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