DISCHARGE MONITORING REPORT -
QUALITY ASSURANCE (DMR-QA) STUDY 18
To Participating Toxicity Laboratories:
In March of 1998, the U.S. Environmental Protection Agency(USEPA) Office of Enforcement and
Compliance Assurance sent announcement letters to designated National Pollutant Discharge Elimination
System (NPDES) permittees, informing them that the USEPA and related state agencies were continuing
the NPDES Discharge Monitoring Report - Quality Assurance (DMR-QA) studies. These announcements
contained the toxicity sample ordering form that you subsequently submitted. The purpose of these studies
is to evaluate the analytical and reporting ability of laboratories routinely performing the inorganic chemistry
and whole-effluent toxicity self-monitoring analysis required in the designated NPDES permits. For further
information, please refer to the Toxicity Laboratory Requirements on page 1 inside.
Participation of NPDES permittees in this program, including proper analyses, reporting and record
retention, is mandatory based on the authority of Section 308(a) of the Clean Water Act. The Agency's legal
opinions, dated August 11, 1977 and January 9, 1989, reaffirm this authority.
If you have technical questions regarding toxicity testing, you may contact John Helm, DMR-QA Toxicity
Coordinator, Office of Wastewater Enforcement and Compliance, telephone (202) 564-4144. Questions
regarding Regional or State NPDES policy should be directed to your permittee(s) who should contact the
appropriate USEPA Regional Office and/or State Agency for guidance. These offices will play an important
role in providing any subsequent study follow-up action. For inquiries regarding ordering and shipping of
toxicity samples and paperwork contact the USEPA Contractor ManTech Environmental, telephone (919)
406-2114. Please include the complete name, address, USEPA Toxicity Lab Code and telephone number
of your laboratory in all correspondence.
Thank you for your cooperation in this national program to improve the quality of NPDES self-monitoring
data.
Enclosures (3):
1) Toxicity Laboratory Instructions
2) NPDES Toxicity Laboratory Data Reporting Form
3) Reference Toxicant(s) (per your request)
-------
TOXICITY LABORATORY INSTRUCTIONS
TABLE OF CONTENTS
Starts on Page:
Toxicity Laboratory Requirements..... 1
Toxicant/Information Replacement Form 2
Method Code/Toxicant Reference Guide ". 3
Dilution Water Instructions .'.. 4
Reporting Toxicity Data:
Completing the NPDES Toxicity Laboratory Data Report Form. 5
What to Do With the Form After Completion 6
Specific Test Instructions by Method Code 7
Statistical Appendix 75
Material Safety Data Sheets (MSDS) .'...' 83
-------
TOXICITY LABORATORY REQUIREMENTS
1. Some tests you normally perform for your permittee may not be included in the DMR-QA Study. You are
only required to perform test types which have been requested by a permittee, provided they are
included in the study. Participating laboratories are required to perform only one of each type of test.
regardless of the number of permittees that request the analysis. Review the toxicity tests described
in Method Code/Toxicant Reference Guide to determine which tests you must perform to satisfy the
DMR-QA reporting requirements for each NPDES Permittee you support. To the extent feasible, all tests
must be performed by the personnel that routinely conduct the self-monitoring required in these NPDES
Permits.
2. Ensure that you have received the appropriate study samples, reporting forms and instructions:
a. Replacement toxicants, reporting forms or instructions may be acquired by returning the
completed Toxicant / Information Replacement Form on the next page. If you have analytical
problems, the available volume of each reference toxicant is usually sufficient to prepare a
second sample.
b. Unused toxicants may be handled in one of three ways:
• Properly dispose Of unused toxicants ensuring compliance with all federal, state and local
regulations governing waste management. For more information regarding waste
disposal, see the MSDS sheets available at the end of these instructions.
Return unopened ampules to the USEPA Contractor, ManTech Environmental, in
accordance with 49 CFR 173.4, and using the return address on the sample shipment
(4907 S. Alston Ave., Durham, NC 27713).
Retain toxicants for internal quality control. If stored at 2°C to 8°C and out of direct light,
unopened toxicants are stable for at least one year. A complete list of "true'Vcalculated
values may be obtained after the results of the study have been distributed to permittees,
which is expected to occur in January, 1999. Your permittees may request a copy by
contacting their Regional/State Coordinator. Users of a Personal Computer w/modem
may use the USEPA Office of Research and Development Electronic Bulletin Board,
modem number (513) 569-7700 or (513) 569-7610. If you need assistance with use of
the Bulletin Board, call (513) 569-7272 (do not call this number for general information
regarding PE Studies).
3. Perform the analyses requested by your permittees, refer to page 3 for specifics regarding required
testing conditions.
4. Report the results of your analyses and additional required information as specified on page 5,
Completing the NPDES Toxicity Data Report Form.
5. Submit your reporting form in accordance with the instructions on. page 6, Toxicity Laboratory
Reporting Procedure.
6. After official evaluation of the study data by the USEPA, laboratories will be asked by the
supported NPDES permittee(s) to explain reported values that were not within the study
acceptance limits. Permittees will provide this explanation to the appropriate USEPA or State Agency.
-------
DISCHARGE MONITORING REPORT - QUALITY ASSURANCE (DMR-QA) STUDY 18
TOXICANT / INFORMATION REPLACEMENT FORM
Copy this form, complete the information and return it, as indicated below.
1. USEPA Toxicrty Laboratory Code: If you are unsure of your assigned lab code, look at the mailing label on the enclosed
Reporting Form or call the USEPA contractor, ManTech Environmental at (919) 406-2114.
(5 digits + 2 digit extension, for example 12345 -12)
2. Address: Mailing and shipping address required.
Laboratory Name
Contact Name Title
Phone(_
State
_Zip Code_
Laboratory Mailing Address (PO Box, if avalable)
Laboratory Shipping Address (No PO Boxes)
city _ .
3. Toxicants and paperwork: Check the appropriate block(s).
Q Reference Toxicant #1 There is sufficient reference toxicant to do all necessary chronic tests with fathead minnows
(Pimephales promelas), and Ceriodaphnia dubia. The glass bottle contains approximately one gram of a solid toxicant.
Q Reference Toxicant #2 There is sufficient reference toxicant to do all necessary chronic tests with Mysidopsis bahia and
sheepshead minnow (Cyprinodon variegatus). The plastic bottle contains approximately 200 grams of a solid toxicant.
D Reference Toxicant #3 There is sufficient reference toxicant to do all necessary acute tests with fathead minnows
(Pimephales promelas), Ceriodaphnia dubia, Daphnia magna, Daphnia pulex, inland silversides (Menidla beryllina),
Mysidopsis bahia and sheepshead minnow (Cyprinodon variegatus). The plastic bottle contains approximately 100
grams of a solid toxicant.
D Study Instructions
Q Data Reporting Form
4. Reason for Request [check the appropriate block(s)]:
Q Broken in shipment
Q Missing in shipment
Q Lab accident
Q Other
5. Fax or send this form to the USEPA Contractor:
Fax: (919)405-2246
ManTech Environmental Technology, Inc.
Mr. Terry Bundy, QAPS Distribution Supervisor
PO Box 12313
2 Triangle Drive
Research Triangle Park, NC 27709
Reminder; Maintain a copy of completed order form for your records.
-------
METHOD CODE / TOXICANT REFERENCE GUIDE
The following table lists the DMR-QA Study organisms, test types, method codes, and toxicant numbers
used for preparing the "simulated effluents" and performing each test.
TEST ORGANISM
Fathead minnow (Pimephales promelas)
Fathead minnow (Pimephales promelas)
Fathead minnow (Pimephales promelas)
Fathead minnow (Pimephales promelas)
Fathead minnow (Pimephales promelas)
Ceriodaphnia dubia
Ceriodaphnia dubia
Ceriodaphnia dubia
Ceriodaphnia dubia
Ceriodaphnia dubia
Ceriodaphnia dubia
Daphnia magna
Daphnia pulex
Daphnia pulex
Mysid (Mysidopsis bahia)
Mysid (Mysidopsis bahia)
Inland silverside (Menidia beryllina)
Sheepshead minnow (Cyprinodon vareigatus)
Sheepshead minnow (Cyprinodon vareigatus)
TEST CONDITIONS
48-h acute, non-renewal,
20°C, moderately-hard synthetic freshwater
48-h acute, non-renewal,
25°C, moderately-hard synthetic freshwater
48-h acute, non-renewal,
25°C, 20% diluted mineral water
7-day short-term chronic, daily renewal,
moderately-hard synthetic freshwater
7-day short-term chronic, daily renewal,
20% diluted mineral water
48-h acute, renewal,
20°C, moderately-hard synthetic freshwater
48-h acute, renewal,
20°C, 20% diluted mineral water
48-h acute, renewal,
25°C, moderately-hard synthetic freshwater
48-h acute, renewal,
25°C, 20% diluted mineral water
7-day short-term chronic, daily renewal,
moderately-hard synthetic freshwater
7-day short-term chronic, daily renewal,
20% diluted mineral water
48-h acute, non-renewal,
20°C, moderately-hard synthetic freshwater
48-h acute, non-renewal,
20°C, moderately-hard synthetic freshwater
48-h acute, non-renewal,
25°C, moderately-hard synthetic freshwater
48-h acute, non-renewal,
20°C, 40 fathoms artificial seawater
7-day short-term chronic, daily renewal,
40 fathoms artificial seawater
48-h acute, non-renewal,
20°C, 40 fathoms artificial seawater
48-h acute, non-renewal,
20°C, 40 fathoms artificial seawater
7-day short-term chronic, daily renewal,
40 fathoms artificial seawater
METHOD
CODE
11
13
14
15
16
17
18
19
20
21
22
32
36
38
42
43
44
46
47
TOXICANT
#
3
3
3
1
1
3
3
3
3
1
1
3
3
3
3
2
3
3
2
-------
DILUTION WATER INSTRUCTIONS
The three types of dilution waters that may be used to prepare the simulated effluents:
1) Moderately-hard synthetic freshwater (MHSF), see EPA/EPA600/4-89/001 for this water preparation.
2) 20% diluted mineral water (DMW), see EPA/EPA600/4-89/001 for this water preparation.
ROUTINE CHEMISTRIES FOR MODERATELY-HARD RECONSTITUTED WATERS
Chemistries
PH
Alkalinity
Hardness
Conductivity
Water Quality for MHSF and DMW
7.4 to 7.8 (MHSF)
7.9 to 8.3 (DMW)
60 to 70 rng/L as CaCO3
80 to 100 mg/L as CaCO3
260 to 300 ^mho/cm (MHSF)
160 to 200 /^mho/cm (DMW)
3) 40 fathoms artificial seawater with a salinity of 25ppt
For one liter of 40 fathoms artificial seawater:
a) Add 25 grams of 40 fathoms sea salts to 900 mL of MILLIPORE MILLI-QR (or equivalent)
deionized water.
b) Mix by stirring or aeration for one hour or until all solid materials are dissolved.
c) Bring the volume to one liter using deionized water.
• Smaller or larger amounts can be prepared by adjusting the volume of deoinized water and the amount
of sea salts that are added, i.e. 10 liters of 40 artificial seawater, is made with 250 grams of 40 fathoms
sea salts into 9 liters of deionized water and adding additional deionized water to bring the final volume
to 10 liters. .
• Laboratories experiencing problems with 40 fathoms artificial seawater should prepare a report of the
nature of the problem(s) and resolution (if any) and submit it along with their data report form.
Toxicity Test Method References
R1 Methods for Measuring the Acute Toxicity of Effluents to Freshwater and Marine Organisms, EPA/600/4-
90/027F.
R2 Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater
Organisms, Second Edition, EPA/600/4-89/001.
R3 Supplement to Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters
to Freshwater Organisms, Second Edition, EPA/600/4-89/001A.
R4 Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Marine And
Estuarine Organisms, EPA/600/4-87/028.
-------
COMPLETING THE NPDES TOXICITY LABORATORY DATA REPORTING FORM
1. Toxicity Laboratory Code: Enter your Toxicity Lab Code on the enclosed NPDES Toxicity Laboratory
Data Report Form in the spaces provided at the top of page 1. The Toxicity Laboratory Code is seven
numbers (5 digits + 2 digit extension) and can be found on the label on page 1 of your Laboratory Data
Report Form. Do not submit a permit number, state laboratory code or certification ID in lieu of the
Toxicity Laboratory Code.
2. Enter the appropriate data in to each field. The headings listed below relate to information on pages
3 & 4 of the Report Form.
a. Analyte Name and No.: Identifies each test type available and their corresponding analyte
numbers (should not be altered).
b. Toxicity Laboratory Code: Enter your Toxicity Lab Code next to each analytical result produced
by your laboratory and on any additional documentation that you may submit.
c. Voluntary Analyte: Toxicity laboratories should not enter anything in this column.
d.. Method Codes: Identifies which method must be used to perform each analysis (should not be
altered).
e. Quantity:
• Report only one value for each endpoint
• Each box utilized must contain only a number or a decimal (no dashes, letters or
symbols for any reason). A decimal point requires its own box.
• Report your data to three significant digits, i.e., O.XXX, X.XX, XX.X or XXX, if method
allows.
• Right Justify each result in the spaces provided.
• Report all data in the units specified on the data report form.
3. Complete the Checklist and Certification Statement on page 1 of the Toxicity Laboratory Data Report
Form. The Certification Statement must be signed by the Director or an authorized representative of the
laboratory. Most important, the laboratory is responsible for certifying the truth, accuracy, and
completeness of the data and that all work has been performed by the personnel that routinely
conduct the self-monitoring required for each permittee they report to. Reference to this section
may be found in 40 CFR Part 122.22.
4. Please provide the analytical details for all the tests you conducted, as requested on the last page of the
Data Report Form. By "holding density", we mean the total number of fish in the tank from which
you got your fish for testing, including the fish you used, divided by the gallons of water in that tank. If
you used fish from more than one tank, "holding density" would be the total number of fish in all tanks
used, divided by the total gallons of water in those tanks.
-------
TOXIC1TY LABORATORY REPORTING PROCEDURE
You must submit the results of all testing performed in your laboratory on the enclosed Laboratory Data
Report Form to the USEPA Contractor (ManTech Environmental). You must ALSO send your NPDES
Permittees whatever results they requested. Results reported by the laboratories to the USEPA Contractor
will be used to calculate the study acceptance limits, but will not satisfy the reporting requirements of any
permittee.
(Permittees will transfer appropriate results
received from inhouse and/or contract labs to
the NPDES Permittee Data Reporting Form and
submit it to the USEPA Contractor to represent
the quality of their routine DMR data.
/
1 Contract
Toxicity laboratory
"\
,k, NPDES Toxicltv Laboratory
I Data Report Form
\ CLeopyJ
\
\MpnFS 1
r\ Permittee j~
1 i
NPDES Permittee
Data Report Form
(original +1 CODV)
USEPA
Contractor
ManTech
Environmental
NPDES Toxicitv Laboratory
Data Report Form
(original +1 copy)
Toxicity Labs should report to:
:1) each NPDES permittee that they support and
21 directly to the USEPA Contractor
Make photo copies of the completed NPDES Toxicity Laboratory Data Report Form (green colored).
1) Send 1 copy to each NPDES permittee you support.
2) Send the original form and brie copy to the USEPA Contractor:
ManTech Environmental Technology, inc.
Mr. Terry Bundy* QAPS Distribution Supervisor
PO Box 13213
2 Tftangle Drive
Research Triangle Park, IMC 27709
<". •• , <•» , , < ' MW '
This form must be received by the NPDES Permittees you support and the USEPA Contractor on or
before August 17.1998 (this date will allow each NPDES Permittee enough time to meet their reporting
deadline of September $0, 1998).
Notes:
To ensure receipt of your data, use a mail carrier which supplies proof of delivery.
Facsimile transmissions are not acceptable.
Keep a copy of all submitted data and related analytical information/results for your records.
-------
U. S. ENVIRONMENTAL PROTECTION AGENCY
DMR-QA LABORATORY PERFORMANCE EVALUATION Study 18
INSTRUCTIONS FOR THE FATHEAD MINNOW (P1MEPHALES PROMELAS)
48-H ACUTE. NON-RENEWAL. TOXICITY TEST
(METHOD CODE 11)
CAUTION: Read Instructions Carefully Before Opening The Reference Toxicants.
PREPARATION OF "SIMULATED" EFFLUENT
The reference toxicant, labeled REFERENCE TOXICANT #3, is supplied in solid form in a glass
bottle. A stock solution of REFERENCE TOXICANT #3 is prepared by adding 2.50 grams of the toxicant material to
approximately 90 mL of distilled water. Stir with a mechanical stirrer or by hand shaking until dissolved. Bring the final
volume to 100 mL with distilled water. The stock solution can be used for more than one method code that uses
REFERENCE TOXICANT #3. A fresh stock solution must be prepared each time a test (or set of tests) using
REFERENCE TOXICANT #3 is started or renewed. For each fathead minnow acute toxicity test described below,
prepare a "simulated" effluent (hereafter referred to as the effluent) from the stock solution. Prepare the effluent by
adding 16.0 mL of the stock solution to 900 mL of Moderately-hard synthetic freshwater (MHSF) (20°C test). Stir
with a mechanical stirrer or by hand shaking until completely mixed. Bring the final volume to 1L with MHSF. Larger or
smaller volumes of effluent can be prepared by using proportionally larger or smaller amounts of the REFERENCE
TOXICANT #3 stock solution and MHSF. MHSF is prepared from MILLIPORE MILLI-Q" (or equivalent) deionized water
and reagent grade chemicals, as specified in Tables 6 and 7, p. 35, EPA/600/4-90/027.
The effluent prepared according to these instructions represents the sample ready for testing, 100%
effluent This 100% effluent sample is split into two 0.5 L aliquots. The first aliquot is your 100% effluent for testing.
The second aliquot is diluted with 0.5 liter of MHSF and mixed. This becomes your 50% effluent sample. Continue
diluting half of each sample with the same volume of MHSF to make your 25%, 12.5%, and 6.25% effluent dilutions,
which represent all five test dilutions.
ACUTE DEFINITIVE TOXICITY TEST
The 48-h definitive test is performed using the test conditions listed in the following table
(MANDATORY TEST CONDITIONS FOR FATHEAD MINNOW (PIMEPHALES PROMELAS) 48-HOUR ACUTE, NON-
RENEWAL, TOXICITY TESTS).
TEST DATA
Biological and physical/chemical measurements are performed and the data recorded as specified in
EPA/600/4-90/027. Records of these measurements must be maintained on file in the laboratory and made available to
USEPA if requested.
-------
U. S. ENVIRONMENTAL PROTECTION AGENCY
DMR-QA LABORATORY PERFORMANCE EVALUATION Study 18
INSTRUCTIONS FOR THE FATHEAD MINNOW (PIMEPHALES PROMELAS)
48-H ACUTE. NON-RENEWAL. TOXICITY TEST
(METHOD CODE 11)
Biological Data
In the 48-h definitive test, data on test organism survival in each replicate chamber for the controls
and effluent concentrations are recorded daily as described on p. 70 and in Figure 4, p. 72, EPA/600/4-90/027.
Physical/Chemical Data
In the 48-h non-renewal, definitive test, dissolved oxygen (DO), pH, total alkalinity, total hardness, conductivity, and
temperature are determined at each concentration of effluent used in the dilution series, and in the control, at the
beginning of the test, and the DO, pH, conductivity, and temperature are determined at the end of the test, or when a
replicate sample experiences 100% mortality, and recorded as described on pp. 70 and 71, and in Figure 4, p. 72,
EPA/600/4-90/027.
BIOLOGICAL DATA ANALYSIS
Mortality data from the 48-h definitive test are used to determine the LC50, expressed as percent
effluent. Follow the flowchart in Figure 1 in the Statistical Appendix to determine the LC50. For additional information
on data analysis, see the Statistical Appendix at the end of this instruction packet.
REPORTING THE TEST RESULTS
The 48-h LC50 (expressed as percent effluent) is entered on the Report Form located at the end of
the package of instructions. Record all results the same as they are recorded for DMR reporting. NOTE: Do not
report LCSOs for tests which fail to meet the acceptability criteria listed with the test conditions. Be sure you
enter your data into the portion of the Report Form that corresponds to the exact test conditions you used. Make sure
you also give the Method Code (see listing among "The Instructions To Toxicity Test Laboratories") appropriate for the
exact test conditions you used.
8
-------
METHOD CODE 11
MANDATORY TEST CONDITIONS FOR FATHEAD MINNOWS (PIMEPHALES PROMELAS)
48-H ACUTE, NON-RENEWAL TOXICITY TESTS
1. Test type:
2. Test duration:
3. Temperature:
4. Light quality:
5. Light intensity:
(Ambient laboratory levels)
6. Photoperiod:
7. Volume of Stock Solution
8. Test chamber size:
9. Test solution volume:
10. Renewal of test solutions:
11. Age of test organisms:
12. No. organisms/test chamber:
13. No. replicate test
chambers/concentration:
14. No. organisms/concentration:
15. Feeding regime:
16. Test chamber cleaning:
17. Aeration:
18. Dilution water:
19. Test concentrations:
20. Dilution series:
21. Endpoint (expressed as %effluent):
22. Test acceptability criterion:
Static, non-renewal
48 h
20±1°C(MHSF)
Ambient laboratory illumination
10-20 uE/m2/s (50-100 ft-c)
16 h light, 8 h darkness
16.0 mL Stock/1 L
250 mL (minimum)
200 mL (minimum)
None
1-14 days; 24 h maximum range in age
10
2
20
Not fed
Cleaning not required
None
Moderately-hard synthetic freshwater (MHSF) (80-100
mg CaCO3, EPA/600/4-90/027) prepared with
MILLIPORE MILLI-QR (or equivalent) deionized water
and reagent grade chemicals
5 effluent concentrations and a control
0.5
LC50
90% or greater survival in controls
-------
-------
U. S. ENVIRONMENTAL PROTECTION AGENCY
DMR-QA LABORATORY PERFORMANCE EVALUATION Study 18
INSTRUCTIONS FOR THE FATHEAD MINNOW (PIMEPHALES PROMELAS)
48-H ACUTE. NON-RENEWAL. TOXICITY TEST
(METHOD CODE 13)
CAUTION: Read Instructions Carefully Before Opening The Reference Toxicants.
PREPARATION OF "SIMULATED" EFFLUENT
The reference toxicant, labeled REFERENCE TOXICANT #3, is supplied in solid form in a glass bottle. A
stock solution of REFERENCE TOXICANT #3 is prepared by adding 2.50 grams of the toxicant material to
approximately 90 mL of distilled water. Stir with a mechanical stirrer or by hand shaking until dissolved.
Bring the final volume to 100 mL with distilled water. The stock solution can be used for more than one
method code that uses REFERENCE TOXICANT #3. A fresh stock solution must be prepared each time a
test (or set of tests) using REFERENCE TOXICANT #3 is started or renewed. For each fathead minnow
acute toxicity test described below, prepare a "simulated" effluent (hereafter referred to as the effluent)
from the stock solution. Prepare the effluent by adding 8.0 mL of the stock solution to 900 mL of
Moderately-hard synthetic freshwater (MHSF) (25°C test). Stir with a mechanical stirrer or by hand
shaking until completely mixed. Bring the final volume to 1L with MHSF. Larger or smaller volumes of
effluent can be prepared by using proportionally larger or smaller amounts of the REFERENCE TOXICANT
#3 stock solution and MHSF. MHSF is prepared from MILLIPORE MILLI-Q" (or equivalent) deionized
water and reagent grade chemicals (or mineral water), as specified in Tables 6 and 7, p. 35, EPA/600/4-
90/027.
The effluent prepared according to these instructions represents the sample ready for testing,
100% effluent. This 100% effluent sample is split into two 0.5 L aliquots. The first aliquot is your 100%
effluent for testing. The second aliquot is diluted with 0.5 liter of MHSF and mixed. This becomes your 50%
effluent sample. Continue diluting half of each sample with the same volume of MHSF to make your 25%,
12.5%, and 6.25% effluent dilutions, which represent all five test dilutions.
ACUTE DEFINITIVE TOXICITY TEST
The 48-h definitive test is performed using the test conditions listed in the following table
(MANDATORY TEST CONDITIONS FOR FATHEAD MINNOW (PIMEPHALES PROMELAS) 48-HOUR
ACUTE, NON-RENEWAL, TOXICITY TESTS).
TEST DATA
Biological and physical/chemical measurements are performed and the data recorded as specified
in EPA/600/4-90/027. Records of these measurements must be maintained on file in the laboratory and
made available to USEPA if requested.
11
-------
U. S. ENVIRONMENTAL PROTECTION AGENCY
DMR-QA LABORATORY PERFORMANCE EVALUATION Study 18
INSTRUCTIONS FOR THE FATHEAD MINNOW (PIMEPHALES PROMELAS)
48-H ACUTE. NON-RENEWAL. TOXICITY TEST
(METHOD CODE 13)
Biological Data
In the 48-h definitive test, data on test organism survival in each replicate chamber for the controls
and effluent concentrations are recorded daily as described on p. 70 and in Figure 4, p. 72, EPA/600/4-
90/027.
Physical/Chemical Data
In the 48-h non-renewal, definitive test, dissolved oxygen (DO), pH, total alkalinity, total hardness,
conductivity, and temperature are determined at each concentration of effluent used in the dilution series,
and in the control, at the beginning of the test, and the DO, pH, conductivity, and temperature are
determined at the end of the test, or when a replicate sample experiences 100% mortality, and recorded as
described on pp. 70 and 71, and in Figure 4, p. 72, EPA/600/4-90/027.
BIOLOGICAL DATA ANALYSIS
Mortality data from the 48-h definitive test are used to determine the LC50, expressed as percent
effluent. Follow the flowchart in Figure 1 in the Statistical Appendix to determine the LC50. For additional
information on data analysis, see the Statistical Appendix at the end of this instruction packet.
REPORTING THE TEST RESULTS
The 48-h LC50 (expressed as percent effluent) is entered on the Report Form located at the end of
the package of instructions. Record all results the same as they are recorded for DMR reporting. NOTE:
Do not report LCSOs for tests which fail to meet the acceptability criteria listed with the test
conditions. Be sure you enter your data into the portion of the Report Form that corresponds to the exact
test conditions you used. Make sure you also give the Method Code (see listing among "The Instructions
To Toxicity Test Laboratories") appropriate for the exact test conditions you used.
12
-------
METHOD CODE 13
MANDATORY TEST CONDITIONS FOR FATHEAD MINNOWS (PIMEPHALES PROMELAS)
48-H ACUTE, NON-RENEWAL TOXICITY TESTS
1. Test type:
2. Test duration:
3. Temperature:
4. Light quality:
5. Light intensity:
6. Photoperiod:
7. Volume of Stock Solution
8. Test chamber size:
9. Test solution volume:
10. Renewal of test solutions:
11. Age of test organisms:
12. No. organisms/test chamber:
13. No. replicate test
chambers/concentration:
14. No. organisms/concentration:
15. Feeding regime:
16. Test chamber cleaning:
17. Aeration:
18. Dilution water:
19. Test concentrations:
20. Dilution series:
21. Endpoint (expressed as %effluent):
22. Test acceptability criterion:
Static, non-renewal
48 h
25±1°C(MHSF)
Ambient laboratory illumination
10-20 uE/m2/s (50-100 ft-c)
(Ambient laboratory levels)
16 h light, 8 h darkness
8 mL Stock/1 L
250 mL (minimum)
200 mL (minimum)
None
1-14 days; 24 h maximum range in age
10
2
20
Not fed
Cleaning not required
None
Moderately-hard synthetic freshwater (MHSF)(80-
100 mg CaCO3, EPA/600/4-90/027) prepared with
MILLIPORE MILLI-Q" (or equivalent) deionized
water and reagent grade chemicals
5 effluent concentrations and a
control
0.5
LC50
90% or greater survival in controls
13
-------
-------
U. S. ENVIRONMENTAL PROTECTION AGENCY
DMR-QA LABORATORY PERFORMANCE EVALUATION Study 18
INSTRUCTIONS FOR THE FATHEAD MINNOW (PIMEPHALES PROMELAS)
48-H ACUTE. NON-RENEWAL. TOXICITY TEST
(METHOD CODE 14)
CAUTION: Read Instructions Carefully Before Opening The Reference Toxicants.
PREPARATION OF "SIMULATED" EFFLUENT
The reference toxicant, labeled REFERENCE TOXICANT #3, is supplied in solid form in a glass bottle. A stock
solution of REFERENCE TOXICANT #3 is prepared by adding 2.50 grams of the toxicant material to
approximately 90 ml of distilled water. Stir with a mechanical stirrer or by hand shaking until dissolved. Bring the
final volume to 100 ml with distilled water. The stock solution can be used for more than one method code that
uses REFERENCE TOXICANT #3. A fresh stock solution must be prepared each time a test (or set of tests)
using REFERENCE TOXICANT #3 is started or renewed. For each fathead minnow acute toxicity test described
below, prepare a "simulated" effluent (hereafter referred to as the effluent) from the stock solution Prepare the
effluent by adding 10.0 ml of the stock solution to 900 mL of Diluted Mineral Water (DMW)(25°C test). Stir
with a mechanical stirrer or by hand shaking until completely mixed. Bring the final volume to 1L with DMW.
Larger or smaller volumes of effluent can be prepared by using proportionally larger or smaller amounts of the
REFERENCE TOXICANT #3 stock solution and DMW. DMW is prepared from MILLIPORE MILLI-Q" (or
equivalent) deionized water and reagent grade chemicals (or mineral water), as specified in Tables 6 and 7, p.
35, EPA/600/4-90/027.
The effluent prepared according to these instructions represents the sample ready for testing, 100%
effluent. This 100% effluent sample is split into two 0.5 L aliquots. The first aliquot is your 100% effluent for
testing. The second aliquot is diluted with 0.5 liter of DMW and mixed. This becomes your 50% effluent sample.
Continue diluting half of each sample with the same volume of DMW to make your 25%, 12.5%, and 6.25%
effluent dilutions, which represent all five test dilutions.
ACUTE DEFINITIVE TOXICITY TEST
The 48-h definitive test is performed using the test conditions listed in the following table (MANDATORY
TEST CONDITIONS FOR FATHEAD MINNOW (PIMEPHALES PROMELAS) 48-HOUR ACUTE, NON-
RENEWAL, TOXICITY TESTS).
TEST DATA
Biological and physical/chemical measurements are performed and the data recorded as specified in
EPA/600/4-90/027. Records of these measurements must be maintained on file in the laboratory and made
available to USEPA if requested.
15
-------
U. S. ENVIRONMENTAL PROTECTION AGENCY
DMR-QA LABORATORY PERFORMANCE EVALUATION Study 18
INSTRUCTIONS FOR THE FATHEAD MINNOW (PIMEPHALES PROMELAS)
48-H ACUTE. NON-RENEWAL. TOXICITY TEST
(METHOD CODE 14)
Biological Data
In the 48-h definitive test, data on test organism survival in each replicate chamber for the controls and
effluent concentrations are recorded daily as described on p. 70 and in Figure 4, p. 72, EPA/600/4-90/027.
Physical/Chemical Data
In the 48-h non-renewal, definitive test, dissolved oxygen (DO), pH, total alkalinity, total hardness,
conductivity, and temperature are determined at each concentration of effluent used in the dilution series, and in
the control, at the beginning of the test, and the DO, pH, conductivity, and temperature are determined at the end
of the test, or when a replicate sample experiences 100% mortality, and recorded as described on pp. 70 and 71,
and in Figure 4, p. 72, EPA/600/4-90/027.
BIOLOGICAL DATA ANALYSIS
Mortality data from the 48-h definitive test are used to determine the LC50, expressed as percent
effluent. Follow the flowchart in Figure 1 in the Statistical Appendix to determine the LC50. For additional
information on data analysis, see the Statistical Appendix at the end of this instruction packet.
REPORTING THE TEST RESULTS
The 48-h LCSO (expressed as percent effluent) is entered on the Report Form located at the end of the
package of instructions. Record all results the same as they are recorded for DMR reporting. NOTE: Do not
report LCSOs for tests which fail to meet the acceptability criteria listed with the test conditions. Be sure
you enter your data into the portion of the Report Form that corresponds to the exact test conditions you used.
Make sure you also give the Method Code (see listing among "The Instructions To Toxicity Test Laboratories")
appropriate for the exact test conditions you used.
16
-------
METHOD CODE 14
MANDATORY TEST CONDITIONS FOR FATHEAD MINNOWS (PIMEPHALES PROMELAS)
48-H ACUTE, NON-RENEWAL TOXICITY TESTS
1. Test type:
2. Test duration:
3. Temperature:
4. Light quality:
5. Light intensity:
6. Photoperiod:
7. Volume of Stock Solution
8. Test chamber size:
9. Testsolution volume:
10. Renewal of test solutions:
11. Age of test organisms:
12. No. organisms/test chamber:
13. No. replicate test
chambers/concentration:
14. No. organisms/concentration:
15. Feeding regime:
16. Test chamber cleaning:
17. Aeration:
18. Dilution water:
i
19. Test concentrations:
20. Dilution series:
21. Endpoint (expressed as %effluent):
22. Test acceptability criterion:
Static, non-renewal
48 h
25±1°C(DMW)
Ambient laboratory illumination
10-20 uE/m2/s (50-100 ft-c)
(Ambient laboratory levels)
16 h light, 8 h darkness
10 mL Stock/1 L
250 mL (minimum)
200 mL (minimum)
None
1-14 days; 24 h maximum range in age
10
2
20
Not fed
Cleaning not required
None
20% Diluted Mineral Water (DMW) (80-100 mg
CaCO3, EPA/600/4-90/027) prepared with
MILLIPORE MILLI-Q" (or equivalent) deionized
water and mineral water
5 effluent concentrations and a
control
0.5
LC50
90% or greater survival in controls
17
-------
-------
U. S. ENVIRONMENTAL PROTECTION AGENCY
DMR-QA LABORATORY PERFORMANCE EVALUATION Study 18
INSTRUCTIONS FOR THE FATHEAD MINNOW (PIMEPHALES PROMELAS)
7-DAY. DAILY RENEWAL. SURVIVAL AND GROWTH TEST
(METHOD CODE 15)
CAUTION: Read Instructions Carefully Before Opening The Reference Toxicants.
PREPARATION OF "SIMULATED" EFFLUENT
The reference toxicant, labeled REFERENCE TOXICANT #1, is supplied in solid form in a glass bottle. A
stock solution of REFERENCE TOXICANT #1 is prepared by adding 0.441 grams of the toxicant material to
approximately 90 mL of distilled water. Stir with a mechanical stirrer or by hand shaking until dissolved. Bring the
final volume to 100 mL with distilled water. The 100 mL stock solution is made one time and is used for the 7
days of the test(s). The stock solution can be used for more than one method code that uses REFERENCE
TOXICANT #1. For each fathead minnow 7-day survival and growth test described below, each day prepare a
fresh solution of "simulated" effluent (hereafter referred to as the effluent) from the stock solution. For tests
using moderately-hard synthetic freshwater (MHSF) as the dilution water, add 1.5 mL of stock solution to
approximately 2.9 L of MHSF. Stir with a mechanical stirrer or by hand shaking until completely mixed. Bring
the final volume to 3 L with moderately-hard synthetic freshwater. Smaller volumes of effluent can be prepared
by using proportionally smaller amounts of the REFERENCE TOXICANT #1 stock solution and moderately-hard
synthetic freshwater. Moderately-hard synthetic freshwater is prepared from MILLIPORE MILLI-QR (or
equivalent) deionized water and reagent grade chemicals or mineral water, as specified in Table 1, p. 26,
EPA/600/4-89/001.
The effluent prepared according to these instructions represents the sample ready for testing, 100% effluent.
This 100% effluent sample is split into two 1.5 L aliquots. The first aliquot is your 100% effluent for testing. The
second aliquot is diluted with 1.5 L of moderately-hard synthetic freshwater and mixed. This becomes your 50%
effluent sample. Continue diluting half of each sample with the same volume of moderately-hard synthetic
freshwater to make your 25%, 12.5%, and 6.25% effluent dilutions, which represent all five test dilutions.
7-DAY. SHORT-TERM. CHRONIC TOXICITY TEST
The 7-day survival and growth test is performed using the test conditions listed in the following table
(MANDATORY TEST CONDITIONS FOR FATHEAD MINNOW (PIMEPHALES PROMELAS) 7-DAY, DAILY
RENEWAL, SURVIVAL AND GROWTH TESTS).
TEST DATA
Daily biological and physical/chemical measurements are performed and the data recorded as specified in
EPA/600/4-89/001. Records of these measurements must be maintained on file in the laboratory and made
available to USEPA if requested.
19
-------
U. S. ENVIRONMENTAL PROTECTION AGENCY
DMR-QA LABORATORY PERFORMANCE EVALUATION Study 18
INSTRUCTIONS FOR THE FATHEAD MINNOW (PIMEPHALES PROMELAS)
7-DAY. DAILY RENEWAL. SURVIVAL AND GROWTH TEST
(METHOD CODE 15)
Biological Data
Test organism survival in each test chamber is determined daily as described on pp. 39-40, and recorded as
described in Figures 2 and 4, pp. 45 and 47, EPA/600/4-89/001. Growth (mean dry weight per surviving
organism) is determined at the end of the test as described on p. 40 and recorded as described in Figures 3 and
4, pp. 46-47, EPA/600/4-89/001.
Physical/Chemical Data
Dissolved oxygen (DO), pH, total alkalinity, total hardness, conductivity, and temperature are determined as
described on p. 39, EPA/600/4-89/001, and recorded as described in Figures 1 and 4, pp. 43 and 47,
EPA/600/4-89/001.
BIOLOGICAL DATA ANALYSIS
The endpoints (expressed as percent effluent) determined from the test data are as follows:
(1) NOEC for survival - see pp. 48-58, EPA/600/4-89/001.
(2) IC25 for growth - see EPA/600/4-89/001 A (Supplement, Sept. I989).
(3) NOEC for growth - see pp. 62-71, EPA/600/4-89/001.
The IC25 for growth can be determined manually using the guidelines in EPA/600/4-89/001 A. For additional
information on data analysis, see the Statistical Appendix at the end of this instruction packet.
REPORTING THE TEST RESULTS
The NOEC for survival, and the IC25 and NOEC for growth, expressed as percent effluent, are entered on
the Report Form located at the end of the instruction package. Record all results the same as they are recorded
for DMR reporting. NOTE: Do not report endpoints for tests which fail to meet the acceptability criteria
listed with the test conditions. Be sure you enter your data into the portion of the Report Form that
corresponds to the exact test conditions you used. Make sure you also give the Method Code (see listing among
"The Instructions To Toxicity Test Laboratories") appropriate for the exact test conditions you used.
20
-------
METHOD CODE 15
MANDATORY TEST CONDITIONS FOR FATHEAD MINNOWS (PIMEPHALES PROMELAS)
7-DAY, DAILY RENEWAL, SURVIVAL AND GROWTH TESTS
1: Test type:
2. Test duration:
3. Temperature:
4. Light quality:
5. Light intensity:
6. Photoperiod:
7. Volume of Stock Solution
8. Test chamber size:
9. Test solution volume:
10. Renewal of test solutions:
11. Age of test organisms:
12. No. organisms/test chamber:
13. No. replicate test
chambers/concentration:
14. No. organisms/concentration:
15. Feeding regime:
16. Test chamber cleaning:
17. Aeration:
18. Dilution water:
19. Test concentrations:
20. Dilution series:
21. Endpoints (expressed as
percent effluent):
22. Test acceptability criteria:
Static, renewal
7 days
25±1°C(MHSF)
Ambient laboratory illumination
10-20 |jE/m2/s (50-100 ft-c)
(Ambient laboratory levels)
16 h light, 8 h darkness
1.5mL/3L
500 mL (minimum)
250 mL (minimum)
Daily
<24h
15 (minimum of 10)
4 (minimum of 3)
60 (minimum of 30)
Feed as described in EPA/600/4-89/001
Siphon daily, immediately before test
solution renewal
None
Moderately-hard synthetic freshwater (MHSF) (80-100 mg
CaC03, EPA/600/4-89/001) prepared with MILLIPORE MlLLI-QR
(or equivalent) deionized water and reagent grade chemicals
5 effluent concentrations and a control
0.5
NOEC -Survival, IC25 - Growth, NOEC - Growth
(1) 80% or greater survival in controls
(2) The average dry weight of control larvae
surviving at the end of the test must equal
or exceed 0.25 mg
21
-------
-------
U. S. ENVIRONMENTAL PROTECTION AGENCY
DMR-QA LABORATORY PERFORMANCE EVALUATION Study 18
INSTRUCTIONS FOR THE FATHEAD MINNOW (PIMEPHALES PROMELAS)
7-DAY. DAILY RENEWAL. SURVIVAL AND GROWTH TEST
(METHOD CODE 16)
CAUTION: Read Instructions Carefully Before Opening The Reference Toxicants.
PREPARATION OF "SIMULATED" EFFLUENT
The reference toxicant, labeled REFERENCE TOXICANT #1, is supplied in solid form in a glass bottle. A
stock solution of REFERENCE TOXICANT #1 is prepared by adding 0.441 grams of the toxicant material to
approximately 90 mL of distilled water. Stir with a mechanical stirrer or by hand shaking until dissolved. Bring the
final volume to 100 mL with distilled water. The 100 mL stock solution is made one time and is used for the 7
days of the test(s). The stock solution can be used for more than one method code that uses REFERENCE
TOXICANT #1. For each fathead minnow 7-day survival and growth test described below, each day prepare a
fresh solution of "simulated" effluent (hereafter referred to as the effluent) from the stock solution. For tests
using 20% diluted mineral water (DMW) as the dilution water, add 3.0 mL of the stock solution to
approximately 2.9 L of DMW. Stir with a mechanical stirrer or by hand shaking until completely mixed. Bring
the final volume to 3 L with 20% diluted mineral water. Smaller volumes of effluent can be prepared by using
proportionally smaller amounts of the REFERENCE TOXICANT #1 stock solution and 20% diluted mineral
water. Diluted mineral water is prepared from MILLIPORE MILLI-QR (or equivalent) deionized water and mineral
water, as specified in Table 1, p. 26, EPA/600/4-89/001.
The effluent prepared according to these instructions represents the sample ready for testing, 100% effluent.
This 100% effluent sample is split into two 1.5 L aliquots. The first aliquot is your 100% effluent for testing. The
second aliquot is diluted with 1.5 L of 20% diluted mineral water and mixed. This becomes your 50% effluent
sample. Continue diluting half of each sample with the same volume of 20% diluted mineral water to make your
25%, 12.5%, and 6.25% effluent dilutions, which represent all five test dilutions.
7-DAY. SHORT-TERM. CHRONIC TOXICITY TEST
The 7-day survival and growth test is performed using the test conditions listed in the following table
(MANDATORY TEST CONDITIONS FOR FATHEAD MINNOW (PIMEPHALES PROMELAS) 7-DAY, DAILY
RENEWAL, SURVIVAL AND GROWTH TESTS).
TEST DATA
Daily biological and physical/chemical measurements are performed and the data recorded as specified in
EPA/600/4-89/001. Records of these measurements must be maintained on file in the laboratory and made
available to USEPA if requested.
23
-------
U. S. ENVIRONMENTAL PROTECTION AGENCY
DMR-QA LABORATORY PERFORMANCE EVALUATION Study 18
INSTRUCTIONS FOR THE FATHEAD MINNOW (PIMEPHALES PROMELAS)
7-DAY. DAILY RENEWAL. SURVIVAL AND GROWTH TEST
(METHOD CODE 16)
Biological Data
Test organism survival in each test chamber is determined daily as described on pp. 39-40, and recorded as
described in Figures 2 and 4, pp. 45 and 47, EPA/600/4-89/001. Growth (mean dry weight per surviving
organism) is determined at the end of the test as described on p. 40 and recorded as described in Figures 3 and
4, pp. 46-47, EPA/6QO/4-89/001.
Physical/Chemical Data
Dissolved oxygen (DO), pH, total alkalinity, total hardness, conductivity, and temperature are determined as
described on p. 39, EPA/600/4-89/001, and recorded as described in Figures 1 and 4, pp. 43 and 47,
EPA/600/4-89/001.
BIOLOGICAL DATA ANALYSIS
The endpoints (expressed as percent effluent) determined from the test data are as follows:
(1) NOEC for survival - see pp. 48-58, EPA/600/4-89/001.
(2) IC25 for growth - see EPA/600/4-89/001 A (Supplement, Sept. I989).
(3) NOEC for growth - see pp. 62-71, EPA/600/4-89/001.
The IC25 for growth can be determined manually using the guidelines in EPA/600/4-89/001 A. For additional
information on data analysis, see the Statistical Appendix at the end of this instruction packet.
REPORTING THE TEST RESULTS
The NOEC for survival, and the IC25 and NOEC for growth, expressed as percent effluent, are entered on
the Report Form located at the end of the instruction package. Record all results the same as they are recorded
for DMR reporting. NOTE: Do not report endpoints for tests which fail to meet the acceptability criteria
listed with the test conditions. Be sure you enter your data into the portion of the Report Form that
corresponds to the exact test conditions you used. Make sure you also give the Method Code (see listing among
"The Instructions To Toxicity Test Laboratories") appropriate for the exact test conditions you used.
24
-------
METHOD CODE 16
MANDATORY TEST CONDITIONS FOR FATHEAD MINNOWS (PIMEPHALES PROMELAS)
7-DAY, DAILY RENEWAL, SURVIVAL AND GROWTH TESTS
1. Test type:
2. Test duration:
3. Temperature:
4. Light quality:
5. Light intensity:
6. Photoperiod:
7. Volume of Stock Solution
8. Test chamber size:
9. Test solution volume:
10. Renewal of test solutions:
11. Age of test organisms:
12. No. organisms/test chamber:
13. No. replicate test
chambers/concentration:
14. No. organisms/concentration:
15. Feeding regime:
16. Test chamber cleaning:
17. Aeration:
18. Dilution water:
19. Test concentrations:
20. Dilution series:
21. Endpoints (expressed as
percent effluent):
22. Test acceptability criteria:
Static, renewal
7 days
25±1°C(DMW)
Ambient laboratory illumination
10-20 uE/m2/s (50-100 ft-c)
(Ambient laboratory levels)
16 h light, 8 h darkness
3.0 mU3L
500 mL (minimum)
250 mL (minimum)
Daily
<24h
15 (minimum of 10)
4 (minimum of 3)
60 (minimum of 30)
Feed as described in EPA/600/4-89/001
Siphon daily, immediately before test
solution renewal
None
20% diluted mineral water (DMW)(80-100 mg CaCO3,
EPA/600/4-89/001) prepared with MILLIPORE MILLI-QR (or
equivalent) deionized water mineral water
5 effluent concentrations and a control
0.5
NOEC -Survival, IC25 - Growth, NOEC - Growth
(1) 80% or greater survival in controls
(2) The average dry weight of control larvae
surviving at the end of the test must equal
or exceed 0.25 mg
25
-------
-------
U. S. ENVIRONMENTAL PROTECTION AGENCY
DMR-QA LABORATORY PERFORMANCE EVALUATION Study 18
INSTRUCTIONS FOR THE CERIODAPHNIA DUBIA 48-H ACUTE.
DAILY RENEWAL. TOXICITY TEST
(METHOD CODE 17)
CAUTION: Read Instructions Carefully Before Opening The Reference Toxicants.
PREPARATION OF "SIMULATED" EFFLUENT
The reference toxicant, labeled REFERENCE TOXICANT #3, is supplied in solid form in a glass bottle. A
stock solution of REFERENCE TOXICANT #3 is prepared by adding 2.5 grams of the toxicant material to
approximately 90 mL of distilled water. Stir with a mechanical stirrer or by hand shaking until dissolved. Bring the
final volume to 100 mL with distilled water. The stock solution can be used for more than one method code that
uses REFERENCE TOXICANT #3. A fresh stock solution must be prepared each time a test (or set of tests)
using REFERENCE TOXICANT #3 is started or renewed. For each Ceriodaphnia dubia acute toxicity test
described below, each day prepare a "simulated" effluent (hereafter referred to as the effluent) from the stock
solution. Prepare the effluent by adding 1.4 mL of the stock solution to 900 mL of Moderately-hard
synthetic freshwater (MHSF)(20°C test). Stir with a mechanical stirrer or by hand shaking until completely
mixed. Bring the final volume to 1L with MHSF. MHSF is prepared from MILLIPORE MILLI-Q" (or equivalent)
deionized water and reagent grade chemicals, as specified in Tables 6 and 7, p. 35, EPA/600/4-90/027. If
smaller, volumes of effluent are prepared, proportionally smaller amounts of the REFERENCE TOXICANT #3
stock solution and MHSF would be required.
The effluent prepared according to these instructions represents the sample ready for testing, 100% effluent.
This 100% effluent sample is split into two 0.5 L aliquots. The first aliquot is your 100% effluent for testing. The
second aliquot is diluted with 0.5 L of MHSF and mixed. This becomes your 50% effluent sample. Continue
diluting half of each sample with the same volume of MHSF to make your 25%, 12.5%, and 6.25% effluent
dilutions, which represent all five test dilutions.
ACUTE DEFINITIVE TOXICITY TEST
Definitive Test
The 48-hour definitive test is performed using the test conditions listed in the following table (MANDATORY
TEST CONDITIONS FOR CERIODAPHNIA DUBIA 48-HOUR ACUTE, DAILY RENEWAL, TOXICITY TESTS).
TEST DATA
Biological and physical/chemical measurements are performed and the data recorded as specified in
EPA/600/4-90/027. Records of these measurements must be maintained on file in the laboratory and made
available to USEPA if requested.
27
-------
U. S. ENVIRONMENTAL PROTECTION AGENCY
DMR-QA LABORATORY PERFORMANCE EVALUATION study is
./I, i i " ' ' i •; ,
INSTRUCTIONS FOR THE CERIODAPHNIA DUBIA 48-H ACUTE.
DAILY RENEWAL. TOXICITY TEST
(METHOD CODE 17)
Biological Data
In the 48-h definitive test, data on organism survival in each replicate chamber for the controls and effluent
concentrations are recorded daily as described on p. 70 and in Figure 4, p. 72, EPA/600/4-90/027.
Physical/Chemical Data
In the 48-h renewed, definitive test, dissolved oxygen (DO), pH, total alkalinity, total hardness, conductivity,
and temperature are determined at each concentration of effluent used in the dilution series, and in the control,
at the beginning of the test, and DO, pH, conductivity, and temperature are determined after 24 h and at the end
of the test, and recorded as described pp. 70 and 71, and in Figure 4, p. 72, EPA/600/4-90/027.
BIOLOGICAL DATA ANALYSIS
Mortality data from the 48-h definitive test are used to determine the LC50, expressed as percent effluent.
Follow the flowchart in Figure 1 in the Statistical Appendix to determine the LC50. For additional information on
data analysis, see the Statistical Appendix at the end of this instruction packet.
REPORTING THE TEST RESULTS
The 48-h LC50 (expressed as percent effluent) is entered on the Report Form located at the end of the
package of instructions. Record all results the same as they are recorded for DMR reporting. NOTE: Do not
report LCSOs for tests which fail to meet the acceptability criteria listed with the test conditions. Be sure
you enter your data into the portion of the Report Form that corresponds to the exact test conditions you used.
Make sure you also give the Method Code (see listing among "The Instructions To Toxicity Test Laboratories")
appropriate for the exact test conditions you used.
28
-------
METHOD CODE 17
MANDATORY TEST CONDITIONS FOR CERIODAPHNIA DUBIA 48-H ACUTE,
DAILY RENEWAL, TOXICITY TESTS
1. Test type:
2. Test duration:
3. Temperature:
4. Light quality:
5. Light intensity:
6. Photoperiod:
7. Volume of Stock Solution
8. Test chamber size:
9. Test solution volume:
10. Renewal of test solutions:
11. Age of test organisms:
12. No. organisms/test chamber:
13. No. replicate test
chambers/concentration:
14. No. organisms/concentration:
15. Feeding regime:
16. Test chamber cleaning:
17. Aeration:
18. Dilution water:
19. Test concentrations:
20. Dilution series:
21. Endpoint (expressed as
percent effluent):
22. Test acceptability criterion:
Static, renewal
48 h
20±1°C(MHSF)
Ambient laboratory illumination
10-20 uE/m2/s (50-100 ft-c)
(Ambient laboratory levels)
16 h light, 8 h darkness
1.4 mL Stock/1 L
30 mL (minimum)
15 mL (minimum)
Daily
<24h
5
4
20
Feed while holding prior to test
Cleaning not required
None
Moderately-hard synthetic freshwater (MHSF) (80-100 mg
CaCO3, EPA/600/4-90/027) prepared with MILLIPORE MILLI-Q"
(or equivalent) deionized water and reagent grade chemicals
5 effluent concentrations and a control
0.5
LC50
90% or greater survival in controls
29
-------
-------
U. S. ENVIRONMENTAL PROTECTION AGENCY
DMR-QA LABORATORY PERFORMANCE EVALUATION Study 18
INSTRUCTIONS FOR THE CERIODAPHNIA DUBIA 48-H ACUTE.
DAILY RENEWAL. TOXICITY TEST
(METHOD CODE 18)
CAUTION: Read Instructions Carefully Before Opening The Reference Toxicants.
PREPARATION OF "SIMULATED" EFFLUENT
The reference toxicant, labeled REFERENCE TOXICANT #3, is supplied in solid form in a glass bottle. A
stock solution of REFERENCE TOXICANT #3 is prepared by adding 2.5 grams of the toxicant material to
approximately 90 mL of distilled water. Stir with a mechanical stirrer or by hand shaking until dissolved. Bring the
final volume to 100 mL with distilled water. The stock solution can be used for more than one method code that
uses REFERENCE TOXICANT #3. A fresh stock solution must be prepared each time a test (or set of tests)
using REFERENCE TOXICANT #3 is started or renewed. For each Ceriodaphnia dubia acute toxicity test
described below, each day prepare a "simulated" effluent (hereafter referred to as the effluent) from the stock
solution. Prepare the effluent by adding 4.8 mL of the stock solution to 900 mL of 20% diluted mineral
water (DMW) (20°C test). Stir with a mechanical stirrer or by hand shaking until completely mixed. Bring the
final volume to 1L with DMW. DMW is prepared from MILLIPORE MILLI-QR (or equivalent) deionized water and
mineral water, as specified in Tables 6 and 7, p. 35, EPA/600/4-90/027. If smaller volumes of effluent are
prepared, proportionally smaller amounts of the REFERENCE TOXICANT #3 stock solution and DMW would be
required.
The effluent prepared according to these instructions represents the sample ready for testing, 100% effluent
This 100% effluent sample is split into two 0.5 L aliquots. The first aliquot is your 100% effluent for testing. The
second aliquot is diluted with 0.5 L of DMW and mixed. This becomes your 50% effluent sample. Continue
diluting half of each sample with the same volume of DMW to make your 25%, 12.5%, and 6.25% effluent
dilutions, which represent all five test dilutions.
ACUTE DEFINITIVE TOXICITY TEST
Definitive Test
The 48-hour definitive test is performed using the test conditions listed in the following table (MANDATORY
TEST CONDITIONS FOR CERIODAPHNIA DUBIA 48-HOUR ACUTE, DAILY RENEWAL, TOXICITY TESTS).
TEST DATA
Biological and physical/chemical measurements are performed and the data recorded as specified in
EPA/600/4-90/027. Records of these measurements must be maintained on file in the laboratory and made
available to USEPA if requested.
31
-------
U. S. ENVIRONMENTAL PROTECTION AGENCY
DMR-QA LABORATORY PERFORMANCE EVALUATION Study 18
'•?"': ' 'i "', ' ' ' i. „ • ,• • ' ' ' , " .''i •
"I', j 'i , •' '.,» , : • ,.,,•! .' ,; ' ' , i,1; • s •' . , ;,
INSTRUCTIONS FOR THE CERIODAPHNIA DUBIA 48-H ACUTE.
DAILY RENEWAL. TOXICITY TEST
(METHOD CODE 18)
Biological Data
In the 48-h definitive test, data on organism survival in each replicate chamber for the controls and effluent
concentrations are recorded daily as described on p. 70 and in Figure 4, p. 72, EPA/600/4-90/027.
Physical/Chemical Data
In the 48-h renewed, definitive test, dissolved oxygen (DO), pH, total alkalinity, total hardness, conductivity,
and temperature are determined at each concentration of effluent used in the dilution series, and in the control,
at the beginning of the test, and DO, pH, conductivity, and temperature are determined after 24 h and at the end
of the test, and recorded as described pp. 70 and 71, and in Figure 4, p. 72, EPA/600/4-90/027.
BIOLOGICAL DATA ANALYSIS
Mortality data from the 48-h definitive test are used to determine the LC50, expressed as percent effluent.
Follow the flowchart in Figure 1 in the Statistical Appendix to determine the LC50. For additional information on
data analysis, see the Statistical Appendix at the end of this instruction packet.
REPORTING THE TEST RESULTS
The 48-h LC50 (expressed as percent effluent) is entered on the Report Form located at the end of the
package of instructions. Record all results the same as they are recorded for DMR reporting. NOTE: Do not
report LCSOs for tests which fail to meet the acceptability criteria listed with the test conditions. Be sure
you enter your data into the portion of the Report Form that corresponds to the exact test conditions you used.
Make sure you also give the Method Code (see listing among "The Instructions To Toxicity Test Laboratories")
appropriate for the exact test conditions you used.
32
-------
METHOD CODE 18
MANDATORY TEST CONDITIONS FOR CERIODAPHNIA DUBIA 48-H ACUTE,
DAILY RENEWAL, TOXICITY TESTS
1. Test type:
2. Test duration:
3. Temperature:
4. Light quality:
5. Light intensity:
6. Photoperiod:
7. Volume of Stock Solution
8. Test chamber size:
9. Test solution volume:
10. Renewal of test solutions:
11. Age of test organisms:
12. No. organisms/test chamber:
13. No. replicate test
chambers/concentration:
14. No. organisms/concentration:
15. Feeding regime:
16. Test chamber cleaning:
17. Aeration:
18. Dilution water:
19. Test concentrations:
20. Dilution series:
21. Endpoint (expressed as
percent effluent):
22. Test acceptability criterion:
Static, renewal
48 h
20 ± 1°C (DMW)
Ambient laboratory illumination
10-20 uE/m2/s (50-100 ft-c)
(Ambient laboratory levels)
16 h light, 8 h darkness
4.8 mL Stock/1 L
30 mL (minimum)
15 mL (minimum)
Daily
<24h
5
4
20
Feed while holding prior to test
Cleaning not required
None
20% Diluted mineral water (80-100 mg CaCO3, EPA/600/4-
90/027) prepared with MILLIPORE MlLLI-QR (or equivalent)
deionized water mineral water
5 effluent concentrations and a control
0.5
LC50
90% or greater survival in controls
33
-------
-------
U. S. ENVIRONMENTAL PROTECTION AGENCY
DMR-QA LABORATORY PERFORMANCE EVALUATION Study 18
INSTRUCTIONS FOR THE CERIODAPHNIA DUBIA 48-H ACUTE.
DAILY RENEWAL. TOXICITY TEST
(METHOD CODE 19)
CAUTION: Read Instructions Carefully Before Opening The Reference Toxicants.
PREPARATION OF "SIMULATED" EFFLUENT
The reference toxicant, labeled REFERENCE TOXICANT #3, is supplied in solid form in a glass bottle. A
stock solution of REFERENCE TOXICANT #3 is prepared by adding 2.5 grams of the toxicant material to
approximately 90 mL of distilled water. Stir with a mechanical stirrer or by hand shaking until dissolved. Bring the
final volume to 100 mL with distilled water. The stock solution can be used for more than one method code that
uses REFERENCE TOXICANT #3. A fresh stock solution must be prepared each time a test (or set of tests)
using REFERENCE TOXICANT #3 is started or renewed. For each Ceriodaphnia dubia acute toxicity test
described below, each day prepare a "simulated" effluent (hereafter referred to as the effluent) from the stock
solution. Prepare the effluent by adding 0.6 mL of the stock solution to 900 mL Moderately-hard synthetic
freshwater (MHSF) (25°C). Stir with a mechanical stirrer or by hand shaking until completely mixed. Bring the
final volume to 1L with MHSF. MHSF is prepared from MILLIPORE MILLI-Q" (or equivalent) deionized water and
reagent grade chemicals, as specified in Tables 6 and 7, p. 35, EPA/600/4-90/027. If smaller volumes of effluent
are prepared, proportionally smaller amounts of the REFERENCE TOXICANT #3 stock solution and MHSF
would be required.
The effluent prepared according to these instructions represents the sample ready for testing, 100% effluent.
This 100% effluent sample is split into two 0.5 L aliquots. The first aliquot is your 100% effluent for testing. The
second aliquot is diluted with 0.5 L of MHSF and mixed. This becomes your 50% effluent sample. Continue
diluting half of each sample with the same volume of MHSF to make your 25%, 12.5%, and 6.25% effluent
dilutions, which represent all five test dilutions.
ACUTE DEFINITIVE TOXICITY TEST
Definitive Test
The 48-hour definitive test is performed using the test conditions listed in the following table (MANDATORY
TEST CONDITIONS FOR CERIODAPHNIA DUBIA 48-HOUR ACUTE, DAILY RENEWAL, TOXICITY TESTS).
TEST DATA
Biological and physical/chemical measurements are performed and the data recorded as specified in
EPA/600/4-90/027. Records of these measurements must be maintained on file in the laboratory and made
available to USEPA if requested.
35
-------
U. S. ENVIRONMENTAL PROTECTION AGENCY
DMR-QA LABORATORY PERFORMANCE EVALUATION Study 18
INSTRUCTIONS FOR THE CERIODAPHNIA DUBIA 48-H ACUTE.
DAILY RENEWAL. TOXICITY TEST
(METHOD CODE 19)
Biological Data
In the 48-h definitive test, data on organism survival in each replicate chamber for the controls and effluent
concentrations are recorded daily as described on p. 70 and in Figure 4, p. 72, EPA/600/4-90/027.
Physical/Chemical Data
In the 48-h renewed, definitive test, dissolved oxygen (DO), pH, total alkalinity, total hardness, conductivity,
and temperature are determined at each concentration of effluent used in the dilution series, and in the control,
at the beginning of the test, and DO, pH, conductivity, and temperature are determined after 24 h and at the end
of the test, and recorded as described pp. 70 and 71, and in Figure 4, p. 72, EPA/600/4-90/027.
BIOLOGICAL DATA ANALYSIS
Mortality data from the 48-h definitive test are used to determine the LC50, expressed as percent effluent.
Follow the flowchart in Figure 1 in the Statistical Appendix to determine the LC50. For additional information on
data analysis, see the Statistical Appendix at the end of this instruction packet.
REPORTING THE TEST RESULTS
The 48-h LC50 (expressed as percent effluent) is entered on the Report Form located at the end of the
package of instructions. Record all results the same as they are recorded for DMR reporting. NOTE: Do not
report LCSOs for tests which fail to meet the acceptability criteria listed with the test conditions. Be sure
you enter your data into the portion of the Report Form that corresponds to the exact test conditions you used.
Make sure you also give the Method Code (see listing among "The Instructions To Toxicity Test Laboratories")
appropriate for the exact test conditions you used.
36
-------
METHOD CODE 19
MANDATORY TEST CONDITIONS FOR CERIODAPHNIA DUBIA 48-H ACUTE,
DAILY RENEWAL, TOXICITY TESTS
1. Test type:
2. Test duration:
3. Temperature:
4. Light quality:
5. Light intensity:
6. Photoperiod:
7. Volume of Stock Solution
8. Test chamber size:
9. Test solution volume:
10. Renewal of test solutions:
11. Age of test organisms:
12. No. organisms/test chamber:
13. No. replicate test
chambers/concentration:
14. No. organisms/concentration:
15. Feeding regime:
16. Test chamber cleaning:
17. Aeration:
18. Dilution water:
19. Test concentrations:
20. Dilution series:
21. Endpoint (expressed as
percent effluent):
22. Test acceptability criterion:
Static, renewal
48 h
25±1°C(MHSF)
Ambient laboratory illumination
10-20 uE/nf/s (50-100 ft-c)
(Ambient laboratory levels)
16 h light, 8 h darkness
0.6 mL Stock/1 L
30 mL (minimum)
15 mL (minimum)
Daily
<24h
5
4
20
Feed while holding prior to test
Cleaning not required
None
Moderately-hard synthetic freshwater (MHSF) (80-100 mg
CaCO3, EPA/600/4-90/027) prepared with MILLIPORE MILLI-Q*
(or equivalent) deionized water and reagent grade chemicals
5 effluent concentrations and a control
0.5
LC50
90% or greater survival in controls
37
-------
-------
U. S. ENVIRONMENTAL PROTECTION AGENCY
DMR-QA LABORATORY PERFORMANCE EVALUATION Study 18
INSTRUCTIONS FOR THE CERIODAPHNIA DUBIA 48-H ACUTE.
DAILY RENEWAL. TOXICITY TEST
(METHOD CODE 20)
CAUTION: Read Instructions Carefully Before Opening The Reference Toxicants.
PREPARATION OF "SIMULATED" EFFLUENT
The reference toxicant, labeled REFERENCE TOXICANT #3, is supplied in solid form in a glass bottle. A
stock solution of REFERENCE TOXICANT #3 is prepared by adding 2.5 grams of the toxicant material to
approximately 90 mL of distilled water. Stir with a mechanical stirrer or by hand shaking until dissolved. Bring the
final volume to 100 mL with distilled water. The stock solution can be used for more than one method code that
uses REFERENCE TOXICANT #3. A fresh stock solution must be prepared each time a test (or set of tests)
using REFERENCE TOXICANT #3 is started or renewed. For each Ceriodaphnia dubia acute toxicity test
described below, each day prepare a "simulated" effluent (hereafter referred to as the effluent) from the stock
solution. Prepare the effluent by adding 2.4 mL of the stock solution to 900 mL 20% Diluted Mineral Water
(DMW) (25°C). Stir with a mechanical stirrer or by hand shaking until completely mixed. Bring the final volume
to 1L with DMW. DMW is prepared from MILLIPORE MILLI-Q" (or equivalent) deionized water and reagent
grade chemicals, as specified in Tables 6 and 7, p. 35, EPA/600/4-90/027. If smaller volumes of effluent are
prepared, proportionally smaller amounts of the REFERENCE TOXICANT #3 stock solution and DMW would be
required.
The effluent prepared according to these instructions represents the sample ready for testing, 100% effluent.
This 100% effluent sample is split into two 0.5 L aliquots. The first aliquot is your 100% effluent for testing. The
second aliquot is diluted with 0.5 L of DMW and mixed. This becomes your 50% effluent sample. Continue
diluting half of each sample with the same volume of DMW to make your 25%, 12.5%, and 6.25% effluent
dilutions, which represent all five test dilutions.
ACUTE DEFINITIVE TOXICITY TEST
Definitive Test
The 48-hour definitive test is performed using the test conditions listed in the following table (MANDATORY
TEST CONDITIONS FOR CERIODAPHNIA DUBIA 48-HOUR ACUTE, DAILY RENEWAL, TOXICITY TESTS).
TEST DATA
Biological and physical/chemical measurements are performed and the data recorded as specified in
EPA/600/4-90/027. Records of these measurements must be maintained on file in the laboratory and made
available to USEPA if requested.
39
-------
U. S. ENVIRONMENTAL PROTECTION AGENCY
DMR-QA LABORATORY PERFORMANCE EVALUATION Study 18
INSTRUCTIONS FOR THE CERIODAPHNIA DUBIA 48-H ACUTE.
DAILY RENEWAL. TOXICITY TEST
(METHOD CODE 20)
Biological Data
In the 48-h definitive test, data on organism survival in each replicate chamber for the controls and effluent
concentrations are recorded daily as described on p. 70 and in Figure 4, p. 72, EPA/600/4-90/027.
Physical/Chemical Data
In the 48-h renewed, definitive test, dissolved oxygen (DO), pH, total alkalinity, total hardness, conductivity,
and temperature are determined at each concentration of effluent used in the dilution series, and in the control,
at the beginning of the test, and DO, pH, conductivity, and temperature are determined after 24 h and at the end
of the test, and recorded as described pp. 70 and 71, and in Figure 4, p. 72, EPA/600/4-90/027.
BIOLOGICAL DATA ANALYSIS
Mortality data from the 48-h definitive test are used to determine the LC50, expressed as percent effluent.
Follow the flowchart in Figure 1 in the Statistical Appendix to determine the LC50. For additional information on
data analysis, see the Statistical Appendix at the end of this instruction packet.
REPORTING THE TEST RESULTS
The 48-h LC50 (expressed as percent effluent) is entered on the Report Form located at the end of the
package of instructions. Record all results the same as they are recorded for DMR reporting. NOTE: Do not
report LCSOs for tests which fail to meet the acceptability criteria listed with the test conditions. Be sure
you enter your data into the portion of the Report Form that corresponds to the exact test conditions you used.
Make sure you also give the Method Code (see listing among "The Instructions To Toxicity Test Laboratories")
appropriate for the exact test conditions you used.
40
-------
METHOD CODE 20
MANDATORY TEST CONDITIONS FOR CERIODAPHNIA DUBIA 48-H ACUTE,
DAILY RENEWAL, TOXICITY TESTS
1. Test type:
2. Test duration:
3. Temperature:
4. Light quality:
5. Light intensity:
6. Photoperiod:
7. Volume of Stock Solution
8. Test chamber size:
9. Test solution volume:
10. Renewal of test solutions:
11. Age of test organisms:
12. No. organisms/test chamber:
13. No. replicate test
chambers/concentration:
14. No. organisms/concentration:
15. Feeding regime:
16. Test chamber cleaning:
17. Aeration:
18. Dilution water:
19. Test concentrations:
20. Dilution series:
21. Endpoint (expressed as
percent effluent):
22. Test acceptability criterion:
Static, renewal
48 h
25±1°C(DMW)
Ambient laboratory illumination
10-20 uE/m2/s (50-100 ft-c)
(Ambient laboratory levels)
16 h light, 8 h darkness
2.4 mL Stock/1 L
30 mL (minimum)
15 mL (minimum)
Daily
<24 h
5
4
20
Feed while holding prior to test
Cleaning not required
None
20% Diluted mineral water (80-100 mg CaCO3, EPA/600/4-
90/027) prepared with MILLIPORE MILLI-Q" (or equivalent)
deionized water and mineral water
5 effluent concentrations and a control
0.5
LC50
90% or greater survival in controls
41
-------
-------
U.S. ENVIRONMENTAL PROTECTION AGENCY
DMR-QA LABORATORY PERFORMANCE EVALUATION Study 18
INSTRUCTIONS FOR THE CERIODAPHNIA DUBIA 7-DAY. DAILY RENEWAL.
SURVIVAL AND REPRODUCTION TEST
(METHOD CODES 21 and 22)
CAUTION: Read Instructions Carefully Before Opening The Reference Toxicants.
PREPARATION OF "SIMULATED" EFFLUENT
The reference toxicant, labeled REFERENCE TOXICANT #1, is supplied in solid form in a glass bottle. A
stock solution of REFERENCE TOXICANT #1 is prepared by adding 0.441 grams of the toxicant material to
approximately 90 mL of distilled water. Stir with a mechanical stirrer or by hand shaking until dissolved. Bring the
final volume to 100 mL with distilled water. The 100 mL stock solution is made one time and is used for the 7
days of the test(s). The stock solution can be used for more than one method code that uses REFERENCE
TOXICANT #1. For each Ceriodaphnia dubia 7-day survival and reproduction test described below, each day
prepare a fresh solution of "simulated" effluent (hereafter referred to as the effluent) from the stock solution.
Prepare the effluent by adding 0.5 mL of the stock solution to 900 mL of Moderately-hard synthetic
freshwater (MHSF) or 20% diluted Mineral Water (DMW). Stir with a mechanical stirrer or by hand shaking
until completely mixed. Bring the final volume to 1 L with moderately-hard synthetic freshwater or 20% diluted
mineral water. Moderately-hard synthetic freshwater or 20% diluted mineral water are prepared from
MILLIPORE MILLI-QR (or equivalent) deionized water and reagent grade chemicals or mineral water, as specified
in Table 1, p. 26, EPA/600/4-89/001. Smaller volumes of effluent can be prepared by using proportionally
smaller amounts of the REFERENCE TOXICANT #1 stock solution and moderately-hard synthetic freshwater or
20% diluted mineral water.
The effluent prepared according to these instructions represents the sample ready for testing, 100% effluent.
This 100% effluent sample is split into two 0.5 L aliquots. The first aliquot is your 100% effluent for testing. The
second aliquot is diluted with 0.5 L of moderately-hard synthetic freshwater or 20% diluted mineral water and
mixed. This becomes your 50% effluent sample. Continue diluting half of each sample with the same volume of
moderately-hard synthetic freshwater or 20% diluted mineral water to make your 25%, 12.5%, and 6.25%
effluent dilutions, which represent all five test dilutions.
7-DAY. SHORT-TERM. CHRONIC TOX1CITY TEST
The 7-day survival and reproduction test is performed using the test conditions listed in the following table
(MANDATORY TEST CONDITIONS FOR CERIODAPHNIA DUBIA 7-DAY, DAILY RENEWAL, SURVIVAL AND
REPRODUCTION TESTS).
TEST DATA
Daily biological and physical/chemical measurements are performed and the data recorded as specified in
EPA/600/4-89/001. Records of these measurements must be maintained on file in the laboratory and made
available to USEPA if requested.
43
-------
U.S. ENVIRONMENTAL PROTECTION AGENCY
DMR-QA LABORATORY PERFORMANCE EVALUATION Study 18
INSTRUCTIONS FOR THE CERIODAPHNIA DUBIA 7-DAY. DAILY RENEWAL.
SURVIVAL AND REPRODUCTION TEST
(METHOD CODES 21 AND 22)
Biological Data
Data on test organism survival and reproduction are recorded daily for each test chamber as described on
pp. 121 -122, and in Figures 2-3, pp. 125-127, EPA/600/4-89/001.
Physical/Chemical Data
Dissolved oxygen (DO), pH, total alkalinity, total hardness, conductivity, and temperature are determined as
described on p. 121, EPA/600/4-89/001, and recorded as described in Figure 1, pp. 43-44, EPA/600/4-89/001.
BIOLOGICAL DATA ANALYSIS
The endpoints (expressed as percent effluent) determined from the test data are as follows:
(1) NOEC for survival - see pp. 128-131, EPA/600/4-89/001.
(2) IC25 for reproduction - see EPA/600/4-89/001A (Suppl., Sept. I989).
(3) NOEC for reproduction - see pp. 132-143, EPA/600/4-89/001.
The IC25 for reproduction can be determined manually using the guidelines in EPA/600/4-89/001 A. For
additional information on data analysis, see the Statistical Appendix at the end of this instruction packet.
REPORTING THE TEST RESULTS
The NOEC for survival, and the IC25 and NOEC for reproduction, expressed as percent effluent, are entered
on the Report Form located at the end of the instruction package. Record all results the same as they are
recorded for DMR reporting. NOTE: Do not report endpoints for tests which fail to meet the acceptability
criteria listed with the test conditions. Be sure you enter your data into the portion of the Report Form that
corresponds to the exact test conditions you used. Make sure you also give the Method Code (see listing among
"The Instructions To Toxicity Test Laboratories") appropriate for the exact test conditions you used.
44
-------
METHOD CODES 21 AND 22
MANDATORY TEST CONDITIONS FOR CERIODAPHNIA DUBIA 7-DAY, DAILY RENEWAL,
SURVIVAL AND REPRODUCTION TESTS
1. Test type:
2. Test duration:
3. Temperature:
4. Light quality:
5. Light intensity:
6. Photoperiod:
7. Volume of Stock Solution
8. Test chamber size:
9. Test solution volume:
10. Renewal of test solutions:
11. Age of test organisms:
12. No. organisms/test chamber:
13. No. replicate test
chambers/concentration:
14. No. organisms/concentration:
15. Feeding regime:
16. Test chamber cleaning:
17. Aeration:
18. Dilution water:
19. Test concentrations:
20. Dilution series:
21. Endpoints (expressed as percent
effluent):
22. Test acceptability criteria:
Static, renewal
Until 60% of control females have three
broods (may require more or less than 7 days)
25±1°C
Ambient laboratory illumination
10-20 uE/m2/s (50-100 ft-c)
(Ambient laboratory levels)
16 h light, 8 h darkness
0.5 mL Stock/1 L
30 mL (minimum)
15 mL (minimum)
Daily
<24 h; 8 h maximum range in age
1
10
10
Feed as described in EPA/600/4-89/001
None
None
20% Diluted Mineral Water (DMW) or Moderately-hard synthetic
freshwater (MHSF)(80-100 mg CaCO3, EPA/600/4-89/001)
prepared with MILLIPORE MILLI-QR (or equivalent) deionized
water and reagent grade chemicals or mineral water
5 effluent concentrations and a control
0.5
NOEC-Survival, IC25-Reprod., NOEC-Reprod.
(1) 80% or greater survival in controls
(2) Reproduction of surviving females in control chambers at the
end of the test must average 15 or more young
45
-------
-------
U. S. ENVIRONMENTAL PROTECTION AGENCY
DMR-QA LABORATORY PERFORMANCE EVALUATION Study 18
INSTRUCTIONS FOR THE DAPHNIA MAGNA & DAPHNIA PULEX
48-H ACUTE. NON-RENEWAL. TOXICITY TEST
(METHOD CODES 32 & 38)
CAUTION: Read Instructions Carefully Before Opening The Reference Toxicants.
PREPARATION OF "SIMULATED" EFFLUENT
The reference toxicant, labeled REFERENCE TOXICANT #3, is supplied in solid form in a glass bottle. A
stock solution of REFERENCE TOXICANT #3 is prepared by adding 2.5 grams of the toxicant material to
approximately 90 mL of distilled water. Stir with a mechanical stirrer or by hand shaking until dissolved. Bring the
final volume to 100 mL with distilled water. The stock solution can be used for more than one method code that
uses REFERENCE TOXICANT #3. A fresh stock solution must be prepared each time a test (or set of tests)
using REFERENCE TOXICANT #3 is started or renewed. For each Daphnia magna and Daphnia pulex acute
toxicity test described below, prepare a "simulated" effluent (hereafter referred to as the effluent) from the stock
solution. Prepare the effluent by adding 1.6 mL of the stock solution to 900 mL of Moderately-hard
synthetic freshwater (MHSF)(20°C or 25°C test). Stir with a mechanical stirrer or by hand shaking until
completely mixed. Bring the final volume to 1L with MHSF. MHSF is prepared from MILLIPORE MILLI-QR (or
equivalent) deionized water and reagent grade chemicals, as specified in Tables 6 and 7, p. 35, EPA/600/4-
90/027. If smaller volumes of effluent are prepared, proportionally smaller amounts of REFERENCE TOXICANT
#3 and MHSF would be required.
The effluent prepared according to these instructions represents the sample ready for testing, 100% effluent.
This 100% effluent sample is split into two 0.5 L aliquots. The first aliquot is your 100% effluent for testing. The
second aliquot is diluted with 0.5 L of MHSF and mixed. This becomes your 50% effluent sample. Continue
diluting half of each sample with the same volume of MHSF to make your 25%, 12.5%, and 6.25% effluent
dilutions, which represent all five test dilutions.
ACUTE DEFINITIVE TOXICITY TEST
The 48-h definitive test is performed using the test conditions listed in the following table (MANDATORY
TEST CONDITIONS FOR DAPHNIA MAGNA & DAPHNIA PULEX 48-HOUR ACUTE, NON-RENEWAL,
TOXICITY TESTS).
TEST DATA
Biological and physical/chemical measurements are performed and the data recorded as specified in
EPA/600/4-90/027. Records of these measurements must be maintained on file in the laboratory and made
available to USEPA if requested.
47
-------
U. S. ENVIRONMENTAL PROTECTION AGENCY
DMR-QA LABORATORY PERFORMANCE EVALUATION Study 18
INSTRUCTIONS FOR THE DAPHNIA MAGNA & DAPHNIA PULEX
48-H ACUTE. NON-RENEWAL. TOXICITY TEST
(METHOD CODE 32 & 38)
Biological Data
In the 48-h definitive test, data on organism survival in each replicate chamber for the controls and effluent
concentrations are recorded daily as described on p. 70 and in Figure 4, p. 72, EPA/600/4-90/027.
Physical/Chemical Data
In the 48-h non-renewal, definitive test, dissolved oxygen (DO), pH, total alkalinity, total hardness,
conductivity and temperature are determined at each concentration of effluent used in the dilution series, and in
the control at the beginning of the test, and the DO, pH, conductivity, and temperature are determined at the end
of the test, or when a replicate sample experiences 100% mortality, and recorded as described on pp. 70 and 71,
and in Figure 4, p. 72, EPA/600/4-90/027.
BIOLOGICAL DATA ANALYSIS
Mortality data from the 48-h definitive test are used to determine the LC50, expressed as percent effluent.
Follow the flowchart in Figure 1 in the Statistical Appendix to determine the LC50. For additional information on
data analysis, see the Statistical Appendix at the end of this instruction packet.
'" '". • • ,.'''. i.
REPORTING THE TEST RESULTS
The 48-h LC50 (expressed as percent effluent) is entered on the Report Form located at the end of the
package of instructions. Record all results the same as they are recorded for DMR reporting. NOTE: Do not
report LCSQs for tests which fail to meet the acceptability criteria listed with the test conditions. Be sure
you enter your data into the portion of the Report Form that corresponds to the exact test conditions you used.
Make sure you also give the Method Code (see listing among "The Instructions To Toxicity Test Laboratories")
appropriate for the exact test conditions you used.
48
-------
METHOD CODE 32 & 38
MANDATORY TEST CONDITIONS FOR DAPHNIA MAGNA & DAPHNIA PULEX
48-H ACUTE, NON-RENEWAL, TOXICITY TESTS
1. Test type:
2. Test duration:
3. Temperature:
4. Light quality:
5. Light intensity:
(Ambient laboratory levels)
6. Photoperiod:
7. Volume of Stock Solution
8. Test chamber size:
9. Test solution volume:
10. Renewal of test solutions:
11. Age of test organisms:
12. No, organisms/test chamber:
13. No. replicate test
chambers/concentration:
14. No. organisms/concentration:
15. Feeding regime:
16. Test chamber cleaning:
17. Aeration:
18. Dilution water:
19. Test concentrations:
20. Dilution series:
21. Endpoint (expressed as
percent effluent):
22. Test acceptability criterion:
Static, non-renewal
48 h
20 ± 1°C (D. magna, Method Code 32)
25 ± 1°C (D. pulex, Method Code 38)
Ambient laboratory illumination
10-20 nE/m2/s (50-100 ft-c)
16 h light, 8 h darkness
1.6 mL Stock/1 L
30 mL (minimum)
25 mL (minimum)
None
<24h
5
4
20
Feed while holding prior to test
Cleaning not required
None
Moderately-hard synthetic freshwater (MHSF) (80-100 mg
CaCO3, EPA/600/4-90/027) prepared with MILLIPORE MILLI-Q"
(or equivalent) deionized water and reagent grade chemicals
5 effluent concentrations and a control
0.5
LC50
90% or greater survival in controls
49
-------
-------
U. S. ENVIRONMENTAL PROTECTION AGENCY
DMR-QA LABORATORY PERFORMANCE EVALUATION Study 18
INSTRUCTIONS FOR THE DAPHNIA PULEX
48-H ACUTE. NON-RENEWAL. TOXICITY TEST
(METHOD CODE 36)
CAUTION: Read Instructions Carefully Before Opening The Reference Toxicants.
PREPARATION OF "SIMULATED" EFFLUENT
The reference toxicant, labeled REFERENCE TOXICANT #3, is supplied in solid form in a glass bottle.
A stock solution of REFERENCE TOXICANT #3 is prepared by adding 2.5 grams of the toxicant material to
approximately 90 mL of distilled water. Stir with a mechanical stirrer or by hand shaking until dissolved.
Bring the final volume to 100 mL with distilled water. The stock solution can be used for more than one
method code that uses REFERENCE TOXICANT #3. A fresh stock solution must be prepared each time a
test (or set of tests) using REFERENCE TOXICANT #3 is started or renewed. For each Daphnia pulex
acute toxicity test described below, prepare a "simulated" effluent (hereafter referred to as the effluent)
from the stock solution. Prepare the effluent by adding 3.2 mL of the stock solution to 900 mL of
Moderately-hard synthetic freshwater (MHSF)(20°C test). Stir with a mechanical stirrer or by hand
shaking until completely mixed. Bring the final volume to 1L with MHSF. MHSF is prepared from
MILLIPORE MILLI-QR (or equivalent) deionized water and reagent grade chemicals, as specified in Tables
6 and 7, p. 35, EPA/600/4-90/027. If smaller volumes of effluent are prepared, proportionally smaller
amounts of REFERENCE TOXICANT #3 and MHSF.
The effluent prepared according to these instructions represents the sample ready for testing, 100%
effluent. This 100% effluent sample is split into two 0.5 L aliquots. The first aliquot is your 100% effluent for
testing. The second aliquot is diluted with 0.5 L of MHSF and mixed. This becomes your 50% effluent
sample. Continue diluting half of each sample with the same volume of MHSF to make your 25%, 12.5%,
and 6.25% effluent dilutions, which represent all five test dilutions.
ACUTE DEFINITIVE TOXICITY TEST
The 48-h definitive test is performed using the test conditions listed in the following table (MANDATORY
TEST CONDITIONS FOR DAPHNIA PULEX 48-HOUR ACUTE, NON-RENEWAL, TOXICITY TESTS).
TEST DATA
Biological and physical/chemical measurements are performed and the data recorded as specified in
EPA/600/4-90/027. Records of these measurements must be maintained on file in the laboratory and
made available to USEPA if requested.
51
-------
U. S. ENVIRONMENTAL PROTECTION AGENCY
DMR-QA LABORATORY PERFORMANCE EVALUATION Study 18
INSTRUCTIONS FOR THE DAPHNIA PULEX
48-H ACUTE. NON-RENEWAL. TOXICITY TEST
(METHOD CODE 36)
Biological Data
In the 48-h definitive test, data on organism survival in each replicate chamber for the controls and
effluent concentrations are recorded daily as described on p. 70 and in Figure 4, p. 72, EPA/600/4-90/027.
Physical/Chemical Data
In the 48-h non-renewal, definitive test, dissolved oxygen (DO), pH, total alkalinity, total hardness,
conductivity, and temperature are determined at each concentration of effluent used in the dilution series,
and in the control, at the beginning of the test, and the DO, pH, conductivity, and temperature are
determined at the end of the test, or when a replicate sample experiences 100% mortality, and recorded as
described on pp. 70 and 71, and in Figure 4, p. 72, EPA/600/4-90/027.
BIOLOGICAL DATA ANALYSIS
Mortality data from the 48-h definitive test are used to determine the LC50, expressed as percent
effluent. Follow the flowchart in Figure 1 in the Statistical Appendix to determine the LC50. For additional
information on data analysis, see the Statistical Appendix at the end of this instruction packet.
REPORTING THE TEST RESULTS
The 48-h LC50 (expressed as percent effluent) is entered on the Report Form located at the end of the
package of instructions. Record all results the same as they are recorded for DMR reporting. NOTE: Do
not report LCSOs for tests which fail to meet the acceptability criteria listed with the test conditions.
Be sure you enter your data into the portion of the Report Form that corresponds to the exact test
conditions you used. Make sure you also give the Method Code (see listing among "The Instructions To
Toxicity Test Laboratories") appropriate for the exact test conditions you used.
52
-------
METHOD CODE 36
MANDATORY TEST CONDITIONS FOR DAPHNIA PULEX
48-H ACUTE, NON-RENEWAL, TOXICITY TESTS
1. Test type:
2. Test duration:
3. Temperature:
4. Light quality:
5. Light intensity:
(Ambient laboratory levels)
6. Photoperiod:
7. Volume of Stock Solution
8. Test chamber size:
9. Test solution volume:
10. Renewal of test solutions:
11. Age of test organisms:
12. No. organisms/test chamber:
13. No. replicate test
chambers/concentration:
14. No. organisms/concentration:
15. Feeding regime:
16. Test chamber cleaning:
17. Aeration:
18. Dilution water:
19. Test concentrations:
20. Dilution series:
21. Endpoint (expressed as
percent effluent):
22. Test acceptability criterion:
Static, non-renewal
48 h
20±1°C
Ambient laboratory illumination
10-20 uE/m2/s (50-100 ft-c)
16 h light, 8 h darkness
3.2 mL Stock/1 L
30 mL (minimum)
25 mL (minimum)
none
<24h
5
4
20
Feed while holding prior to test
Cleaning not required
None
Moderately-hard synthetic freshwater (MHSF) (80-100 mg
CaC03, (EPA/600/4-90/027) prepared with MILLIPORE MILLI-QR
(or equivalent) deionized water and reagent grade chemicals
5 effluent concentrations and a control
0.5
LC50
90% or greater survival in controls
53
-------
-------
U. S. ENVIRONMENTAL PROTECTION AGENCY
DMR-QA LABORATORY PERFORMANCE EVALUATION Study 18
INSTRUCTIONS FOR THE MYSID IMYSIDOPSIS BAHIA)
48-H ACUTE. NON-RENEWAL. TOXICITY TEST
(METHOD CODE 42)
CAUTION: Read Instructions Carefully Before Opening The Reference Toxicants.
PREPARATION OF "SIMULATED" EFFLUENT
The reference toxicant, labeled REFERENCE TOXICANT #3, is supplied in solid form in a glass bottle.
A stock solution of REFERENCE TOXICANT #3 is prepared by adding 2.5 grams of the toxicant material to
approximately 90 mL of distilled water. Stir with a mechanical stirrer or by hand shaking until dissolved.
Bring the final volume to 100 mL with distilled water. The stock solution can be used for more than one
method code that uses REFERENCE TOXICANT #3. A fresh stock solution must be prepared each time a
test (or set of tests) using REFERENCE TOXICANT #3 is started or renewed. For each mysid
(mysidopsis bahia) acute toxicity test described below, prepare a "simulated" effluent (hereafter
referred to as the effluent) by adding 3.2 mL of stock solution to approximately 900 mL of 40
fathoms artificial seawater, with a salinity of 25 ppt, used as dilution water. Stir with a mechanical
stirrer or by hand shaking until dissolved. Stir with a mechanical stirrer or by hand shaking until dissolved.
Bring the final volume to 1000 mL with 40 fathoms artificial seawater. 40 fathoms artificial seawater, with a
salinity of 25 ppt, is prepared from MILLIPORE MILLI-QR (or equivalent) deionized water and 40 fathoms
artificial sea salts, as specified in the text preceding the test instructions. Smaller volumes of effluent can
be prepared by using proportionally smaller volumes of the REFERENCE TOXICANT #3 stock solution and
40 fathoms artificial seawater.
The effluent prepared according to these instructions represents the sample ready for testing, 100%
effluent. This 100% effluent sample is split into two 0.5 L aliquots. The first aliquot is your 100% effluent for
testing. The second aliquot is diluted with 0.5 L of 40 fathoms artificial seawater and mixed. This becomes
your 50% effluent sample. Continue diluting half of each sample with the same volume of artificial
seawater to make your 25%, 12.5%, and 6.25% effluent dilutions, which represent all five test dilutions.
ACUTE DEFINITIVE TOXICITY TEST
The 48-h definitive test is performed using the test conditions listed in the following table
(MANDATORY TEST CONDITIONS FOR MYSID (MYSIDOPSIS BAHIA) 48-H ACUTE, NON-RENEWAL,
TOXICITY TESTS).
TEST DATA
Biological and physical/chemical measurements are performed and the data recorded as specified in
EPA/600/4-90/027. Records of these measurements must be maintained on file in the laboratory and
made available to USEPA if requested.
55
-------
U. S. ENVIRONMENTAL PROTECTION AGENCY
DMR-QA LABORATORY PERFORMANCE EVALUATION Study 18
INSTRUCTIONS FOR THE MYSlD (MYSlDOPSIS BAHIA}
48-H ACUTE. NON-RENEWAL. TOXICITY TEST
(METHOD CODE 42)
Biological Data
In the 48-h definitive test, data on test organism survival in each replicate chamber for the controls and
effluent concentrations are recorded daily as described on p. 70, and in Figure 4, p. 72, EPA/600/4-90/027.
Physical/Chemical Data
In the 48-h definitive test, dissolved oxygen (DO), pH, salinity, and temperature are determined at each
concentration of effluent used in the dilution series, and in the control, at the beginning of the test, and DO,
pH, salinity, and temperature are determined at the end of the test, or when a replicate sample experiences
100% mortality, and recorded as described pp. 70 and 71, and in Figure 4, p. 72, EPA/600/4-90/027.
BIOLOGICAL DATA ANALYSIS
Mortality data from the 48-h definitive test are used to determine the LC50, expressed as percent
effluent. Follow the flowchart in Figure 1 in the Statistical Appendix to determine the LC50. For additional
information on data analysis, see the Statistical Appendix at the end of this instruction packet.
REPORTING THE TEST RESULTS
The 48-h LC50, expressed as percent effluent, is reported on the Report Form located at the end of
the package of instructions. Record all results the same as they are recorded for DMR reporting. Note:
Do not report LCSOs for tests which fail to meet the acceptability criteria listed with the test
conditions. Be sure you enter your data into the portion of the Report Form that corresponds to the exact
test conditions you used. Make sure you also give the Method Code (see listing among "The Instructions
To Toxicity Test Laboratories") appropriate for the exact test conditions you used.
56
-------
METHOD CODE 42
MANDATORY TEST CONDITIONS FOR MYSID (MYSIDOPSIS BAHIA)
48-H ACUTE, NON-RENEWAL, TOXICITY TESTS
1. Test type:
2. Test duration:
3. Temperature:
4. Light quality:
5. Light intensity:
6. Photoperiod:
7. Amount of Material to Test
8. Test chamber size:
9. Test solution volume:
10. Renewal of test solutions:
11. Age of test organisms:
12. No. organisms/test chamber:
13. No. replicate test
chambers/concentration:
14. No. organisms/concentration:
15. Feeding regime:
16. Test chamber cleaning:
17. Aeration:
18. Dilution water:
19. Test concentrations:
20. Dilution series:
21. Endpoint (expressed as percent
effluent):
22. Test acceptability criterion:
Static, non-renewal
48 h
20±1°C
Ambient laboratory illumination
10-20 uE/m2/s (50-100 ft-c)
(Ambient laboratory levels)
16 h light, 8 h darkness
3.2 mL Stock/1 L
250 mL (minimum)
200 mL (minimum)
None
1-5 days; 24 h maximum range in age
10
2
20
0.2 mL of concentrated brine shrimp nauplii
suspension once daily (approximately 100
nauplii per mysid)
Cleaning not required
None
40 fathoms artificial seawater, with a salinity of 25 ppt, prepared
with MILLIPORE MILLI-QR (or equivalent) deionized water
5 effluent concentrations and a control
0.5
LC50
90% or greater survival in controls
57
-------
-------
U.S. ENVIRONMENTAL PROTECTION AGENCY
DMR-QA LABORATORY PERFORMANCE EVALUATION Study 18
INSTRUCTIONS FOR THE MYS1DOPSIS BAHIA 7-DAY. DAILY RENEWAL.
SURVIVAL AND GROWTH TEST
(METHOD CODE 43)
CAUTION: Read Instructions Carefully Before Opening The Reference Toxicants.
PREPARATION OF "SIMULATED" EFFLUENT
The reference toxicant, labeled REFERENCE TOXICANT #2, is supplied in solid form in a plastic
bottle. For each M, bahia 7-day survival and growth toxicity test described below, each day prepare
a fresh solution of "simulated" effluent (hereafter referred to as the effluent) by adding 2.0 grams of
REFERENCE TOXICANT #2 to approximately 3.9 L of 40 fathoms artificial seawater, with a salinity of
25 ppt, used as dilution water. Stir with a mechanical stirrer or by hand shaking until dissolved. Bring the
final volume to 4 L with 40 fathoms artificial seawater. 40 fathoms artificial seawater, with a salinity of 25
ppt, is prepared from MILLIPORE MILLI-QR (or equivalent) deionized water and 40 fathoms artificial sea
salts, as specified in the text preceding the test instructions. Smaller volumes of effluent can be prepared
by using proportionally smaller volumes of REFERENCE TOXICANT #2 and 40 fathoms artificial seawater.
The effluent prepared according to these instructions represents the sample ready for testing, 100%
effluent. This 100% effluent sample is split into two 2 L aliquots. The first aliquot is your 100% effluent for
testing. The second aliquot is diluted with 2 L of 40 fathoms artificial seawater and mixed. This becomes
your 50% effluent sample. Continue diluting half of each sample with the same volume of artificial
seawater to make your 25%, 12.5%, and 6.25% effluent dilutions, which represent all five test dilutions.
7-DAY. SHORT-TERM. CHRONIC TOXICITY TEST
The 7-day survival and growth test is performed using the test conditions listed in the following table
(MANDATORY TEST CONDITIONS FOR MYSIDOPSIS BAHIA 7-DAY, DAILY RENEWAL, SURVIVAL
AND GROWTH TESTS).
TEST DATA
Daily biological* and physical/chemical measurements are performed and the data recorded as
specified in EPA/600/4-87/028. Records of these measurements must be maintained on file in the
laboratory and made available to USEPA if requested.
The fecundity portion of this test is not part of the DMR-QA Study 18.
59
-------
U.S. ENVIRONMENTAL PROTECTION AGENCY
DMR-QA LABORATORY PERFORMANCE EVALUATION Study 1 8
INSTRUCTIONS FOR THE MYSIDOPSIS BAHIA 7-DAY. DAILY RENEWAL.
SURVIVAL AND GROWTH TEST
(METHOD CODE 43)
Biological Data
Data on effluent concentrations and test organism survival are recorded daily for each test chamber as
described on p. 187 and in Figures 15, pp. 235, EPA/600/4-87/028. Growth (mean dry weight per surviving
organism) is determined at the end of the test as described on pp. 187 and I92, and recorded as described
in Figures 15 and 16, pp. 236-237 EPA/600/4-87/028.
1 ill ! .
..»! ' , ' . ' ..... :
Physical/Chemical Data
Dissolved oxygen (DO), pH, salinity, and temperature are determined as described on p. 186, and
recorded as described in Figure 14, p. 234, EPA/600/4-87/028.
BIOLOGICAL DATA ANALYSIS
The endpoints (expressed as percent effluent) determined from the test data are as follows:
(1) NOEC for survival - see pp. 192-203, EPA/600/4-87/028.
(2) IC25 for growth - see EPA/600/4-89/001 A (Suppl., Sept. I989).
(3) NOEC for growth -see pp. 206-215, EPA/600/4-87/028.
The 1C25 for growth can be determined manually using the guidelines in EPA/600/4-89/001 A. For
additional information on data analysis, see the Statistical Appendix at the end of this instruction packet.
REPORTING THE TEST RESULTS
The NOEC for survival, the IC25 and NOEC for growth, are expressed as percent effluent, are entered
on the Report Form located at the end of the package of instructions. Record all results the same as they
are recorded for DMR reporting. NOTE: Do not report endpoints for tests which fail to meet the
acceptability criteria listed with the test conditions. Be sure you enter your data into the portion of the
Report Form that corresponds to the exact test conditions you used. Make sure you also give the Method
Code (see listing among "The Instructions To Toxicity Test Laboratories") appropriate for the exact test
conditions you used.
60
-------
METHOD CODE 43
MANDATORY TEST CONDITIONS FOR MYS1DOPSIS BAHIA 7-DAY, DAILY RENEWAL,
SURVIVAL AND GROWTH TESTS
1. Test type:
2. Test duration:
3. Temperature:
4. Light quality:
5. Light intensity:
6. Photoperiod:
7. Amount of Material to Test
8. Test chamber size:
9. Test solution volume:
10. Renewal of test solutions:
11. Age of test organisms:
12. No. organisms/test chamber:
13. No. replicate test
chambers/concentration:
14. No. organisms/concentration:
15. Feeding regime:
16. Test chamber cleaning:
17. Aeration:
18. Dilution water:
19. Test concentrations:
20. Dilution series:
21. Endpoints (expressed as percent
effluent):
22. Test acceptability criteria:
Static, renewal
7 days
26-27°C
Ambient laboratory illumination
10-20 |JE/m2/s (50-100 ft-c)
(Ambient laboratory levels)
16 h light, 8 h darkness
2.0 gm Toxicant /4L
250 mL (minimum)
150 mL (minimum)
Daily
7 days; 24 h maximum range in age
5
8
40
Feed as described in EPA/600/4-87/028
Pipette excess food from cups daily
None
40 fathoms artificial seawater, with a salinity of 25 ppt, prepared
with MILLIPORE MILLI-Q" (or equivalent) deionized water
5 effluent concentrations and a control
0.5
NOEC - Survival
IC25 - Growth
NOEC - Growth
(1) 80% or greater survival in controls
(2) Average dry weight of surviving mysids in control chambers is
at least 0.20 mg
61
-------
-------
U. S. ENVIRONMENTAL PROTECTION AGENCY
DMR-QA LABORATORY PERFORMANCE EVALUATION Study 18
INSTRUCTIONS FOR THE INLAND SILVERSIDE IMENIDIA BERYLLINA)
48-H ACUTE. NON-RENEWAL. TOXICITY TEST
(METHOD CODE 44)
CAUTION: Read Instructions Carefully Before Opening The Reference Toxicants.
PREPARATION OF "SIMULATED" EFFLUENT
The reference toxicant, labeled REFERENCE TOXICANT #3, is supplied in solid form in a glass bottle.
A stock solution of REFERENCE TOXICANT #3 is prepared by adding 2.5 grams of the toxicant material to
approximately 90 mL of distilled water. Stir with a mechanical stirrer or by hand shaking until dissolved.
Bring the final volume to 100 mL with distilled water. The stock solution can be used for more than one
method code that uses REFERENCE TOXICANT #3. A fresh stock solution must be prepared each time a
test (or set of tests) using REFERENCE TOXICANT #3 is started or renewed. For each Menidia beryllina
acute toxicity test described below, prepare a "simulated" effluent (hereafter referred to as the
effluent) by adding 2.4 mL of stock solution to approximately 900 mL of 40 fathoms artificial
seawater, with a salinity of 25 ppt, used as dilution water. Stir with a mechanical stirrer or by hand
shaking until dissolved. Bring the final volume to 1 L with 40 fathoms artificial seawater. 40 fathoms
artificial seawater, with a salinity of 25 ppt, is prepared from MILLIPORE MILLI-QR (or equivalent) deionized
water and 40 fathoms artificial sea salts, as specified in the text preceding the test instructions. Smaller
volumes of effluent can be prepared by using proportionally smaller volumes of the REFERENCE
TOXICANT #3 stock solution and 40 fathoms artificial seawater.
The effluent prepared according to these instructions represents the sample ready for testing, 100%
effluent. This 100% effluent sample is split into two 0.5 L aliquots. The first aliquot is your 100% effluent for
testing. The second aliquot is diluted with 0.5 L of 40 fathoms artificial sea water and mixed. This becomes
your 50% effluent sample. Continue diluting half of each sample with the same volume of artificial
seawater to make your 25%, 12.5%, and 6.25% effluent dilutions, which represent all five test dilutions.
ACUTE DEFINITIVE TOXICITY TEST
The 48-h definitive test is performed using the test conditions listed in the following table
(MANDATORY TEST CONDITIONS FOR INLAND SILVERSIDE (MENIDIA BERYLLINA) 48-H ACUTE,
NON-RENEWAL, TOXICITY TESTS).
LABORATORY TEST DATA
Biological and physical/chemical measurements are performed and the data recorded as specified in
EPA/600/4-90/027. Records of these measurements must be maintained on file in the laboratory and
made available to USEPA if requested.
63
-------
U. S. ENVIRONMENTAL PROTECTION AGENCY
DMR-QA LABORATORY PERFORMANCE EVALUATION Study 18
INSTRUCTIONS FOR THE INLAND SILVERSIDE (MENIDIA BERYLLINA)
48-H ACUTE. NON-RENEWAL. TOXICITY TEST
(METHOD CODE 44)
Biological Data
In the 48-h definitive test, data on test organism survival in each replicate chamber for the controls and
effluent concentrations are recorded daily as described on p. 70, and in Figure 4, p. 72, EPA/600/4-90/027.
Physical/Chemical Data
In the 48-h definitive test, dissolved oxygen (DO), pH, salinity, and temperature are determined at each
concentration of effluent used in the dilution series, and in the control, at the beginning of the test, and DO,
pH, salinity, and temperature are determined at the end of the test, or when a replicate sample experiences
100% mortality, and recorded as described pp. 70 and 71, and in Figure 4, p. 72, EPA/600/4-90/027.
BIOLOGICAL DATA ANALYSIS
Mortality data from the 48-h definitive test are used to determine the LC50, expressed as percent
effluent. Follow the flowchart in Figure 1 in the Statistical Appendix to determine the LC50. For additional
Information on data analysis, see the Statistical Appendix at the end of this instruction packet.
REPORTING THE TEST RESULTS
The 48-h LC50, expressed as percent effluent, is entered on the Report Form located at the end of the
instructions. Record all results the same as they are recorded for DMR reporting. Note: Do not report
LCSOs for tests that fail to meet the acceptability criteria listed with the test conditions. Be sure you
enter your data into the portion of the Report Form that corresponds to the exact test conditions you used.
Make sure you also give the Method Code (see listing among "The Instructions To Toxicity Test
Laboratories") appropriate for the exact test conditions you used.
64
-------
METHOD CODE 44
MANDATORY TEST CONDITIONS FOR INLAND SILVERSIDE (MENIDIA BERYLLINA)
48-H ACUTE, NON-RENEWAL, TOXICITY TESTS
1. Test type:
2. Test duration:
3. Temperature:
4. Light quality:
5. Light intensity:
(Ambient laboratory levels)
6. Photoperiod:
7. Amount of Material to Test
8. Test chamber size:
9. Test solution volume:
10. Renewal of test solutions:
11. Age of test organisms:
12. No. organisms/test chamber:
13. No. replicate test
chambers/concentration:
14. No. organisms/concentration:
15. Feeding regime:
16. Test chamber cleaning:
17. Aeration:
18. Dilution water:
19. Test concentrations:
20. Dilution series:
21. Endpoint (expressed as percent
effluent):
22. Test acceptability criterion:
Static, non-renewal
48 h
20 + 1°C
Ambient laboratory illumination
10-20 uE/m2/s (50-100 ft-c)
16 h light, 8 h darkness
2.4 mL Stock/1 L
250 mL (minimum)
200 mL (minimum)
None
9-14 days; 24 h maximum range in age
10
2
20
Feed while holding before test
Cleaning not required
None
40 fathoms artificial seawater, with a salinity of 25 ppt prepared
with MILLIPORE MILLI-QR (or equivalent) deionized water
5 effluent concentrations and a control
0.5
LC50
90% or greater survival in controls
65
-------
-------
U. S. ENVIRONMENTAL PROTECTION AGENCY
DMR-QA LABORATORY PERFORMANCE EVALUATION Study 18
INSTRUCTIONS FOR THE SHEEPSHEAD MINNOW (CYPRINODON VARIEGATUS]
48-H ACUTE. NON-RENEWAL. TOXICITY TEST
(METHOD CODE 46)
CAUTION: Read Instructions Carefully Before Opening The Reference Toxicants.
PREPARATION OF "SIMULATED" EFFLUENT
The reference toxicant, labeled REFERENCE TOXICANT #3, is supplied in solid form in a glass bottle.
A stock solution of REFERENCE TOXICANT #3 is prepared by adding 2.5 grams of the toxicant material to
approximately 90 mL of distilled water. Stir with a mechanical stirrer or by hand shaking until dissolved.
Bring the final volume to 100 mL with distilled water. The stock solution can be used for more than one
method code that uses REFERENCE TOXICANT #3. A fresh stock solution must be prepared each time a
test (or set of tests) using REFERENCE TOXICANT #3 is started or renewed. For each C. variegatus
acute toxicity test described below, prepare a "simulated" effluent (hereafter referred to as the
effluent) by adding 32 mL of the stock solution to approximately 900 mL of 40 fathoms artificial
seawater, with a salinity of 25 ppt, used as dilution water. Stir with a mechanical stirrer or by hand
shaking until dissolved. Bring the final volume to 1 L with 40 fathoms artificial seawater. 40 fathoms
artificial seawater, with a salinity of 25 ppt, is prepared from MILLIPORE MILLI-Q" (or equivalent) deionized
water and 40 fathoms artificial sea salts, as specified in the text preceding the test instructions. Smaller
volumes of effluent can be prepared by using proportionally smaller amounts of the REFERENCE
TOXICANT #3 stock solution and 40 fathoms artificial seawater.
The effluent prepared according to these instructions represents the sample ready for testing, 100%
effluent. This 100% effluent sample is split into two 0.5 L aliquots. The first aliquot is your 100% effluent for
testing. The second aliquot is diluted with 0.5 L of 40 fathoms artificial seawater and mixed. This becomes
your 50% effluent sample. Continue diluting half of each sample with the same volume of artificial
seawater to make your 25%, 12.5%, and 6.25% effluent dilutions, which represent all five test dilutions.
ACUTE DEFINITIVE TOXICITY TEST
The 48-h definitive test is performed using the test conditions listed in the following table
(MANDATORY TEST CONDITIONS FOR SHEEPSHEAD MINNOW (CYPRINODON VARIEGATUS) 48-H
ACUTE, NON-RENEWAL, TOXICITY TESTS).
LABORATORY TEST DATA
Biological and physical/chemical measurements are performed and the data recorded as specified in
EPA/600/4-90/027. Records of these measurements must be maintained on file in the laboratory and
made available to USEPA if requested.
67
-------
U. S. ENVIRONMENTAL PROTECTION AGENCY
DRJIR-QA LABORATORY PERFORMANCE EVALUATION Study 18
INSTRUCTIONS FOR THE SHEEPSHEAD MINNOW (CYPRINODON VARIEGATUS]
48-H ACUTE. NON-RENEWAL TOXICITY TEST
(METHOD CODE 46)
Biological Data
In the 48-h definitive test, data on test organism survival in each replicate chamber for the controls and
effluent concentrations are recorded daily as described on p. 70, and in Figure 4, p. 72, EPA/600/4-90/027.
Physical/Chemical Data
In the 48-h definitive test, dissolved oxygen (DO), pH, salinity, and temperature are determined at each
concentration of effluent used in the dilution series, and in the control, at the beginning of the test, and DO,
pH, salinity, and temperature are determined at the end of the test, or when a replicate sample experiences
100% mortality, and recorded as described pp. 70 and 71, and in Figure 4, p. 72, EPA/600/4-90/027.
BIOLOGICAL DATA ANALYSIS
Mortality data from the 48-h definitive test are used to determine the LC50, expressed as percent
effluent. Follow the flowchart in Figure 1 in the Statistical Appendix to determine the LC50. For additional
information on data analysis, see the Statistical Appendix at the end of this instruction packet.
REPORTING THE TEST RESULTS
The 48-h LC50, expressed as percent effluent, is entered on the Report Form located at the end of the
Instructions. Record all results the same as they are recorded for DMR reporting. Note: Do not report
LCSQs for tests that fail to meet the acceptability criteria listed with the test conditions. Be sure you
enter your data into the portion of the Report Form that corresponds to the exact test conditions you used.
Make sure you also give the Method Code (see listing among "The Instructions To Toxicity Test
Laboratories") appropriate for the exact test conditions you used.
68
-------
METHOD CODE 46
MANDATORY TEST CONDITIONS FOR SHEEPSHEAD MINNOW (CYPRINODON VARIEGATUS)
48-H ACUTE, NON-RENEWAL, TOXICITY TESTS
1. Test type:
2. Test duration:
3. Temperature:
4. Light quality:
5. Light intensity:
6. Photoperiod:
7. Amount of Material to Test
8. Test chamber size:
9. Test solution volume:
10. Renewal of test solutions:
11. Age of test organisms:
12. No. organisms/test chamber:
13. No. replicate test
chambers/concentration:
14. No. organisms/concentration:
15. Feeding regime:
16. Test chamber cleaning:
17. Aeration:
18. Dilution water:
19. Test concentrations:
20. Dilution series:
21. Endpoint (expressed as percent
effluent):
22. Test acceptability criterion:
Static, non-renewal
48 h
20±1°C
Ambient laboratory
illumination
10-20 uE/m2/s (50-100 ft-c)
(Ambient laboratory levels)
16 h light, 8 h darkness
32 mL Stock/1 L
250 mL (Minimum)
200 mL (Minimum)
None
1-14 days, 24 h-maximum range in age
10
2
20
Feed while holding before test
Cleaning not required
None
40 fathoms artificial seawater, with a salinity of 25 ppt salinity,
prepared with MILLIPORE MILLI-Q" (or equivalent) deionized
water
5 effluent concentrations and a control
0.5
LC50
90% or greater survival in controls
69
-------
-------
U.S. ENVIRONMENTAL PROTECTION AGENCY
DMR-QA LABORATORY PERFORMANCE EVALUATION Study 18
INSTRUCTIONS FOR THE SHEEPSHEAD MINNOW (CYPRINODON VARIEGATUS)
7-DAY. DAILY RENEWAL. SURVIVAL AND GROWTH TEST
(METHOD CODE 47)
CAUTION: Read Instructions Carefully Before Opening The Reference Toxicants.
PREPARATION OF "SIMULATED" EFFLUENT
The reference toxicant, labeled REFERENCE TOXICANT #2, is supplied in solid form in a plastic
bottle. For each C. variegatus 7-day survival and growth test described below, each day prepare a
fresh solution of "simulated" effluent (hereafter referred to as the effluent) by adding 9.00 grams of
REFERENCE TOXICANT #2 to approximately 2.9 L of 40 fathoms artificial seawater, with a salinity
of 25 ppt, as dilution water. Stir with a mechanical stirrer or by hand shaking until well mixed. Bring the
final volume to 3 L with 40 fathoms artificial seawater. 40 fathoms artificial seawater with a salinity of 25 ppt
is prepared from MILLIPORE MILLI-Q" (or equivalent) deionized water and 40 fathoms artificial sea salts,
as specified in the text preceding the test instructions. Smaller volumes of effluent can be prepared by
using proportionally smaller amounts of REFERENCE TOXICANT #2 and 40 fathoms artificial seawater.
The effluent prepared according to these instructions represents the sample ready for testing, 100%.
This 100% effluent sample is split into two 1.5 L aliquots. The first aliquot is your 100% effluent for testing.
The second aliquot is diluted with 1.5 L of 40 fathoms artificial seawater and mixed. This becomes your
50% effluent sample. Continue diluting half of each sample with the same volume of artificial seawater to
make your 25%, 12.5%, and 6.25% effluent dilutions, which represent all five test dilutions.
7-DAY. SHORT-TERM. CHRONIC TOXICITY TEST
The 7-day survival and growth test is performed using the test conditions listed in the following table
(MANDATORY TEST CONDITIONS FOR SHEEPSHEAD MINNOW (CYPRINODON VARIEGATUS) 7-
DAY, DAILY RENEWAL, SURVIVAL AND GROWTH TESTS).
TEST DATA
Daily biological and physical/chemical measurements are performed and the data recorded as
specified in EPA/600/4-87/028. Records of these measurements must be maintained on file in the
laboratory and made available to USEPA if requested.
71
-------
U.S. ENVIRONMENTAL PROTECTION AGENCY
DMR-QA LABORATORY PERFORMANCE EVALUATION Study 18
INSTRUCTIONS FOR THE SHEEPSHEAD MINNOW (CYPRINODON VARIEGATUS)
7-DAY. DAILY RENEWAL. SURVIVAL AND GROWTH TEST
(METHOD CODE 47)
Biological Data
Data on test organism survival are recorded daily for each test chamber as described on p. 138, and in
Figures 7 and 9, pp. 167 and 170, EPA/600/4-87/028. Growth (mean dry weight per surviving organism) is
determined at the end of the test and recorded as described on p. 138 and in Figures 7-9, pp. 167-170,
EPA/600/4-87/028.
Physical/Chemical Data
Dissolved oxygen (DO), pH, salinity, and temperature are measured as described on pp. 137-138,
EPA/600/4-87/028, and recorded as described in Figures 7 and 9, pp. 167-168 and 170, EPA/600/4-
87/028.
BIOLOGICAL DATA ANALYSIS
The endpoints (expressed as percent effluent) determined from the test data are as follows:
(1) NOEC for survival - see pp. 139-151, EPA/600/4-87/028.
(2) IC25 for growth - see EPA/600/4-89/001 A (Suppl., Sept. I989).
(3) NOEC for growth - see pp. 155-164, EPA/600/4-87/028.
The IC25 for growth can be determined manually using the guidelines in EPA/600/4-89/001 A. For
additional information on data analysis, see the Statistical Appendix at the end of this instruction packet.
REPORTING THE RESULTS
The NOEC for survival, and the IC25 and NOEC for growth, expressed as percent effluent, are entered
on the Report Form located at the end of the instruction package. Record all results the same as they are
recorded for DMR reporting. NOTE: Do not report endpoints for tests which fail to meet the
acceptability criteria listed with the test conditions. Be sure you enter your data into the portion of the
Report Form that corresponds to the exact test conditions you used. Make sure you also give the Method
Code (see listing among "The Instructions To Toxicity Test Laboratories") appropriate for the exact test
conditions you used.
72
-------
METHOD CODE 47
MANDATORY TEST CONDITIONS FOR SHEEPSHEAD MINNOW (CYPRINODON VARIEGATUS)
7-DAY, DAILY RENEWAL, SURVIVAL AND GROWTH TESTS
1. Test type:
2. Test duration:
3. Temperature:
4. Light quality:
5. Light intensity(Ambient levels):
6. Photoperiod:
7. Amount of Material to Test
8. Test chamber size:
9. Test solution volume:
10. Renewal of test solutions:
11. Age of test organisms:
12. No. organisms/test chamber:
13. No. replicate test
chambers/concentration:
14. No. organisms/concentration:
15. Feeding regime:
16. Test chamber cleaning:
17. Aeration:
18. Dilution Water:
19. Test concentrations:
20. Dilution series:
21. Endpoints (expressed as percent
effluent):
22. Test acceptability criteria:
Static, renewal
7 days
25 ± 1°C
Ambient laboratory illumination
10-20 uE/m2/s (50-100 ft-c)
14 h light, 10 h darkness (May use 16/8)
9.0 gm Toxicant/3L
300 mL (minimum)
250 mL (minimum)
Daily
< 24 h old
15 (minimum of 10)
4 (minimum of 3)
60 (minimum of 30)
Feed as described in EPA/600/4-87/028
Pipette excess food from chambers daily
None
40 fathoms artificial seawater, with a salinity of 25 ppt, prepared
with MILLiPORE MILLI-Q" (or equivalent) deionized water
5 effluent concentrations and a control
0.5
NOEC - Survival, IC25 - Growth, NOEC - Growth
(1) 80% or greater survival in controls
(2) The average dry weight of surviving organisms in control
chambers at the end of the test should be > 0.60 mg if
preserved in formalin, and > 0.50 mg if preserved in ethanol
73
-------
-------
STATISTICAL APPENDIX
STATISTICAL PROGRAMS
A disk containing USEPA computer programs to perform many of the statistical procedures outlined in this
appendix is available from NERL-Cincinnati. To obtain a copy of the program disk, contact Dr. James
Lazorchak, NERL-Cincinnati, at (513) 569-7076 (voice), (513) 569-7078 (fax) or
lazorchak.jim@epamail.epa.gov (E-mail).
The disk contains the following programs:
PROBIT calculates test LC50 and 95% confidence interval using the Probit Method.
TSK calculates test LC50 and 95% confidence interval using the Spearman-Karber or the Trimmed
Spearman-Karber Method.
DUNNETT determines test NOEC and LOEC using Dunnett's Procedure or the t test with the Bonferroni
adjustment. This program also performs Bartlett's test for homogeneity of variance.
ICPIIM calculates test IC25 and 95% confidence interval using the Linear Interpolation and Bootstrap
methods.
CALCULATION OF ACUTE TEST LCSOs
GENERAL COMMENTS
Mortality data from acute definitive tests are used to determine the LC50, expressed as percent effluent.
The recommended statistical analysis of survival data from acute toxicity tests with aquatic organisms
follows a decision process illustrated in the flowchart in Figure 1. The methods used to calculate the
LC50s are explained below. For detailed explanations of these methods, including numerical examples,
refer to pages 75-91, EPA/600/4-90/027F.
PROBIT METHOD
The Probit Method a parametric statistical procedure for estimating the LC50 and the associated 95%
confidence interval. The procedure consists of transforming the observed proportion mortalities with a
probit transformation and transforming the effluent concentrations to Iog10. Given the assumption of
normality for the Iog10 of the tolerances, the relationship between the transformed variables is
approximately linear. This relationship allows estimation of linear regression parameters using an iterative
approach. These parameters are used to calculate the LC50 and the associated 95% confidence interval.
Abbott's procedure is used to adjust the concentration response proportions for mortality occurring in the
control replicates. The Probit Method does not require that the response proportions be monotonically
non-decreasing with increasing concentration (constant or steadily increasing with concentration) so no
smoothing is necessary.
To calculate a reasonably precise estimate of the LC50 using the Probit Method, the adjusted, observed
proportion mortalities must bracket 0.5. It is assumed that the Iog10 of the tolerances is normally
distributed. At least two partial mortalities are needed to calculate confidence limits. To calculate the
LC50 using the Probit Method, run the USEPA PROBIT program.
75
-------
STATISTICAL APPENDIX (CONTINUED)
PROBIT METHOD (CONTINUED)
The appropriateness of the Probit model is checked with a chi-square test. If this test is significant, the
Probit Method should not be used to calculate the LC50. For a numerical example of the Probit Method,
see pages 85-91, EPA/600/4-90/027F.
SPEARMAN-KARBER METHOD
The Spearman-Karber Method is a nonparametric statistical procedure for estimating the LC50 and the
associated 95% confidence interval. The procedure estimates the mean of the distribution of the Iog10 of
the tolerance. If the Iog10 tolerance distribution is symmetric, this estimate of the mean is equivalent to an
estimate of the median of the Iog10 tolerance distribution.
If the response proportions are not monotonically non-decreasing with increasing concentration (constant
or steadily increasing with concentration), the data must be smoothed. Abbott's procedure is used to
adjust the concentration response proportions for mortality occurring in the control replicates.
To calculate the LC50 using the Spearman-Karber Method, the smoothed, adjusted, observed proportion
mortalities must bracket 0.5. The USEPA TSK program may be used to calculate LCSOs with the
Spearman-Karber Method. For instructions on smoothing response proportions and performing Abbott's
adjustment, see section 11.2.2.3, page 78, EPA/600/4-90/027F. For a numerical example of the
Spearman-Karber Method, see pages 81-83, EPA/600/4-90/027F.
TRIMMED SPEARMAN-KARBER METHOD
The Trimmed Spearman-Karber Method is a modification of the Spearman-Karber Method, a
nonparametric statistical procedure for estimating the LC50 and the associated 95% confidence interval.
The Trimmed Spearman-Karber Method estimates the trimmed mean of the distribution of the Iog10 of the
tolerance. If the Iog10 tolerance distribution is symmetric, this estimate of the trimmed
mean is equivalent to an estimate of the median of the Iog10 tolerance distribution.
If the response proportions are not monotonically non-decreasing with increasing concentration (constant
or steadily increasing with concentration), the data must be smoothed. Abbott's procedure is used to
adjust the concentration response proportions for mortality occurring in the control replicates.
To calculate the LC50 using the Trimmed Spearman-Karber Method, the smoothed, adjusted, observed
proportion mortalities must bracket 0.5. The USEPA TSK program may be used to calculate LCSOs with
the Trimmed Spearman-Karber Method. For instructions on smoothing response proportions and
performing Abbott's adjustment, see section 11.2.2.3, page 78, EPA/600/4-90/027F. For a numerical
example of the Trimmed Spearman-Karber Method, see pages 83-87, EPA/600/4-90/027F.
76
-------
STATISTICAL APPENDIX (CONTINUED)
GRAPHICAL METHOD
The Graphical Method is used to calculate the LC50 if partial mortality is not observed at any effluent
concentration. It is a mathematical procedure which estimates the LC50 by linearly interpolating between
points of a plot of observed percent mortality versus the base 10 logarithm (Iog10) of percent effluent
concentration. The only requirement for the Graphical Method is that the observed percent mortalities
bracket 50%.
For an analysis using the Graphical Method the data must first be smoothed and adjusted for mortality in
the control replicates. For a complete example of the Graphical Method, including data smoothing and
Abbott's adjustment, see pages 78-80, EPA/600/4-90/027F.
CALCULATION OF CHRONIC TEST ENDPOINTS
GENERAL COMMENTS
The recommended statistical analysis of most data from chronic toxicity tests with aquatic organisms
follows a decision process illustrated in the flowchart in Figure 2. A few key points to note are listed below:
* In the analysis of weight data for growth tests, the final dried weight of the organisms in the replicate
is divided by the number of survivors to determine the average dried weights used in the determination of
both the growth NOEC and IC25.
* In the statistical analysis of data by hypothesis testing, concentrations that have a significant toxic
effect on one of the observed parameters are not subsequently tested for an effect on some other
parameter. This means that concentrations showing a statistically significant reduction in survival would be
excluded from a subsequent statistical analysis for effects on another parameter such as growth or
reproduction. The exclusion of such concentrations usually results in a more powerful and appropriate
statistical analysis.
* When calculating the chronic point estimate, the IC25, an all-data approach is used, where
growth or reproduction data from all effluent concentrations, including those with significantly lower
survival than the controls, are included in the calculation of the endpoint.
* For the Ceriodaphnia dubia survival test, male organisms are included in the calculations for
determining the survival NOEC. Male organisms, of course, are removed from consideration in the
reproduction portion of the test and the numbers of replicates are adjusted accordingly.
* For the Ceriodaphnia dubia reproduction test, the total young for each female are analyzed. If a
female dies before producing young, a zero is entered for that replicate's total young. If an organism dies
after producing six young, that replicate's total young is entered as six.
Brief descriptions of all the statistical analysis techniques used in determining the DMR-QA Study 15
chronic test endpoints are provided below. For detailed explanations of the method calculations, including
numerical examples, refer to the appropriate appendices in EPA/600/4-89/001 (A) or EPA/600/4-87/028.
FOR ALL CHRONIC POINT ESTIMATION: LINEAR INTERPOLATION METHOD
The Linear Interpolation Method is used to calculate a point estimate of the effluent or other toxicant
concentration that causes a given percent reduction (e.g., 25%, 50%, etc.) in the reproduction or growth of
the test organisms (Inhibition Concentration, or 1C).
77
-------
STATISTICAL APPENDIX (CONTINUED)
The Linear Interpolation Method assumes that the responses (1) are monotonically non-increasing, where
the mean response for each higher concentration is less than or equal to the mean response for the
previous concentration, (2) follow a piecewise linear response function, and (3) are from a random,
independent, and representative sample of test data. If the data are not monotonically non-increasing, they
are adjusted by smoothing (averaging). In cases where the responses at the low toxicant concentrations
are much higher than in the controls, the smoothing process may result in a large upward adjustment in the
control mean. Also, no assumption is made about the distribution of the data except that the data within a
concentration are independent and identically distributed. The USEPA ICPIN program may be used to
calculate test IC25s. Instructions for performing the Linear Interpolation Method, along with a numerical
example, can be found in Appendix J, EPA/600/4-89/001 A.
C. DUBIA SURVIVAL ANALYSIS: FISHER'S EXACT TEST
Fisher's Exact Test is a statistical method based on the hypergeometric probability distribution that can be
used to test if the proportion of successes is the same in two Bernoulli (binomial) populations. When used
with the Ceriodaphnia dubia data, it provides a conservative test of the equality of any two survival
proportions assuming only the independence of responses from a Bernoulli population. Additionally, since
it is a conservative test, a pair-wise comparison error rate of 0.05 is
suggested rather than an experiment-wise error rate of 0.05. Instructions for performing Fisher's Exact
Test, along with a numerical example, can be found in Appendix G, EPA/600/4-89/001.
ANALYSIS OF OTHER CHRONIC DATA: VALIDATING NORMALITY AND HOMOGENEITY OF
VARIANCE ASSUMPTIONS
GENERAL COMMENTS
Dunnett's Procedure and the t test with Bonferroni's adjustment are parametric procedures based on the
assumptions that the observations within treatments are independent and normally distributed, and that the
variance of the observations is homogeneous across all toxicant concentrations and the control. These
assumptions should be checked prior to using these tests, to determine if they have been met. Tests for
validating the assumptions are provided in the following discussion. If the tests fail (if the data do not meet
the assumptions), a nonparametric procedure such as Steel's Many-one Rank Test may be more
appropriate.
TESTS FOR NORMAL DISTRIBUTION OF DATA: THE SHAPIRO-WILK'S TEST:
One formal test for normality is the Shapiro-Wilk's Test. The test statistic is obtained by dividing the
square of an appropriate linear combination of the sample order statistics by the usual symmetric estimate
of variance. The calculated W must be greater than zero and less than or equal to one. This test is
recommended for sample sizes of 50 or less.
In general, if the data fail the test for normality, a transformation such as to log values may normalize the
data. After transforming the data, repeat the appropriate test for normality. Instructions for performing the
Shapiro-Wilk's Test, including a numerical example, can be found in Appendix B, EPA/600/4-89/001 or
Appendix B, EPA/600/4-87/028.
78
-------
STATISTICAL APPENDIX (CONTINUED)
TEST FOR HOMOGENEITY OF VARIANCE: BARTLETT'S TEST
For Dunnett's Procedure and the t test with Bonferroni's adjustment, the variances of the data
obtained from each toxicant concentration and the control are assumed to be equal. Bartlett's Test is a
formal test of this assumption. In using this test, it is assumed that the data are normally distributed.
Instructions for performing Bartlett's Test, including a numerical example, can be found in Appendix B,
EPA/600/4-89/001 or Appendix B, EPA/600/4-87/028. This test is performed when the USEPA DUNNETT
program is run.
TRANSFORMATIONS OF THE DATA
GENERAL COMMENTS
When the assumptions of normality and/or homogeneity of variance are not met, transformations of the
data may remedy the problem, so that the data can be analyzed by parametric procedures, rather than by a
nonparametric technique such as Steel's Many-one Rank Test or Wilcoxon's Rank Sum Test. Examples of
transformations include log, square root, arc sine square root, and reciprocals. After the data have been
transformed, Shapiro-Wilk's and Bartlett's tests should be performed on the transformed observations to
determine whether the assumptions of normality and/or homogeneity of variance are met.
ARC SINE SQUARE ROOT TRANSFORMATION
For data consisting of proportions from a binomial (response/no response; live/dead) response variable,
the variance within the l-th treatment is proportional to P, (1 - Pj), where P, is the expected proportion for
the treatment. This clearly violates the homogeneity of variance assumption required by parametric
procedures such as Dunnett's Procedure or the t-test with Bonferroni's adjustment, since the existence of a
treatment effect implies different values of P, for different treatments, I. Also, when the observed
proportions are based on small samples, or when Pj is close to zero or one, the normality assumption may
be invalid. The arc sine square root (arc sine V P ) transformation is commonly used for such data to
stabilize the variance and satisfy the normality requirement.
Arc sine transformation consists of determining the angle (in radians) represented by a sine value. In the
case of arc sine square root transformation of mortality data, the proportion of dead (or affected)
organisms is taken as the sine value, the square root of the sine value is calculated, and the angle (in
radians) for the square root of the sine value is determined. Whenever the proportion dead is 0 or 1, a
special modification of the arc sine square root transformation must be used (Bartlett, 1937). An
explanation of the arc sine square root transformation and the modification can be found in Appendix B,
EPA/600/4-89/001 or Appendix B, EPA/600/4-87/028.
DUNNETT'S PROCEDURE
Dunnett's Procedure is used to compare each concentration mean with the control mean to decide if any of
the concentrations differ from the control. This test has an overall error rate of alpha, which accounts for
the multiple comparisons with the control. It is based on the assumptions that the observations are
independent and normally distributed and that the variance of the observations is homogeneous across all
concentrations and the control. Dunnett's Procedure uses a pooled estimate of the variance, which is
equal to the error value calculated in an analysis of variance. Dunnett's Procedure can only be used when
the same number of replicate test vessels have been used at each concentration and the control. When
this condition is not met, a t test with Bonferroni's adjustment is used. To determine the test
79
-------
STATISTICAL APPENDIX (CONTINUED)
NOEC using Dunnett's procedure, run the USEPA DUNMETT program. Instructions for performing
Dunnett's Procedure, along with a numerical example, can be found in Appendix C, EPA/600/4-89/001 or
Appendix C, EPA/600/4-87/028.
t TEST WITH BONFERRONI'S ADJUSTMENT
The t test with Bonferroni's adjustment is used as an alternative to Dunnett's Procedure when the number
of replicates is not the same for all concentrations. This test sets an upper bound of alpha on the overall
error rate, in contrast to Dunnett's Procedure, for which the overall error rate is fixed at alpha. Thus,
Dunnett's Procedure is a more powerful test.
The t test with Bonferroni's adjustment is based on the same assumptions of normality and homogeneity of
variance as Dunnett's Procedure, and, like Dunnett's Procedure, uses a pooled estimate of the variance,
which is equal to the error value calculated in an analysis of variance. To determine the test NOEC using a
t test with Bonferroni's adjustment, run the USEPA DUNNETT program. Instructions for performing the t
test with Bonferroni's adjustment, including a numerical example, can be found in Appendix D, EPA/600/4-
89/001 or Appendix D, EPA/600/4-87/028.
STEEL'S MANY-ONE RANK TEST
Steel's Many-one Rank Test is a nonparametric test for comparing treatments with a control. This test is
an alternative to Dunnett's Procedure, and may be applied to the data when the normality assumption has
not been met. Steel's Test requires equal variances across the treatments and the control, but it is thought
to be fairly insensitive to deviations from this condition. The tables for Steel's Many-one Rank Test require
an equal number of replicates at each concentration. If this is not the case, use Wilcoxon's Rank Sum
Test with Bonferroni's adjustment. Instructions for performing Steel's Test, along with a numerical
example, can be found in Appendix E, EPA/600/4-89/001 or Appendix E, EPA/600/4-87/028.
WILCOXON RANK SUM TEST
Wilcoxon's Rank Sum Test is a nonparametric test, to be used as an alternative to Steel's Many-one Rank
Test when the number of replicates are not the same at each concentration. A Bonferroni's adjustment of
the pairwise error rate for comparison of each concentration versus the control is used to set an upper
bound of alpha on the overall error rate, in contrast to Steel's Many-one Rank Test, for which the overall
error rate is fixed at alpha! Thus, Steel's Test is a more powerful test. Instructions for performing the
Wilcoxon Rank Sum Jest with Bonferroni's adjustment can be found in Appendix F, EPA/600/4-89/001 or
Appendix F, EPA/600/4-87/027.
80
-------
MORTALITY DATA
#DEAD
1
TWO OR MORE
PARTIAL MORTALITIES?
W YES
IS PROBIT MODEL N
APPROPRIATE? -|
(SIGNIFICANT X2 TEST)
i
. YES
'
PROBIT METHOD
1
NO
1
O ONE OR MORE 'NO
^ PARTIAL MORTALITIES? ~^
W YES
ZERO MORTALITY IN THE
LOWEST EFFLUENT CONG.
AND 100% MORTALITY IN THE
HIGHEST EFFLUENT CONG.?
f YES
SPEARMAN-KARBER
METHOD
t
LC50AND95%
^" CONFIDENCE ~^~
INTERVAL
GRAPHICAL METHOD
LC50
NO
I
TRIMMED SPEARMAN-
KARBER METHOD
Figure 1. DMR-QA Acute Data Analysis Flowchart.
81
-------
CHRONfC ENDPCHNT (SURVIVAL. GROWTH, REPRODUCTION, ETC.) NEEDED?
YES
LINEAR
INTERPOLATION
YES
POINT ESTIMATE NEEDED?
ENDPOiNT NEEDED FOR
C. DUB1A SURVIVAL DATA?
YES
FISHER'S
EXACT TEST
SHAPIRO-WJUCS
TEST
1
ENDPOINT ESTIMATE
(NOEQ
NORMAL DISTRIBLmON
HOMOGENEOUS VARIANCE
NON-NORMAL DISTRIBUTION
BARTLETTS TES
HETEROGENEOUS
VARIANCE
•—»•
YES
i
HAVE APPROPRIATE
TRANSFORMATION
OPTIONS BEEN
CONSIDERED?
NO
NO
1
t
4 OR MORE
REPLICATES?
vee 1
NO
NO STATISTICAL
ANALYSIS
RECOMMENDED
FOR THIS ENDPOINT
EQUAL NUMBER OF
REPLICATES?
YES
TTESTWITH
BONFERRONI
ADJUSTMENT
(EQUAL NUMBER OF
REPLICATES?
NO
YES
DUNNETTS
TEST
STEEL'S MANY-pNE
RANK TEST
I
WILCOXON RANK SUM
TEST WITH
BONFERRONI ADJUSTMENT
ENDPOINT ESTIMATE
(NOEC)
Figure 2. DMR-QA Chronic Data Analysis Flowchart.
82
-------
MATERIAL SAFETY DATA SHEET
**USEPA
PERFORMANCE EVALUATION SAMPLE: REFERENCE TOXICANTS - 1 and 2
DMR-QA/NPDES Study 18
DATE: April, 1998
The following information is believed to be accurate and represents the best
information currently available. Specific identification of the material has been
intentionally omitted by invoking the "Trade Secrets" provision, Section (i) of the
"OSHA Hazard Communication Standard, August 24, 1987". Proper evaluation of the
laboratories performance depends on the compound remaining an "unknown". The chemical
poses minor hazard to the user and any potential hazard and its proper handling are
provided below.
**RESPONSIBLE PARTY
USE PA
Environmental Monitoring Systems Laboratory
Cincinnati, OH 45268
(513) 569-7325
CERCLA RATINGS (Scale 0-3), HEALTH = 2 FIRE = 0 REACTIVITY = 0
PERSISTENCE = 0
NFPA RATINGS (Scale 0-4), HEALTH = 2 FIRE = 0 REACTIVITY 0
EXPOSURE LIMITS: No occupational exposure limits established by OSHA, ACGIH,
or NIOSH.
***** TOXICITY *****
LOCAL EFFECTS: Irritant - eyes
TARGET EFFECTS: Poisoning may affect the electrolyte level with resultant
disturbances in the heart rhythm.
AT INCREASED RISK FROM EXPOSURE: Persons with renal, cardiac, pancreatic and adrenal
insufficiencies; persons with esophageal, pyloric or duodenal stenosis.
ADDITIONAL DATA: Interactions with medications have been reported.
***** HEALTH HAZARD *****
ACUTE EFFECTS: May be harmful by inhalation, ingestion or skin absorption. Causes
eye and skin irritation. Material is irritating to mucous membranes and upper
respiratory tract. To the best of our knowledge, the chemical, physical, and
toxicological properties have not been thoroughly investigated.
FIRST AID: In case of contact, immediately flush eyes with copious amounts of water
for at least 15 minutes.
In case of contact, immediately wash skin with soap and copious amounts
of water.
If inhaled, remove to fresh air. If not breathing give artificial respiration. If
breathing is difficult, give oxygen. Call a physician.
Wash contaminated clothing before reuse.
ADDITIONAL INFORMATION: Ingestion of large quantities can cause weakness,
gastrointestinal irritation and circulatory disturbances.
83
-------
***** PHYSICAL DATA *****
DESCRIPTION: Odorless, colorless to white crystals or granular powder. pH: 5.4-8.6
",; , , (@5% 'so In) .
SOLUBILITY: soluble in glycerol, alkalies, ether; slightly soluble in alcohol;
insoluble in acetone.
***** FIRE AND EXPLOSION DATA *****
FIRE AND EXPLOSION HAZARD: Negligible fire hazard when exposed to heat or flame.
FIREFIGHTING MEDIA: Dry chemical, carbon dioxide, water spray or foam. For larger
fires, use water spray, fog or alcohol foam.
***** REACTIVITY *****
REACTIVITY: Stable under normal temperatures and pressures.
INCOMPATIBILITIES: bromine trifluoride: may react violently. Sulfuric acid and
potassium permanganate; possible explosion.
DECOMPOSITION: Thermal decomposition products may include toxic and corrosive fumes.
POLYMERIZATION: Hazardous polymerization has not been reported to occur under normal
temperatures and pressures.
***** SPILL OR LEAK PROCEDURES *****
Steps to be taken if material is released or spilled:
Wear protective clothing. Sweep up, place in a bag and hold for waste disposal. Avoid
raising dust. Ventilate area and wash spill site after material pickup is complete.
Waste disposal method: Add in small quantities to a large quantity of water, while
stirring.Adjust pH to neutral. Separate any insoluble solids or liquids and package
them for hazardous-waste disposal. Flush the aqueous solution down the drain with
plenty of water. The hydrolysis and neutralization reactions may generate heat and
fumes which can be controlled by the rate of addition. Observe all federal, state and
local laws.
PRECAUTIONS TO BE TAKEN IN HANDLING AND STORAGE
Chemical safety goggles
Rubber gloves
NIOSH/MSHA-approved respirator
Safety shower and eye bath
Mechanical exhaust required
i Avoid contact and inhalation
Do not get in eyes, on skin, or clothing
Wash thoroughly after handling
Harmful solid
Irritant
Keep tightly closed
Hygroscopic
Store in a cool dry place
84
-------
PERFORMANCE EVALUATION SAMPLE:
DMR-QA/NPDES Study 18
DATE: April, 1998
**USEPA
REFERENCE TOXICANT
- 3
The following information is believed to be accurate and represents the best
information currently available. Specific identification of the material has been
intentionally omitted by invoking the "Trade Secrets" provision, Section (i) of the
"OSHA Hazard Communication Standard, August 24, 1987". Proper evaluation of the
laboratories performance depends on the compound remaining an "unknown". The chemical
poses minor hazard to the user and any potential, hazard and its proper handling are
provided below.
** RESPONSIBLE PARTY
USE PA
Environmental Monitoring Systems Laboratory
Cincinnati, OH 45268
(513) 569-7325
PRECAUTIONARY LABELING
BAKER SAF-T-DATA(TM) SYSTEM
HEALTH - 3 SEVERE (LIFE) .
FLAMMABILITY - 2 MODERATE
REACTIVITY - 1 SLIGHT
CONTACT - 4 EXTREME (CORROSIVE)
HAZARD RATINGS ARE 0 TO 4 (0 = NO HAZARD; 4 = EXTREME HAZARD).
LABORATORY PROTECTIVE EQUIPMENT
PRECAUTIONARY LABEL STATEMENTS
POISON DANGER COMBUSTIBLE
CAUSES SEVERE BURNS - RAPIDLY 'ABSORBED THROUGH SKIN .
MAY BE FATAL IF SWALLOWED, INHALED, OR ABSORBED THROUGH SKIN
EXCEPTIONAL HEALTH AND CONTACT HAZARDS - READ MATERIAL SAFETY DATA SHEET
KEEP AWAY FROM HEAT, SPARKS, FLAME. DO NOT GET IN EYES, ON SKIN, ON CLOTHING. DO NOT
BREATHE DUST. KEEP IN TIGHTLY CLOSED CONTAINER. USE WITH ADEQUATE VENTILATION. WASH
THOROUGHLY AFTER HANDLING. IN CASE OF FIRE, SOAK WITH WATER. IN CASE OF SPILL, SWEEP
UP AND REMOVE. FLUSH SPILL AREA WITH WATER.
SAF-T-DATA(TM) STORAGE COLOR CODE:
PHYSICAL DATA
BOILING POINT:
MELTING POINT:
SPECIFIC GRAVITY:
(H20=l)
SOLUBILITY(H20):
182 C
40 C
1.07
360 F)
104 F)
MODERATE (1 TO 10
RED STRIPE (STORE SEPARATELY)
VAPOR PRESSURE(MM HG): 0.35
VAPOR DENSITY(AIR=1): 3.24
EVAPORATION RATE: <1
(BUTYL ACETATE=1)
% VOLATILES BY VOLUME:
100
APPEARANCE & ODOR: COLORLESS CRYSTALS; CHARACTERISTIC ODOR.
FIRE AND EXPLOSION HAZARD DATA
175 F) NFPA 704M RATING: 3-2-0
LOWER - 1.5 %
FLASH POINT (CLOSED CUP 79 C (
FLAMMABLE LIMITS: UPPER - 8.6 %
FIRE EXTINGUISHING MEDIA
USE WATER SPRAY, ALCOHOL FOAM, DRY CHEMICAL OR CARBON DIOXIDE.
SPECIAL FIRE-FIGHTING PROCEDURES F
FIREFIGHTERS SHOULD WEAR PROPER PROTECTIVE EQUIPMENT AND SELF-CONTAINED BREATHING
85
-------
APPARATUS WITH FULL FACEPIECE OPERATED IN POSITIVE PRESSURE MODE. MOVE CONTAINERS
FROM FIRE AREA IF IT CAN BE DONE WITHOUT RISK. USE WATER TO KEEP FIRE-EXPOSED
CONTAINERS COOL.
UNUSUAL FIRE & EXPLOSION HAZARDS
GIVES OFF HEAVY SMOKE. GIVES OFF "FLAMMABLE VAPORS. VAPORS MAY FORM EXPLOSIVE MIXTURE
WITH AIR. CLOSED CONTAINERS EXPOSED TO HEAT MAY EXPLODE. CONTACT WITH STRONG
OXIDIZERS MAY CAUSE FIRE.
TOXIC GASES PRODUCED CARBON MONOXIDE, CARBON DIOXIDE
HEALTH HAZARD DATA
TLV AND PEL LISTED DENOTE (SKIN).
THRESHOLD LIMIT VALUE (TLV/TWA) : 19 MG/M3 ( 5 PPM)
SHORT-TERM EXPOSURE LIMIT (STEL): 38 MG/M3 ( 10 PPM)
PERMISSIBLE EXPOSURE LIMIT (PEL): 19 MG/M3 ( 5 PPM)
TOXICITY: LD50 (ORAL-RAT)(MG/KG) - 384
LD50 (SKN-RAT) (MG/KG) - 669
LD50 (IPR-RAT)(MG/KG) - 250
LC50 (INHL-RAT) (MG/KG) - 316
CARCINOGENICITY: NTP: NO IARC: NO Z LIST: NO OSHA REG: NO
EFFECTS OF OVEREXPOSURE
ACUTE POISONING VIA ALL ROUTES OF EXPOSURE MAY BE SEVERE ENOUGH TO BE FATAL.
INHALATION OF DUST MAY CAUSE HEADACHE, COUGHING, DIFFICULTY IN BREATHING, CHEST PAIN,
SEVERE LUNG IRRITATION, OR PULMONARY EDEMA. CONTACT WITH SKIN OR" EYES MAY CAUSE
SEVERE IRRITATION OR BURNS. SUBSTANCE IS READILY ABSORBED THROUGH THE SKIN.
INGESTION MAY CAUSE NAUSEA, VOMITING, GASTROINTESTINAL IRRITATION, AND BURNS TO MOUTH
AND THROAT. CHRONIC EFFECTS OF OVEREXPOSURE MAY INCLUDE KIDNEY AND/OR LIVER DAMAGE.
TARGET ORGANS LIVER, KIDNEYS, SKIN MEDICAL CONDITIONS GENERALLY AGGRAVATED BY
EXPOSURE KIDNEY DISORDERS
ROUTES OF ENTRY
INHALATION, ABSORPTION, INHALATION, EYE CONTA.CT, SKIN CONTACT
EMERGENCY AND FIRST AID PROCEDURES
CALL A PHYSICIAN.
IF SWALLOWED, DO NOT INDUCE VOMITING; IF CONSCIOUS, GIVE WATER, MILK, OR MILK OF
MAGNESIA. IF INHALED, REMOVE TO FRESH AIR. IF NOT BREATHING, GIVE ARTIFICIAL
RESPIRATION. IF BREATHING IS DIFFICULT, GIVE OXYGEN. IN CASE OF CONTACT, IMMEDIATELY
FLUSH EYES OR SKIN WITH PLENTY OF WATER FOR AT LEAST 15 MINUTES WHILE REMOVING
CONTAMINATED CLOTHING AND SHOES. WASH CLOTHING BEFORE RE-USE.
REACTIVITY DATA
STABILITY: STABLE HAZARDOUS POLYMERIZATION: WILL NOT OCCUR
CONDITIONS TO AVOID: HEAT, FLAME, OTHER SOURCES OF IGNITION, LIGHT,
AIR
INCOMPATIBLES: STRONG OXIDIZING AGENTS, STRONG BASES, ALKALIES,
,:, CALCIUM HYPOCHLORITE
DECOMPOSITION PRODUCTS: CARBON MONOXIDE, CARBON DIOXIDE
SPILL AND DISPOSAL PROCEDURES
STEPS TO BE TAKEN IN THE EVENT OF A SPILL OR DISCHARGE
"WEAR SELF-CONTAINED BREATHING APPARATUS AND FULL PROTECTIVE CLOTHING. SHUT OFF
IGNITION SOURCES; NO FLARES, SMOKING, OR FLAMES IN AREA. WITH CLEAN SHOVEL, CAREFULLY
PLACE MATERIAL INTO CLEAN, DRY CONTAINER AND COVER; REMOVE FROM AREA. FLUSH SPILL
AREA WITH WATER.
DISPOSAL PROCEDURE
DISPOSE IN ACCORDANCE WITH ALL APPLICABLE FEDERAL, STATE, AND LOCAL
ENVIRONMENTAL REGULATIONS.
86
-------
PROTECTIVE EQUIPMENT
VENTILATION: USE GENERAL OR LOCAL EXHAUST VENTILATION TO MEET
TLV REQUIREMENTS.
RESPIRATORY PROTECTION: RESPIRATORY PROTECTION REQUIRED IF AIRBORNE CONCENTRATION
EXCEEDS TLV. AT CONCENTRATIONS UP
TO 50 PPM, A CHEMICAL CARTRIDGE RESPIRATOR WITH ORGANIC VAPOR CARTRIDGE IS
RECOMMENDED. ABOVE THIS LEVEL, A SELF-CONTAINED BREATHING APPARATUS IS RECOMMENDED.
EYE/SKIN PROTECTION: SAFETY GOGGLES AND FACE SHIELD, UNIFORM, PROTECTIVE SUIT,
VITON GLOVES ARE RECOMMENDED.
STORAGE AND HANDLING PRECAUTIONS
SAF-T-DATA(TM) STORAGE COLOR CODE: RED STRIPE (STORE SEPARATELY)
SPECIAL PRECAUTIONS
KEEP CONTAINER TIGHTLY CLOSED. STORE IN A COOL, DRY, WELL-VENTILATED,
FLAMMABLE LIQUID STORAGE AREA OR CABINET.
STORE IN LIGHT-RESISTANT CONTAINERS.
THE ABOVE INFORMATION IS BELIEVED TO BE CORRECT BUT DOES NOT PURPORT TO BE ALL
INCLUSIVE AND SHALL BE USED ONLY AS A GUIDE.
87 -&V.S. GOVERNMENT PRINTING OFFICE: 1998 -650-070/60021
-------
------- |