tube.
Add 5 drops (about 0.25 ml) methyl red reagent.

     4.    Results:

     Distinct red color -  (+) methyl red.  Distinct yellow
color = (-) methyl red.  Mixed shade = questionable results.

Voges-Proskauer Test

     1.    Prepare medium:

     The same tube of MR-VP broth inoculated for the methyl
red tests may be used for the Voges-Proskauer test as well.

     2.    Prepare reagents:

     Naphthol solution;  Dissolve 5 g. purified naphthol
(melting point 92.5 or higher) in 100 ml absolute ethyl alcohol.
Solution should be prepared fresh each day.

     Potassium Hydroxide solution:  Dissolved 40 g. KOH in
100 ml distilled water.

     3.    Procedure:

     Inoculate 5 ml medium.  Incubate at 35 + 0.5°C for 48
hours.  Pipette 1 ml culture into a clean test tube.  Add to
this 0.6 ml naphthol solution and 0.2 ml KOH solution.

     4.    Results:

     Pink to crimson color develops in 2-4 hours -  (+) for V-P.

Sodium Citrate Test

     1.    Prepare medium:

     Add 24.2 g. Simmons Citrate Agar to  1 liter distilled
water.  Mix thoroughly.  Heat with frequent agitation until
medium boils for one minute.  Distribute into screw-capped test
'.ubes.  Autoclave at 3^§-°C for 15 minutes.  Cool tubes in
.1 anted position.    (^'

     2.    Procedure :

     Inoculate the medium with a straight needle, using both
i  stab and a streak.  Incubate 48 hours at 35 + 0.5°C.

     3.    Results:

     Growth on the medium with a blue color (usually)  = ( + )
for citrate utilization.  No growth - (-) test.

-------
                                                            26

             PROCEDURE FOR SALMONELLA ISOLATION

(for shellfish or sediment modifications see note following
Serological Grouping)

Water Samples, Filtration

1.  Set up filter system.  Millipore assembly may be used.
Substitute filter pad for actual filter grid.  On top of pad
(with funnel in place) add one inch of diatomaceous earth
(Celite or equivalent).  Add sterile buffered water to saturate
Celite column.
2.  Filter one liter sample.
3.  Add filter pad plus diatomaceous earth to enrichment broth.
Repeat filtration procedure for each flask of enrichment broth
(a total of six filtrations for two broths and three tempera-
tures) .

Enrichment

1.  Two enrichment broths (tetrathionate and selenite) should
be used at each of three different temperatures  (37°, 41.5°,
43°C).  In case of limited laboratory capability the 43°C incuba-
tion may be omitted.
2.  Incubate 18-24 hours at appropriate temperature.

Isolation

1.  From the enrichment flask make streak plates and plates for
impression smears onto Brilliant Green and XLD agars  (reincubate
enrichment broth).  Incubate plates at same temperature as for
flasks.
2.  Time of incubation for streak plates is indicated in Table 1.
Impression smears should be made on slides after 2-4 hours
incubation.
3.  Smears should be stained and examined as indicated in
fluorescent-antibody staining directions.  If no fluorescing cells
(3+, 4+)  are found, sample may be said to contain no Salmonellae.
If fluorescing cells are present, continue isolation procedure
(steps 4-5) ; streaking from enrichment broths may be repeated
after 2 and 3 days incubation (or up to the point at which no
Salmonella-like colonies are recovered).
4.  From each medium select isolated colonies of Salmonella-like
appearance  (consult Table 1) and restreak until pure cultures
are obtained.  Incubate at 37°C.
5.  Inoculate isolates into differential media in order indicated.

Differential Tests

1.  Using a single colony, inoculate 1/2 into a Triple Sugar Iron
slant and the other 1/2 into urea agar.   Incubate at 37°C (times
are indicated in Table 1).
2.  Those isolates with Salmonella-like reactions in both TSI and
urea should be inoculated into the following:  Brilliant Green

-------
                                                            27

and XLD agar plates (streak); SIM (stab); Lysine Decarboxylase;
Nutrient Agar.  Use material from the TSI slant for these
inoculations.  Incubate at 37°C for times indicated in Table 1.

Serological Grouping

1.  Isolates displaying a Salmonella-like pattern in differential
media may be grouped according to their somatic antigens.  This
agglutination reaction may be lost when cultures are not freshly
isolated.
2.  To prime the isolate for the agglutination test, inoculate
it into a  Brain Heart Infusion slant (or broth).  Incubate
24 hours at 37°C.
3.  Repeat step 2 at least once or twice, ending up with a fresh
BHI slant.
4.  Perform slide agglutination tests as indicated in directions
accompanying Difco Salmonella O Antisera.  Perform the agglu-
tination test for each of the sets of antisera  (Sets A-l, A,
B, C, D, E, F, G).
5.  Final typing for 0 and H antigens may be done at a convenient
typing center (California State Public Health, Berkeley).
MODIFICATIONS FOR SEDIMENT OR SHELLFISH SAMPLES

Sediment

1.  Omit filtration procedure.
2.  Inoculate sediment directly into enrichment broth.
For oily sample, use 1 gm.  For other material, use 10 gnu
3.  Proceed as directed through outline.

Shellfish

1.  Omit filtration procedure.
2.  As directed in Recommended Procedures for the Examination of
Sea Water and Shellfish (1970) , weigh 100 gm  (approximately) of
shellfish meat and liquor and add an equal weight of buffered
dilution water.  Grind in a blender about 2 minutes.  Pipet 20 ml
into each enrichment flask.
3.  Proceed as directed through outline.

-------
                                                            28
Table 1:  Incubation Time and Colonial Appearance for Various
          Organisms in Selected Media
Medium
Brilliant Green
Incubation
   (Hours)

  48
        Colonial Appearance
Salmonella  Shigella  Proteus  Coliforms
pinkish
with red
background
                                                    green
green
XLD
Lysine
Decarboxylase
Indole
Motility
Urease
TSI-slant
-butt
-H2S
24
24
24
24
24
18-48

red with red yellow
black ctr.
+ (purple) -(yellow) -
+ ,- +,-
+ - +
+
Al Al A
AG A AG
*'~ ~ +'~
ye:
+ '
+ ''
+ '
-
A
AG
+ '
 A = acid production (indicator  turns  yellow)
 Al = alkaline  reaction (indicator  turns  red)
 G = gas formation (bubbles  appear  in  agar)


 Rapid Procedure - In addition to and  supplement of Table 1:

    Omit Indole, Motility, Urease,  TSI,  lysine decarboxylase

    Inoculate Improved Enterotube.   Instructions in use of
    Enterotube  accompany package.
    The Enterotube is prepared  by Roche  Diagnostics,  Division of
    Hoffmann - La  Roche,  Inc., Nutley,  New Jersey,  07110.

    Table  II shows parameters and biochemical  reactions  for
    Salmonelleae  (Salmonella, Arizona, Citrobacter).

-------
                                                               29
                       TABLE II SALMONELLEAE
               Parameters and Biochemical Reactions
 TEST OR  SUBSTRATE
                                         SALMONELLEAE
                              Salmonella
Arizona
                                                       Citrobacter
Indol

Methyl Red

Voges - Proskauer

Simmons' Citrate

Hydrogen Sulfide  (TSI)

Urease

KCN

Motility

Gelatin (22°C)

Lysine Decarboxylase

Arginine Dihydrolase

Ornithine Decarboxylase

Phenylalanine Deaminase

Malonate

Gas From Glucose

Lactose

Sucrose

Mannitol

Dulcitol

Salicin
                              (+)  or +
+ or  (+)
                                                         + or  -
                                                             w
d

d
                                                             d

                                                             d



                                                             d

                                                             d
(Continued  next page)

-------
                                                 (Continued)
                                                             30
                     TABLE II SALMONELLEAE
             Parameters and Biochemical Reactions
TEST OR SUBSTRATE
Adonitol
Inositol
Sorbitol
Arabinose
Raf finose
Rhamnose
SALMONELLEAE
Salmonella
-
d
+
+ (D
-
+
Arizona
-
-
+
+
-
+
Citrobacter
-
-
+
+
d
+
 (1)  S_. typhi, S_. cholerae-suis, S. enteritidis bioser. Paratyphi A
     and Pullorum, and a few others ordinarily do not  ferment
     dulcitol promptly,  g^ cholerae~suis does not ferment arabinose.

 + , 90 percent or more positive in 1 or 2 days. -, 90 percent or
 more negative, d, different biochemical types [+,(+),-].
 ( + ) , delayed positive. + or -, majority of cultures positive.
 - or +, majority negative, w, weakly positive reaction.
Compiled by Difco Laboratories, Detroit, Michigan

-------
                                          Standard Method used
                                                by
                                          Region IX Laboratory


                FA  -  SALMONELLA  SCREENING


 1.   From colony or agar  slant,  make  light  saline suspension.
      (Use fresh agar  slant culture and  suspend in a  small  amount  of
     solution made  by mixing  0.85 g NaCl  to 100ml distilled water.
 2.   Prepare smears of  this suspension  on clear glass  FA slides
      (1.0 to 1.1 mm thick).

 3.   Air dry the smears - then fix for  2  minutes in  Kirkpatrick' s
     Fixative.  Rinse briefly in 95%  ethanol.  Allow to dry.
     Do not blot.

 4.   Cover the fixed  smears with one  drop of Salmonella
     polyvalent OH  conjugate.   (use 1:8 dilution of  the conjugate)

*5.   Place slides in  a  moist  chamber  to prevent evaporation of
     the staining reagent.  After 30  minutes, wash away excess
     reagent by dipping slide into buffered saline  (pH 7.5 -  8.0).

 6.   Place slide in second bath  of buffered saline for 10  minutes.

 7.   Remove slides, rinse in  distilled  H^O)  and allow to drain
     dry.

 8.   Place a small  drop of mounting fluid on the smear and cover
     with a No. 1 coverslip.  Examine under fluorescence scope,
     using UG-1  (2  mm)  primary filter and GG-9  (1 mm)  ocular
     filter.  A combination of BG-12  (3mm)  and OG-1  (1 mm) will
     also give satisfactory results.


                           Kirkpatrick's  Fixative

                          60  ml  absolute  ethanol

                          30  ml  chloroform

                          10  ml  formalin


 *Buffered Saline:   Bacto-FA Buffer Dried, prepared by Difco
                    Laboratories  is recommended.   Instructions
                    for  preparation accompany the  package.

                                        F.  Brezenski
                                        Region II

-------
                                                               32
TECHNIQUES USED BY REGION IX FOR SEROLOGICAL GROUPING OF SALMONELLA


1.  After completion of Differential Test, choose an isolated
    colony from Brilliant Green Agar and/or XLD plates which
    were streaked from Triple Sugar Iron slant (to obtain well
    isolated colonies it may be necessary to re-streak several
    times).

2.  From Brilliant Green Agar and/or XLD plates, pick well isolated
    Salmonella-like colony.  Inoculate it into Brain Heart
    Infusion broth.  Incubate 24 hours at 37°C.

3.  Repeat priming of isolate by transferring a loopful  of
    Brain Heart Infusion (BHI)  broth culture into a fresh tube
    of BHI broth.  After 3-4 transfers, inoculate onto BHI
    slant.  Make two slants (always keep one for stock).

4.  Perform slide agglutination test (see also page 27 Salmonella
    Procedures, Serological Grouping, Step 4).

    a.  Prepare a dense suspension of organism from fresh 18-
        hour BHI slant in 0.5 ml of 0.85% sodium chloride
        solution.  Suspension should be homogeneous and at
        least as concentrated as that of Bacto McFarland Barium
        Sulfate Standard #10 (which corresponds to 3x10^ cells/ml).

    b.  Using alcohol-cleaned slide mark slide into 4 sections
        1 cm square.  Using wax pencil, mark heavily (form
        continuous lines) to avoid spilling from one section
        to another.

    c.  Place one drop ( use capillary pipet with rubber bulb)
        of 0.05 ml of the Bacto-Salraonella O Antiserum Poly
        within one square.

    d.  To the square next  to antiserum, place one drop of
        0.85% sodium chloride solution (This serves as negative
        control) .

    e.  Using a clean inoculating loop, transfer a loopful
        (0.05 ml) of bacterial suspension in 0.85% sodium
        chloride prepared in step a and gently mix to emulsify
        thoroughly.

    f.  Transfer another loopful of bacterial suspension to
        section containing  antiserum.

    g.  Gently rock the slide 1-2 minutes watching for agglutination.
        (Using a small inverted fluorescent lamp aids in
        detecting agglutination process.)

-------
                                                            33


        Positive agglutination is  rapid.   Delayed agglutination
        (over 2 minutes)  or partial agglutination should be
        considered negative.

5.   If culture reacts with Bacto-Salmonella O Antiserum Poly
    (step g)  but does not react with the  specific Salmonella 0
    Antiserum groups, it  should be checked with  Bacto-Salmonella
    Vi Antiserum by same  method as described above.   If the
    culture  does not agglutinate with Salmonella Vi  antiserum,
    the culture may be regarded as not of the Salmonella genus.

    If the culture does react with the Vi antiserum,  proceed
    as follows:

    a.  Heat the culture  suspension in a  boiling water bath for
        10 minutes,  cool.

    b.  After cooling the heated culture  should  be re-tested
        with the desired  individual Salmonella 0 antiserum
        groups and the Salmonella  Vi antiserum.

6.   If the organism does  not  react with the Vi antiserum after
    heating,  it is ready  to be confirmed  by a Public  Health
    Laboratory or typing  center.

    a.  Send a pure culture slant.

    b.  List all parameters which  were performed and  results
        obtained.   Send along with culture slant.

    c.  It is preferable  to deliver culture to Public Health
        Laboratory;  however,  if this cannot be done,  the culture
        (in  a screw-capped tube) should be carefully  packed
        inside a metal screw  cap tube and then into  a mailing
        tube properly labelled and sent as registered mail.
Attachment:   Diagram of  slide agglutination

-------
                                             34
Diagram  of  slide  agglutination
 Frosted
Culture
#
"0"
Antiseri:
A-l
(1)
n (3)
(2)
(4)
(Example)
         Wax  pencil  enclosure

.?lide  Section       Dr_qp_ to contain
     #1

     #2

     #3


     #4
Antiserum alone

Antiserum + 0.85% NaCl

Bacterial Suspension in
  0.85% NaCl

Bacterial Suspension in
  0.85% NaCl + Antiserum

-------
ENVIRONMENTAL PROTECTION AGENCY
           Water Quality Office                Indicating conformity with the 13th
         Water Hygiene Division              edition of Standard Methods for the
                                              Examination of Water and Waste -
        Bacteriological Survey for             water (1971).
           Water Laboratories
Survey By
 X = Deviation     U =  Undetermined
            O = Not Used
Laboratory
Location
Date
                        Sampling and Monitoring Response

1.  Location and Frequency
       Representative points on system	
       Frequency of sampling adequate	

2.  Collection Procedure
       Faucets with aerators should not be used	
       Flush tap  1 min. prior to sampling	
       Pump well 1 min. to waste prior to sampling .  .  .  .
       River, stream, lake, or reservoir sampled at least
          6 inches below surface and toward current.  .  .  .
       Minimum  sample not less than 100 ml	
       Ample air space in bottle for mixing	
       Promptly  identify sample legibly and indelibly  .  .  .
3.  Sample Bottles
       Wide mouth,  glass or plastic bottles of	capacity.
       Sample bottles capable of sterilization and rinse  ....
       Closure:
          a.  Glass stoppered bottles protected with metal foil,
             rubberized cloth or kraft type paper	
          b.  Metal or plastic screw cap with leakproof liner .  .
       Sodium thiosulfate added for dechlorination	
              Concentration 100 mg/1 added before sterilization
       Chelation agent for stream samples (optional)	
              Concentration 372 mg/1 added before sterilization
•I.  Transportation and Storage
       Complete and accurate data accompanies sample  ....
       Transit time  for potable water samples should not exceed
          48 hrs, preferably within 30 hrs	.-  .
       Transit time  for source waters,  reservoirs,  and natural
          bathing waters should not exceed 6 hrs	
       All samples examined within 2 hours of arrival	
 EPA-103 (Gin)
 (Itov. 3-71)

-------
Laboratory
Location
Date
4.  Transportation and Storage (Continued)
       Sample refrigeration mandatory on stream samples,
          optional on potable water samples	

5.  Record of Laboratory Examination
       Results assembled and available for inspection  .  .  .
       Number of Tests per year
          MPN Test - Type of sample
              Confirmed (+)	  (-)	  (Total)_
              Completed (+)	  (-)	  (Total)]

          MF Test - Type of sample	
              Direct Count   (+)	  (-)	(Total)
              Verified Count (+)        (-)       (Total)"
       Data processed rapidly through laboratory and engineering sections
       Unsatisfactory sample defined as 3 or more positive tubes per
          MPN test or 5 or more colonies per 100 ml in MF test  ....
       High priority placed on alerting operator to unsatisfactory
          potable water results	,
       Prompt resampling for unsatisfactory samples	,
6.  Laboratory Evaluation Service
       State program to evaluate all laboratories which examine
          potable water supplies	
       Frequency of surveys on a	year basis	
       State survey officer (Name) 	
       Status of laboratory evaluation service ...
          Total	labs known to examine water

               	approved laboratories
               	provisional laboratories

                              Laboratory Apparatus

7.  Incubator
       Manufacturer                              Model
       Sufficient size for daily work load	
       Maintain uniform temperature in all parts (± 0. 5°C)	
       Accurate thermometer with bulb immersed in liquid on
          top and bottom shelves	
       Daily record of temperature or use of recording thermometer
          sensitive to 0. 5°C change	
       Incubator not subject to excessive room temperature variations
          beyond a range of 50 - 80° F	
 EPA-103  (Gin)
 (Rev.  3-71)

-------
Laboratory
Location                       Date
8.  Incubator Room (Optional) Manufacturer
       Well insulated, equipped with properly distributed heating
         and humidifying units for optimum environmental control.
       Shelf areas used for incubation must conform to 35° C ± 0. 5°
         temperature requirement	
       Accurate thermometers with bulb immersed in liquid. .  .  .
       Daily record of temperature at selected areas or use
         recording thermometer sensitive to 0.5°C changes .  .  .
9. Water Bath
       Manufacturer	Model	
       Sufficient size for fecal coliform tests  ....
       Maintain uniform temperature 44.5°C±0.2°C.  ,
       Accurate thermometer immersed in water bath  ,
       Daily record of temperature or use of recording
         thermometer sensitive to 0.2°C  changes .  .  ,
10. Hot Air Sterilizing Oven
       Manufacturer                          Model
       Size sufficient to prevent crowding of interior	
       Constructed to insure a stable sterilizing temperature .  .
       Equipped with accurate thermometer in range of 160-180° C
         or with recording thermometer	
11. Autoclave
       Manufacturer                          Model
       Size sufficient to prevent crowding of interior	
       Constructed to provide uniform temperature up to and
         including 121° C	
       Equipped with accurate thermometer with bulb properly located
         to register minimal temperature within chamber	
       Pressure gage and operational safety valve	
       Steam source from saturated steam line,  or from gas or
         electrically heated steam generator	
       Reach sterilization temperature in 30 min	
       Pressure cooker may be used only if provided with a pressure
         gage and thermometer with bulb  1 in.  above water level .  .
    Thermometers
      Accuracy checked with thermometer certified by National
         Bureau of Standards or one of equivalent accuracy.  .
      Liquid column free of discontinuous sections and graduation
         marks legible	
 EPA-103 (Gin)
 (Rev. 3-71)

-------
Laboratory
Location                       Date
13.  pH Meter
       Manufacturer    	Model	
       Electronic pH meter accurate to 0.1  pH units	

14.  Balance
       Balance with 2 g sensitivity at 150 g load used for general
          media preparations, Type
       Analytical balance with 1 mg sensitivity at 10 g load used
          for weighing quantities less than 2 g ,  Type	
       Appropriate weights of good quality for each balance
15.  Microscope and Lamp
       Preferably binocular wide field,  10 to 15 diameters magnifi-
          cation for MF colony counts, Type   	.
       Fluorescent light source for sheen discernment	

16.  Colony Count
       Quebec colony counter, dark-field model preferred for
          standard plate counts	
17.  Inoculating Equipment
       Wire loop of 22 or 24 gauge chromel, nichrome, or platinum
          iridium,  sterilized by flame	
       Single-service transfer loops of aluminum or stainless steel,  pre-
          sterilized by dry heat or steam	
       Disposable single service hardwood applicators, pre-
          sterilized by dry heat only	
18.  Membrane Filtration Units
       Manufacturer	Type_
       Leak proof during filtration	
       Metal plating not worn to expose base metal

19.  Membrane filters
       Manufacturer	Type
       Full bacterial retention, satisfactory filtration speed
       Stable in use,  glycerin free	
       Grid marked with non-toxic ink	
       Presterilized  or autoclaved 121° C for 10 min. .  .  .
20.  Absorbent Pads
       Manufacturer	Type
       Filter paper free from growth inhibitory substances	
       Thickness uniform to permit 1. 8 - 2. 2 ml medium absorption
       Presterilized or autoclaved with membrane filters	
EPA-103 (Gin)
(Rev. 3-71)

-------
Laboratory
Location                        Date
21.  Forceps
       Preferably round tip without corrugations	
       Forceps are alcohol flamed for use in MF procedure.
                   Glassware, Metal Utensils and Plastic Items

22. Media Preparation Utensils
       Borosilicate glass	
       Stainless steel	
       Utensils clean and free  from foreign residues or
          dried medium	
23.  Pipets
       Brand                                 Type
       Calibration error not exceeding 2. 5%.  .  .  .
       Tips unbroken, graduation distinctly marked
       Deliver accurately and quickly	
       Mouth end plugged with cotton (optional)  .  .
 24.  Pipet Containers
       Box, aluminum or stainless steel	
       Paper wrapping of good quality sulfite paper (optional) •.  .  .  .

 25.  Petri Dishes
       Brand	Type	
       Use 100 mm x 15 mm dishes for pour plates	
       Use 60 mm x 15 mm dishes for MF cultures	
       Clear,  flat bottom, free from bubbles and scratches	
       Plastic dishes may be reused if sterilized in 70% ethanol for
          30 min.  or by ultraviolet radiation	
 26,  Petri Dish Containers
       Aluminum or stainless steel cans with covers,  coarsely woven
          wire baskets, char-resistant paper sacks or wrappings .  .
 27.  Culture Tubes
       Size sufficient for total volume of medium and sample portions
       Borosilicate glass or other corrosive resistant glass  ....

 28.  Dilution Bottles or Tubes
       Borosilicate or other corrosive resistant glass	
       Screw cap with leak-proof liner free from toxic substances
          on sterilization	
       Graduation level indelibly marked on side of bottle or tube
 EPA-103 (Gin)
 (Key. 3-71)

-------
Laboratory
Location
Date
                         Materials and Media Preparation

29.  Cleaning Glassware
       Dishwasher Manufacturer               Model
       Thoroughly washed in detergent at 160°F, cycle time
       Rinse in clean water at 180° F, cycle time
       Final rinse in distilled water t  cycle time
       Detergent brand	
       Washing procedure leaves no toxic residue  ....
       Glassware free from acidity or alkalinity	
30.  Sterilization of Materials
       Dry heat sterilization (1 hr at 170°C)
          Glassware not in metal containers .
       Dry heat sterilization (2 hrs at 170°C)
          Glassware in metal containers.  .  .
          Glass sample bottles	
       Autoclaving at 121°C for 15 min  .  .  .
          Plastic sample bottles	
          Dilution water blanks	
31.  Laboratory Water Quality
       Still manufacturer                   Construction Material
       Demineralizer with	recharge frequency
       Protected storage tank	
       Supply adequate for all laboratory needs.	
       Free from traces of dissolved metals or chlorine	
       Free from bactericidal compounds as measured
          by bacteriological suitability test	
       Bacteriological quality of water measured once each year
          by suitability test or sooner if necessary	
32.  Buffered Dilution Water
       Stock phosphate buffer solution pH 7.2	
       Prepare fresh stock buffer when turbidity appears	
       Stock buffer autoclaved and stored at 5 - 10° C	
       1. 25 ml stock buffer per 1 liter distilled water	
       Dispense to give 99 ± 2 ml or 9  ± 0. 2 ml after autoclaving	
    pTI Measurements
       Calibrate pH meter against appropriate standard buffer prior to use
       Standard buffer brand                      pH
       Check the pH of each sterile medium batch or at least one batch
          from each new medium lot number	
EPA-103  (Gin)
(Rev. 3-71)

-------
 Laboratory
                                         Location
Date
 33.  pH Measurements (Continued)
       Maintain a pH record of each sterile medium batch,
         the date and lot number	
 34.  Sterilization of Media
       Carbohydrate medium sterilized 121° C for 12 min	
       All other media autoclaved 121°C for 15 min	
       Tubes packed loosely in baskets for uniform heating and cooling.
       Timing starts when autoclave reaches 121°C	
       Total exposure  of carbohydrate media to heat not over 45 min.   .
       Media removed and cooled as soon  as possible after sterilization
 35.  Storage
       Dehydrated media bottles kept tightly closed and stored
         at less than 30°C	
       Dehydrated media not used if discolored or caked	
       Sterile culture media stored  in clean area free  from
         contamination and excessive evaporation	
       Sterile batches  used in less than 1 week	
       All media protected from sunlight	
       If media is stored at low temperatures,  it must be incubated
        . overnight and any tubes with air bubbles discarded	
                         Culture Media - Specifications

36.  Lactose Broth
      Manufacturer           	Lot No.
      Single strength composition 13 g per liter distilled  water .
      Single strength pH 6. 9 ± 0. 1,  double strength pH 6. 7 ± 0. 1
      Not less than 10 ml medium per tube	
      Composition of medium after  10 ml  sample is added must
         contain 0. 013 g per ml dry ingredients	
37-  Lauryl Tryptose  Broth
      Manufacturer                                  Lot No.
      Single strength composition 35. 6 g per liter distilled water
      Single strength pH 6. 8 ± 0. 1,  double strength pH 6. 7 ± 0. 1
      Not less than 10 ml medium per tube	
      Composition of medium after  10 ml sample is added must
         contain 0. 0356 g per ml of dry ingredients	
    Brilliant Green Lactose Bile Broth
      Manufacturer                                 Lot No.
      (Gin)
     3-71)

-------
Laboratory
Location                       Date
38.  Brilliant Green Lactose Bile Broth (Continued)
       Correct composition, sterility and pH 7. 2.
       Not less than 10 ml medium per tube .  .  .
39.  Eosin Methylene Blue A gar
       Manufacturer        	. Lot No.
       Medium contains no sucrose,  Cat.  No.	
       Correct composition, sterility and pH 7.1	

40.  Plate Count A gar (Tryptose  Glucose Yeast Agar)
       Manufacturer                                Lot No.
       Correct composition, sterility and pH 7. 0 ± 0.1	
       Free from precipitate	
       Sterile medium not remelted a second time after sterilization.
41.  EC Medium
       Manufacturer	Lot No.
       Correct composition, sterility and pH 6. 9	
       Not less than 10 ml medium per tube	
42.  M-Endo Medium
       Manufacturer                                Lot No.
                    	
       Correct composition and pH 7. 1 - 7. 3	
       Reconstituted in distilled water containing 2% ethanol.
       Heat to boiling point,  promptly remove and cool .  .  .
       Store in dark at 2 - 10° C	
       Unused medium discarded after 96 hrs	
43.  M-FC Broth
       Manufacturer	Lot No.
       Correct composition and pH 7. 4	,
       Reconstituted in 100 ml distilled water containing 1 ml of
          a 1% rosolic acid reagent	,
       Stock solution of rosolic acid discarded after 2 weeks  or
          when red color changes to muddy brown	,
       Heat to boiling point,  promptly remove  and  cool . . .  .  ,
       Store in dark at 2 - 10° C	
       Unused medium discarded after 96 hrs	
44.	 Broth
       Manufacturer                                Lot No.
       Correct composition and pH
45. 	Agar
       Manufacturer      	 Lot No.

EPA-103 (Gin)
(Rev. 3-71)

-------
Laboratory
Location                       Date
45.	Agar (Continued)
       Correct composition and pH	
                           Multiple Tube Coliform Test

46.  Presumptive Procedure
       Lactose broth	lauryl tryptose broth
       Shake sample vigorously
       Potable water:  5 standard portions, either 10 or 100 ml
       Stream monitoring: multiple dilutions	
       Incubate tubes at 35° ± 0. 5°C for 24 ± 2 hr	
       Examine for gas	any gas bubble positive ....
       Return negative tubes to incubator	,
       Examine for gas at 48 ± 3 hr from original incubation . ,
47.  Confirmed Test
       Promptly submit all presumptive tubes showing gas production
         before or at 24 hr and 48 hr periods to Confirmed Test .  .
       a.  Brilliant green lactose broth
          Gently shake presumptive tube or mix by rotating	
          Transfer one loopful of positive broth or one dip of applicator
             from presumptive tube to brilliant green lactose broth.  .  .
          Incubate at 35° ± 0. 5°C and check at  24 hrs for gas production.
          Reincubate negative tubes for additional 24 hrs
             and check for gas production	
          Calculate MPN or report positive tube results	
      b.  Endo or eosin methylene blue agar plates adequate streaking
             to obtain discrete colonies separated by 0. 5 cm	
          Incubate at 35° ± 0. 5°C for 24 ± 2 hr	
          Typical nucleated colonies with or without sheen are coliforms
          If atypical unnucleated pink colonies develop, result is
             doubtful and completed test must be applied	
          If no colonies  or only colorless colonies appear, the
             confirmed test is negative.	
48.  Completed Test
      Applied to all potable water samples or a proportion each three
         months to establish the validity of the confirmed test in
         determining their sanitary quality	
      Applied to positive confirmed tubes or to doubtful colonies
         on differential medium	
      Streak positive confirmed tubes on Endo or EMB plates  for
         colony isolation	
EPA-103 (Gin)
(Rev. 3-71)

-------
Laboratory
Location
Date
48.  Completed Test (Continued)
        Choice of selected isolated colony for verification should be one
          typical or two atypical to lactose or  lauryl tryptose broth and
          to agar slant for Gram stain	
        Incubate at  35° C ± 0. 5°C for 24 hrs or  48 hrs	
        Gram negative rods without spores and gas in lactose tube
          with 48 hrs in positive Completed Test	,
                          Membrane Filter Coliform Test
49.  Application as Standard     ^
        Use as a standard test for determining potability of water after
          demonstration by parallel testing that it yields information
          equal to that from the multiple -tube fermentation procedure

50.  MF Procedure
       Filter funnel and receptacle sterile at start of series	
       Rapid funnel re sterilization by UV, flowing steam or boiling water
          acceptable	,	
       Membrane filter cultures and  technician eyes should not be
          subject to UV radiation leaks	,
       Filtration volume not less than 50 ml for potable water; multiple
          dilutions for stream pollution	
       Rinse funnel by flushing several 20 - 30 ml portions of sterile buffered
          water through MF	
       Remove filter with sterile forceps	
       Roll filter over M-ENDO medium pad or agar so air bubbles
          will not form	
51.  Incubation
       In high humidity or in tight fitting culture dishes
       At 35° C ± 0.5° C for 22 - 24 hrs	
52.  Counting
       All colonies with a metallic yellowish green surface sheen  .  .  .
       If coliforms are found in potable samples, verify by transfers
          to lactose broth, then to BGB broth for evidence of gas
          production at 35°C within 48 hr limit	
       Calculate direct count in coliform density per 100 ml	

^3.  Standard MF test with Enrichment
       Incubate MF after filtration on pad saturated with lauryl tryptose
          broth for 1 1/2 - 2 hr at 35°C  ± 0. 5°C	
EPA-103  (Cin)
(Rev. 3-71)                                                                     10

-------
Laboratory
Location                        Date
53.  Standard MF test with Enrichment (Continued)
       Transfer MF culture to M-Endo medium for a final
         20 - 22 hr incubation at 35°C ± 0. 5°C	
       Count sheen colonies, verify if necessary, and calculate
         direct count in coliform density per 100 ml	
                     Supplementary Bacteriological Methods

54. Standard Plate Count
       Plate not more than 1 or less than 0. 1 ml (sample or dilution)
       Add 10 ml or more liquefied agar medium at a temperature
         between 43 - 45° C	
       Melted medium stored for no more than 3 hr at 43 -  45° C .  .
       Liquid agar and sample  portion thoroughly mixed by  gently
         rotating to spread mixture evenly	
       Count only plates  with between 30 and 300 colonies, exception
         being 1 ml sample  with less than 30  colonies	
       Record only two significant figures and calculate as "standard
         plate  count at 35°C per 1 ml of sample"	
 55.  Fecal Coliform Test
       a.  Multiple Tube Procedure
          Applied as an EC broth confirmation of all positive
             presumptive tubes
          Place EC  tubes in water bath within 30 min of transfers
          Incubate at 44.5° C ± 0.2° C for 24 hrs
          Gas production is  positive test for fecal coliforms
          Calculate  MPN based on combination of positive EC tubes

       b.  Membrane Filter Procedure
         Following filtration place MF over pad  saturated with
             M-FC broth
          Place MF cultures in water-proof plastic bag and submerge
             in water bath within 30 min
          Incubate at 44. 5°C  ± 0. 2° C for 24 hrs
          All blue colonies are fecal coliforms
          Calculate  direct count in density per 100 ml
 •r>(>.  ])claycd-Incubation Coliform Test
       After filtration,  place MF over pad of M-Endo containing 3. 2 ml
         of a 12% sodium benzoate solution per 100 ml of medium.
       Addition of 50 mg cycloheximide per  100 ml of preservative
         medium for fungus  suppression is optional
       Transport culture  by mail service to laboratory within 72 hours


  EPA-103  (Gin)
 (Rev.  3-71)

-------
 Laboratory
Location
Date
 56.  Delayed-Incubation Coliform Test (Continued)
        Transfer MF cultures to standard M-Endo medium
          at laboratory	
        Incubate at 35° C ± 0. 5°C for 20 - 22 hr	
        If at time of transfer, growth is visible, hold in refrigerator
          till end of work day then incubate at 35° overnight
          (16 - 18 hr period)	
        Count sheen colonies, verify if necessary, and calculate
          direct count in coliform density per 100 ml	
57.  Additional Test Capabilities
        Fecal streptococci        	  Method
        Pseudomonas aeruginosa             Method
        Staphylococcus            	  Method
        Salmonellae              	  Method
        Biochemical tests         	  Purpose
        Serological tests          	  Purpose
        Other                    	  Purpose
                           Laboratory Staff and Facilities

58.  Personnel
       Adequately trained or supervised for bacteriological
          examination of water	
       Laboratory staff 	(Total) Prep room staff	(Total)"
59.  Reference Material
       Copy of the current edition of Standard Methods available
          in the laboratory	
       State or federal manuals on bacteriological procedures for
          water available for staff use	
60.  Physical Facilities
       Bench-top area adequate for periods of peak work in
          processing samples	
       Sufficient cabinet space for media and chemical storage .  .
       Office space and equipment available for processing water
          examination reports and mailing sample bottles .  .  .  .
       Facilities clean, with adequate lighting, ventilation and
          reasonably free from dust and drafts	
61.   Laboratory Safety
       Proper receptacles for contaminated glassware and pipettes
 EPA-103 (Gin)
 (Rev.  3-71)                                                                    12

-------
 Laboratory
Location
                                 Date
 61.  Laboratory Safety (Continued)
        Adequately functioning autoclaves with periodic inspection
          and maintenance	
        Accessible facilities for hand washing	
        Proper maintenance  of electrical equipment to prevent fire
          and electrical shock	
        Convenient gas and electric outlets	
        First aid supplies  available and not  out-dated	
 62.  Remarks
EPA-103 (Gin)
(Rev.  3-71)   * GPO 600-246
                                     13

-------