United States
Environmental Protection
Agency
Office of Solid Waste and
Emergency Response
Washington DC 20460
EPA/540/P-91/009
January 1991
&EPA
Compendium of
Toxicity Testing
Procedures
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EPA/540/P-91/009
OSWER Directive 9360.4-08
January 1991
COMPENDIUM OF ERT TOXIOITY TESTING
PROCEDURES
7-Day Standard Reference Toxicity Test using Larval Pimephales Promelas
24-Hour Rangefinding Test using Daphnia Magna or Daphnia Pulex
96-Hour Acute Toxicity Test using Larval Pimephales Promelas
24-Hour Rangefinding Test using Larval Pimephales Promelas
48-Hour Acute Toxicity Test using Daphnia Magna or Daphnia Pulex
7-Day Static Renewal Toxicity Test using Ceriodaphnia Dubia
7-Day Static Toxicity Test using Larval Pimephales Promelas
96-Hour Static Toxicity Test using Selenastrum Capricornutum
10-Day Chronic Toxicity Test using Daphnia Magna or Daphnia Pulex
Interim Final
Environmental Response Team
Emergency Response Division
Office of Emergency and Remedial Response
U.S. Environmental Protection Agency
Washington, DC 20460
Printed on Recycled Paper
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Notice
This document has been reviewed in accordance with U.S. Environmental Protection Agency policy and approved
for publication. Mention of trade names or commercial products does not constitute endorsement or
recommendation for use.
The policies and procedures established in this document are intended solely for the guidance of government
personnel, for use in the Superfund Removal Program. They are not intended, and cannot be relied upon, to
create any rights, substantive or procedural, enforceable by any party in litigation with the United States. The
Agency reserves the right to act at variance with these policies and procedures and to change them at any time
without public notice.
Depending on circumstances and needs, it may not be possible or appropriate to follow these procedures exactly
in all situations due to site conditions, equipment limitations, and limitations of the standard procedures.
Whenever these procedures cannot be followed as written, they may be used as general guidance with any and
all modifications fully documented in either QA Plans, Sampling Plans, or final reports of results.
Each Standard Operating Procedure in this compendium contains a discussion on quality assurance/quality
control (QA/QC). For more information on QA/QC objectives and requirements, refer to the Quality
Assurance/Quality Control Guidance for Removal Activities, OSWER directive 9360.4-1, EPA/540/G-90/004.
Questions, comments, and recommendations are welcomed regarding the Compendium of ERT Toxicity Testing
Procedures. Send remarks to:
Mr. William A. CoaWey .
Removal Program QA Coordinator
U.S. EPA - ERT
Raritan Depot - Building 18, MS-101
2890 Woodbridge Avenue
Edison, NJ 08837-3679
For additional copies of the Compendium of ERT Toxicity Testing Procedures, please contact:
National Technical Information Service (NTIS)
U.S. Department of Commerce
5285 Port Royal Road
Springfield, VA 22161
(703) 487-4600
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Table of Contents
Section
1.0 7-DAY STANDARD REFERENCE TOXICITY TEST USING LARVAL
PIMEPHALES PROMELAS: SOP #2020
1.1 Scope and Application 1
1.2 Method Summary 1
1.3 Sample Preservation, Containers, Handling, and Storage 1
1.4 Interferences and Potential Problems 1
1.5 Equipment/Apparatus 1
1.5.1 Apparatus 1
1.5.2 Test Organisms 2
1.5.3 Equipment for Chemical Analysis 2
1.6 Reagents 2
1.6.1 Dilution Water 2
1.6.2 Test Medium 2
1.7 Procedures 2
1.8 Calculations 3
1.9 Quality Assurance/Quality Control 3
1.10 Data Validation 3
1.11 Health and Safety 4
2.0 24-HOUR RANGEFINDING TEST USING DAPHNIA MAGNA OR DAPHNIA
PULEX: SOP #2021
2.1 Scope and Application 5
2.2 Method Summary 5
2.3 Sample Preservation, Containers, Handling, and Storage 5
2.4 Interferences and Potential Problems 5
2.5 Equipment/Apparatus 5
2.5.1 Apparatus 5
2.5.2 Test Organisms g
2.5.3 Equipment for Chemical Analysis g
2.6 Reagents g
2.6.1 Dilution Water g
2.6.2 Test Medium g
2.7 Procedures g
2.8 Calculations 7
2.9 Quality Assurance/Quality Control 7
2.10 Data Validation 7
2.11 Health and Safety 7
111
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Section
3.0 96-HOUR ACUTE TOXICITY TEST USING LARVAL PIMEPHALES
PROMELAS: SOP #2022
3.1 Scope and Application 9
3.2 Method Summary 9
33 Sample Preservation, Containers, Handling, and Storage 9
3.4 Interferences and Potential Problems 9
3.5 Equipment/Apparatus 9
35.1 Apparatus 9
35.2 Test Organisms 10
35.3 Equipment for Chemical Analysis 10
3.6 Reagents
10
3.6.1 Dilution Water 10
3.6.2 Test Medium 10
3.7 Procedures 10
3.8 Calculations H
3.9 Quality Assurance/Quality Control H
3.10 Data Validation H
3.11 Health and Safety U
4.0 24-HOUR RANGEFINDING TEST USING LARVAL PIMEPHALES
PROMELAS: SOP #2023
4.1 Scope and Application 13
4.2 Method Summary 13
43 Sample Preservation, Containers, Handling, and Storage 13
4.4 Interferences and Potential Problems 13
45 Equipment/Apparatus 13
4.5.1 Apparatus 13
4.5.2 Test Organisms 13
4.53 Equipment for Chemical Analysis 14
4.6 Reagents 14
4.6.1 Dilution Water 14
4.6.2 Test Medium 14
4.7 Procedures 14
4.8 Calculations 15
4.9 Quality Assurance/Quality Control 15
4.10 Data Validation 15
4.11 Health and Safety 15
IV
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Section Page
5.0 48-HOUR ACUTE TOXICITY TEST USING DAPHNIA MAGNA OR
DAPHNIA PULEX: SOP #2024
5.1 Scope and Application 17
5.2 Method Summary 17
5.3 Sample Preservation, Containers, Handling, and Storage 17
5.4. Interferences and Potential Problems 17
5.5 Equipment/Apparatus 17
5.5.1 Apparatus 17
5.5.2 Test Organisms 17
5.5.3 Equipment for Chemical Analysis 18
5.6 Reagents 18
5.6.1 Dilution Water 18
5.6.2 Test Medium 18
5.7 Procedures 18
5.8 Calculations 19
5.9 Quality Assurance/Quality Control 19
5.10 Data Validation 19
5.11 Health and Safety 19
6.0 7-DAY RENEWAL TOXICITY TEST USING CERIODAPHNIA DUBIA:
SOP #2025
6.1 Scope and Application 21
6.2 Method Summary 21
6.3 Sample Preservation, Containers, Handling, and Storage 21
6.4 Interferences and Potential Problems 21
6.5 Equipment/Apparatus 21
6.5.1 Apparatus 21
6.5.2 Test Organisms 22
6.5.3 Equipment for Chemical Analysis 22
6.6 Reagents 22
6.6.1 Dilution Water 22
6.6.2 Test Medium 22
6.7 Procedures 22
6.8 Calculations 23
6.9 Quality Assurance/Quality Control 23
6.10 Data Validation 23
6.11 Health and Safety 23
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Section Page
7.0 7-DAY STATIC TOXICITY TEST USING LARVAL PIMEPHALES PROMELAS:
SOP #20126
7.1 Scope and Application 25
7.2 Method Summary 25
73 Sample Preservation, Containers, Handling, and Storage 25
7.4 Interferences and Potential Problems 25
75 Equipment/Apparatus 25
Apparatus 25
7.5.2 Test Organisms 26
7.5.3 Equipment for Chemical Analysis 26
7.6 Reagents 26
7.6.1 Dilution Water 26
7.6.2 Test Medium 26
7.7 Procedures 26
7.8 Calculations 27
7.9 Quality Assurance/Quality Control 27
7.10 Data Validation 27
7.11 Health and Safety 27
8.0 96-HOUR STATIC TOXICITY TEST USING SELENASTRUM CAPRICORNUTUM:
SOP #2027
8.1 Scope and Application 29
82 Method Summary 29
83 Sample Preservation, Containers, Handling and Storage 29
8.4 Interferences and Potential Problems 29
8.5 Equipment/Apparatus 29
8.5.1 Apparatus 29
8.5.2 Washing Procedure 29
8.5.3 Test Organisms 30
8.5.4 Equipment for Chemical Analysis 30
8.6 Reagents 30
8.6.1 Dilution Water 30
8.6.2 Test Medium 30
8.6.3 Stock Culture Solution 30
8.7 Procedures 31
8.8 Calculations 31
8.9 Quality Assurance/Quality Control 32
8.10 Data Validation 32
8.11 Health and Safety 32
VI
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Section Page
9.0 10-DAY CHRONIC TOXICITY TEST USING DAPHNIA MAGNA OR DAPHNIA
PULEX: SOP #2028
9.1 Scope and Application 33
9.2 Method Summary 33
9.3 Sample Preservation, Containers, Handling, and Storage 33
9.4 Interferences and Potential Problems 33
9.5 Equipment/Apparatus 33
9.5.1 Apparatus 33
9.5.2 Washing Procedure 33
9.5.3 Test Organisms 34
9.5.4 Equipment for Chemical Analysis 34
9.6 Reagents 34
9.6.1 Dilution Water 34
9.62 Test Medium 34
9.7 Procedures 34
9.8 Calculations 35
9.9 Quality Assurance/Quality Control 35
9.10 Data Validation 35
9.11 Health and Safety 36
REFERENCES 37
Vll
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List of Exhibits
Exhibit
SOP
Page
Table 1: Example 1 #2020
Table 2: Summary of Test Conditions for a 7-Day Standard #2020
Reference Toxicity Test using Larval Pimephales
promelas
Table 3: Example 2 #2021
Table 4: Summary of Test Conditions for a 24-Hour #2021
Rangefinding Test using Daphnia magna or
Daphnia pulex
Table 5: Example 3 #2022
Table 6: Summary of Conditions for a 96-Hour Toxicity #2022
Test using Pimephales promelas
Table 7: Example 4 #2023
Table 8: Summary of Test Conditions for a 24-Hour #2023
Rangefinding Toxicity Test using
Pimephales promelas
Table 9: Example 5 #2024
Table 10: Summary of Test Conditions for a #2024
48-Hour Acute Toxicity Test using Daphnia
magna or Daphnia pulex
Table 11: Example 6 #2025
Table 12: Summary of Test Conditions for 7-Day Static Renewal #2025
Toxicity Test using Ceriodaphnia dubia
Table 13: Example 7 #2026
Table 14: Summary of Text Conditions for 7-Day Static Renewal #2026
Toxicity Test using Larval Pimephales promelas
Table 15: Example 8 #2027
Table 16: Summary of Test Conditions for a 96-Hour Static #2027
Toxicity Test using Selenastrum capricomutum
Table 17: Example 9 #2028
Table 18: Summary of Test Conditions for a 10-Day Chronic #2028
Toxicity Text using Daphnia magna or Daphnia pulex
3
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
vui
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Acknowledgments
Preparation of this document was dkected by William A. CoaMey, the Removal Program QA Coordinator of
the Environmental Response Team, Emergency Response Division. Additional support was provided under U.S.
EPA contract #68-03-3482 and U.S. EPA contract #68-WO-0036.
IX
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1.0 7-DAY STANDARD REFERENCE TOXICITY TEST
USING LARVAL PIMEPHALES PROMELAS: SOP #2020
1.1 SCOPE AND APPLICATION
The procedure for conducting a standard reference
toxicity test using sodium pentachlorophenate
(NaPCP) as the toxicant and larval Pimephales
protnelas (fathead minnows) as the test organism is
described below. This test estimates the fitness,
condition, and sensitivity of the organisms used in a
definitive toxicity test. It allows for niter- and intra-
laboratory comparisons of toxicity information and
provides an experimental control (Lee, 1980).
Response of the organisms should be within two
standard deviations from the accepted mortality
values for the definitive test data to be considered
valid (American Public Health Association, 1985).
Other standard reference toxicants may be used if
justified and the appropriate reference cited.
Reference toxicants are available from the U.S.
EPA Environmental Monitoring and Support
Laboratory, Cincinnati, Ohio.
1.2 METHOD SUMMARY
Fathead minnow larva are exposed to several
concentrations of the standard reference toxicant.
This test is conducted following the same
procedures used for the definitive test. The range
of concentrations used in the standard reference
toxicant test are selected to encompass the ECso of
the standard reference toxicant used. The lethal
threshold of NaPCP is 0.1-0.2 mg/L at about 24
hours (Adelman et al., 1980). The U.S. EPA LC^
of NaPCP is 0.08-0.19 mg/L.
containers constructed from materials suitable for
the suspected contaminants. Because surrogate test
species will be exposed to varying concentrations of
the sample material, no chemical preservatives are
to be used. The preservation and storage protocol
is therefore limited to holding the samples on ice at
4°C for the holding tune specified by the analytical
method. Prior to shipping, the laboratory
performing the toxicity tests will be notified of any
potential hazards that may be associated with the
samples.
1.4 INTERFERENCES AND
POTENTIAL PROBLEMS
• When conducting a toxicity test with
NaPCP, the pH needs to be kept above 7.4.
The toxicity of NaPCP increases as the pH
drops, which could give erroneous results
(Lee, 1980).
• Non-target chemicals (e.g., residual
chlorine) may cause adverse effects to the
organisms, giving false results.
• Dissolved oxygen depletion due to
biological oxygen demand and/or chemical
oxygen demand (e.g., metabolic wastes) is
also a potential problem.
• Loss of a toxicant through adsorption to
exposure chambers and volatilization may
occur (Peltier and Weber, 1985).
1.3 SAMPLE PRESERVATION,
CONTAINERS, HANDLING,
AND STORAGE
The selected environmental medium will be sampled
utilizing the methodology detailed in ERT Standard
Operating Procedures (SOPs) #2012, Soil Sampling;
#2013, Surface Water Sampling; #2016, Sediment
Sampling, and any other procedure applicable to the
medium sampled.
Once collected, the samples will be placed in
1.5 EQUIPMENT/APPARATUS
1.5.1 Apparatus
• 12 small cups — glass or plastic
• 12 exposure chambers — glass or plastic,
2 liters
• 3 graduated cylinders ~ 1 liter
• 6 beakers — 250 mL or a larger volumetric
flask -- 2 liters
• 2 mixing buckets or beakers
• pipettes -- 10 mL or smaller
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• plastic tubing — 3/8-inch outside diameter
• plastic screening — a mesh smaller than the
fish
• dilution water - 11 L/day standard
reference toxicant — NaPCP
• wide-bore pipettes — 1.5 times the length
of the fish
• suitable food
1.5.2 Test Organisms
Test organisms may be reared in-house or received
from an outside source. All fathead minnow larva
must be less than 24-hours old. To ensure larva
less than 24-hours old, use eggs that were laid
approximately 3 to 4 days prior to the beginning of
the test. Place the substrate containing the eggs
into a bucket containing dilution water. This allows
the test organisms to become acclimated to the
dilution water, reducing stress. Aerate the eggs
vigorously to avoid fungal growth and use
populations of fish that have less than 5% mortality
(AmericanPublicHealthAssociation, 1985). Peltier
and Weber (1985) and Denny (1987) provide more
detailed information, including culturing, caring for,
handling, and preventing disease in fathead
minnows.
1.5.3 Equipment for Chemical
Analysis
Meters are needed to measure dissolved oxygen,
temperature, pH and conductivity. Calibrate the
meters according to the manufacturer's instructions.
Measure and record alkalinity and hardness
according to a standard method (American Public
Health Association, 1985).
1.6 REAGENTS
1.6.1 Dilution Water
Dilution water is moderately hard, reconstituted
dcionized water unless otherwise specified. See
Horning and Weber (1985) for the preparation of
synthetic fresh water.
1.6.2 Test Medium
As a quality control measure, the accuracy of the
dilutions should be measured on test concentrations
so that results from one test are comparable to
other tests. If the reference toxicant is from the
U.S. EPA, instructions are included on how to
prepare a stock solution. If not, a stock solution
should be prepared in advance to facilitate the
preparation of test concentrations.
1.7 PROCEDURES
1. Choose a range of concentrations that span
those causing zero mortality to those causing
complete mortality (indicated by a total
absence of movement, even when prodded).
Two replicates per concentration and two
control replicates following a geometric or
logarithmic concentration should be used.
Table 1: Example 1 below provides standard
reference concentrations that may be used.
2. Label clean exposure chambers, rinse them in
dilution water, and then place chambers on a
table that will meet test requirements in Table
2. Dilution water must be 25°C ± 2°C.
3. Pour 1 liter of dilution water into each control
exposure chamber. Then prepare the NaPCP
stock solution by diluting 10 mL of NaPCP up
to 100 mL. This will provide a 321 mg/L stock
solution.
4.- To prepare the first exposure chamber as per
Example 1, measure 0.2 mL of the stock
solution into a flask and dilute to 2 liters with
the dilution water. Pour 1 liter each into the
replicate exposure chambers that are labelled
0.03 mg/L.
5. Working in order of increasing concentration,
prepare the remaining exposure solutions.
6. After all exposure chambers are filled, the fish
may be added to the chambers. Using a wide-
bore pipette, select one fish at a time from the
test population and place into a small cup.
Prepare 12 cups containing 10 fish each.
7. After the 12 cups have been prepared,
randomly select a cup for each exposure
chamber. Gently submerge the cup below the
surface of the dilution and gently pour the fish
into the chamber.
8. The addition of the fish signifies the beginning
of the test. Record the start time on a data
sheet.
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9. Measure temperature, pH, conductivity, and
dissolved oxygen directly from the exposure
chamber and measure hardness and alkalinity
from an aliquot removed from a chamber.
Measurement should be conducted after the
fish have been added to the chambers.
10. Note mortality 2 hours after initiation of the
test, and thereafter on a daily basis.
11. Feed larval fish three times daily at 4-hour
intervals (e.g., 0800, 1200, and 1600). Use a
commercially prepared food suitable to larval
fish, or a freshwater-rinsed concentrated
suspension of newly-hatched brine shrimp. If
brine shrimp are used for food, add
approximately 700-1000 nauplii (0.1 mL) to
each chamber.
12. New exposure solutions must be prepared daily.
Draw out the old exposure solution, waste
debris and food as carefully as possible. (Leave
sufficient volume to cover the test fish.)
13. Carefully pour the new solution down the sides
of the test chamber.
14. Steps 9-12 must be conducted every day of the
test.
15. On the last day of the test, renewal of the test
solution is not conducted. Live test fish are
removed, preserved in 4% buffered formalin,
and weighed and measured as required.
1.8 CALCULATIONS
The methods used to determine the ECs, differ
depending on the results of the test. If there are no
partial effects in any replicate (i.e. all alive and
healthy or all dead), then the Moving-Average
Method may be used to determine the EC^. If
there are partial effects within a replicate, then the
Probit Method should be used to calculate the ECs,.
Also the Lowest Observable Effect Concentration
(LOEC), the No Observable Effects Concentration
(NOEC) and the chronic value (CHV) are recorded
(Peltier and Weber, 1985). Measure growth in larva
to determine the effect of the standard reference
toxicant on the life cycle. Compare the dry weight
of the fish in the various concentrations to the dry
weight of the control group of fish raised under the
same conditions.
1.9 QUALITY ASSURANCE/
QUALITY CONTROL
Follow the guidelines in this SOP, which are
summarized in Table 2, for adequate QA/QC.
1.10 DATA VALIDATION
The criteria below provide a basis for rejecting the
results generated under this toxicity test:
• Greater than 20% control mortality.
• Greater than 20% aberrant mortality.
• Temperature variation greater than 2°C.
Table 1: Example 1
Standard Reference
Concentration (mg/L NaPCP)
0
0.03
0.06
0.08
0.16
0.30
Test Dilution Volumes (mL)
Diluent
2000.0
1999.8
1999.6
1999.5
1999.0
1998.0
Test Media
0
0.2
0.4
0.5
1.0
2.0
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• Standard reference toxicant stored more
than 72 hours.
• Criteria in Table 2 not met.
1.11 HEALTH AND SAFETY
When working with potentially hazardous materials,
follow U.S. EPA, OSHA and specific health and
safety procedures.
TABLE 2: Summary of Test Conditions for a 7-Day Standard Reference Toxicity Test
using Larval Pimephales promelas*
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
Test type
Temperature
Light quality
Light intensity
Photoperiod
Test chamber size
Test solution volume
Renewal
Age of test organisms
Number/container
Replicates
Feeding
Aeration
Dilution water
Standard reference
toxicant concentrations
Test duration
Effects
Static, daily renewal
25°C ± 2°C
Ambient laboratory illumination
50-100 foot candles
16 hours light, 8 hours dark
2-L containers
1000 mL/replicate
Daily
Newly-hatched larva (less than 24 hours old)
10 per container
Minimum of 2
Feed 0.1 mL brine shrimp nauplii three tunes per day in each
container
None unless DO concentration falls below 40% saturation, then
<100 bubbles per minute
Moderately hard, reconstituted, deionized water, unless
otherwise specified
Minimum of 5 and 1 control
7 days
Survival and growth
* Based on Horning and Weber, 1985
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2.0 24-HOUR RANGEFINDING TEST USING
DAPHNIA MAGNA OR DAPHNIA PULEX: SOP #2021
2.1 SCOPE AND APPLICATION
The procedure for conducting a 24-hour
rangefinding toxicity test using Daphnia magfia or
Daphnia pulex is described below. This test is
applicable to leachates, effluents, and liquid phases
of sediments. The selection of concentrations to
use in a definitive toxicity test are based on the
results of the rangefinder.
2.2 METHOD AND SUMMARY
Larval daphnids are placed in individual containers
and exposed to a wide range of concentrations of
the test medium. No replicates are needed and only
a few concentrations (i.e. 0%, 1%, 10% and 100%)
are used.
2.3 SAMPLE PRESERVATION,
CONTAINERS, HANDLING,
AND STORAGE
The selected environmental medium will be sampled
utilizing the methodology detailed in ERT Standard
Operating Procedures (SOPs) #2012, Soil Sampling;
#2013, Surface Water Sampling; #2016, Sediment
Sampling; and any other procedure applicable to the
medium sampled.
Once collected, the samples will be placed in
containers constructed from materials suitable for
the suspected contaminants. Because surrogate test
species will be exposed to varying concentrations of
the sample material, no chemical preservatives are
to be used. The preservation and storage protocol
is therefore limited to holding the samples on ice at
4°C for the holding time specified by the analytical
method. Prior to shipping, the laboratory
performing the toxicity tests will be notified of any
potential hazards that may be associated with the
samples.
2.4 INTERFERENCES AND
POTENTIAL PROBLEMS
• The results of a static toxicity test do not
reflect temporal fluctuation in effluent
toxicity (Peltier and Weber, 1985). This is
a preliminary test which provides an
estimate of toxicity and the results are
viewed as such.
• Non-target chemicals (e.g., residual
chlorine) may cause adverse effects to the
organisms giving false results.
• Dissolved oxygen depletion due to
biological oxygen demand and/or chemical
oxygen demand (e.g., metabolic wastes) is
also a potential problem.
• Loss of a toxicant through adsorption to
exposure chambers and volatilization may
occur (Peltier and Weber, 1985).
2.5 EQUIPMENT/APPARATUS
2.5.1 Apparatus
25 larval daphnids — acclimated 24 hours to
dilution water
4 exposure chambers ~ 100 mL/chamber
rinsed in dilution water
tray to hold exposure chambers and glass
covers
wide-bore pipettes — inside diameter 1.5
times the size of a daphnid
graduated cylinders, 250 mL
beakers for chemical measurements
suitable food
test medium ~ 150 mL
diluent - 300 mL
pipette - 1 mL
light table — to assist hi counting the
organisms
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2.5.2 Test Organisms
Test organisms may be reared in-house or obtained
from an outside source. Positive identification of
the species is required before testing begins.
Daphnids must be less than 24-hours old and from
the second to the sixth brood of a healthy adult.
Populations of healthy daphnids have large
individuals, an absence of floaters, an absence of
cphippia, and have an absence of parasites.
Individuals are dark colored and produce large
numbers of young (Biesinger, et al. 1987).
2.5.3 Equipment for Chemical
Analysis
Meters are needed to measure dissolved oxygen,
temperature, pH and conductivity. Calibrate the
meters according to the manufacturer's instructions.
Measure and record alkalinity and hardness
according to a standard method (American Public
Health Association, 1985).
2.6 REAGENTS
2.6.1 Dilution Water
Dilution water is moderately hard, reconstituted
dcionized water unless otherwise specified. See
Horning and Weber (1985) for the preparation of
synthetic fresh water. The dilution water for a test
is the same as the water used to culture daphnids
and the water used to acclimate daphnids before the
beginning of the test.
2.6.2 Test Medium
If the test medium is a liquid, dilutions may be
made directly for the required concentrations. If
the test medium is a sediment, preliminary filtration
and dilutions are required to produce a liquid
phase.
2.7 PROCEDURES
1. Choose a wide range of concentrations to
estimate the toxicity of the test medium. The
concentrations cited in Table 3: Example 2 may
be adjusted to meet the criteria of the specific
situation. A geometric or logarithmic range of
concentrations also may be used (Sprague,
1973). The example provides enough test
medium for three test chambers containing 80
mL each. In addition, 100 mL each of dilution
water and test medium are required for
chemical analyses.
2. Measure temperature, pH, conductivity,
dissolved oxygen, alkalinity and hardness for all
test solutions prior to the start of the test.
3. Label clean exposure chambers and rinse in
dilution water, except for the chamber
containing 100% test medium.
4. To prepare the first test solution, measure 1.0
mL of the test medium into a beaker and dilute
to 100 mL with dilution water.
5. Using a graduated cylinder, pour 80 mL into
the exposure chamber. Mix the remaining
concentrations in the same manner. Always
Table 3: Example 2
Test Media Concentrations
(% test media)
0
1
10
100
Test Dilution Volumes (mL)
Diluent
100
99
90
0
Test Media
0
1
10
100
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work from the lowest concentration to the
highest.
6. Using a wide-bore pipette, randomly select a
daphnid, place the pipette below the surface of
the test solution and gently expel each daphnid
individually into an exposure chamber.
7. The test begins when half of the organisms
have been placed into exposure chambers.
Mortality (indicated by a total absence of
movement, even when prodded) should be
determined at 1 hour and again at 24 hours.
2.8 CALCULATIONS
The methods used to determine the LC^ differ
depending on the results of the test. The
Moving-Average Method is used to determine the
LC50 when there is no partial mortality in any
replicate (i.e. all alive or all dead). If there is
partial mortality, the Probit Method is used to
calculate the LC^. The Lowest Observable Effect
Concentration (LOEC) is recorded and the No
Observable Effect Concentration (NOEC) is
recorded (Peltier and Weber, 1985). Since this is a
simple acute test, only mortality is recorded. Other
methods of estimating the LCSO may be used if
justified and an accepted reference is cited
(Biesinger, et al. 1987).
2.9 QUALITY ASSURANCE/
QUALITY CONTROL
Follow the guidelines in this SOP, which are
summarized in Table 4, for adequate QA/QC.
2.10 DATA VALIDATION
The following criteria provide a basis to reject test
results:
• Greater than 10% control mortality.
• Greater than 10% aberrant mortality in
concentrations.
• Temperature variation greater than 2°C.
• Effluent stored more than 72 hours.
• Criteria in Table 4 not met.
2.11 HEALTH AND SAFETY
When working with potentially hazardous materials,
follow U.S. EPA, OSHA and specific health and
safety procedures.
-------
TABLE 4: Summary of Test Conditions for a 24-Hour Rangefinding Test using Daphnia
magna or Daphnia pulex*
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
Test type
Temperature
Light Quality
Light Intensity
Photoperiod
Test chamber size
Test solution volume
Age of test organisms
Number/container
Feeding
Aeration
Dilution water
Effluent/leachate concentrations
Static, 24 hours
20.0°C ± 2°C
Ambient laboratory illumination
50-100 foot candles
16 hours light, 8 hours dark
100-mL containers
80 mL/replicate
Larval daphnids, less than 24 hours old and within 4 hours of
each other
10 per container
Do not feed during the test
None unless DO concentration falls below 40% saturation,
then < 100 bubbles per minute
Moderately hard, reconstituted, deionized water, unless
otherwise specified
3 and 1 control
*Based on Peltier and Weber, 1975
-------
3.0 96-HOUR ACUTE TOXICITY TEST USING
LARVAL PIMEPHALES PROMELAS: SOP #2022
3.1 SCOPE AND APPLICATION
The procedure for conducting a 96-hour acute
toxicity test using larval Pimephales promelas
(fathead minnows) is described below. This test is
applicable to effluents, leachates, and liquid phases
of sediment which require an acute toxicity estimate.
3.2 METHOD SUMMARY
Larval fathead minnows are exposed to different
concentrations of a test medium over a 96-hour
period. Survival results are used to determine the
LCjo of the test medium. Test concentrations are
renewed daily and mortality is the endpoint of the
test.
3.3 SAMPLE PRESERVATION,
CONTAINERS, HANDLING, AND
STORAGE
The selected environmental medium will be sampled
utilizing the methodology detailed in ERT Standard
Operating Procedures (SOPs) #2012, Soil Sampling;
#2013, Surface Water Sampling; #2016, Sediment
Sampling, and any other procedure applicable to the
medium sampled.
Once collected, the samples will be placed in
containers constructed from materials suitable for
the suspected contaminants. Because surrogate test
species will be exposed to varying concentrations of
the sample material, no chemical preservatives are
to be used. The preservation and storage protocol
is therefore limited to holding the samples on ice at
4°C for the holding time specified by the analytical
method. Prior to shipping, the laboratory
performing the toxicity tests will be notified of any
potential hazards that may be associated with the
samples.
3.4 INTERFERENCES AND
POTENTIAL PROBLEMS
• The results of a static toxicity test do not
reflect temporal changes in effluent toxicity.
This method is less sensitive than a
flow-through toxicity test and the sensitivity
is dependent on the accuracy of the
dilutions (Peltier and Weber, 1985).
• Non-target chemicals (e.g., residual
chlorine) may cause adverse effects to the
organisms giving false results.
• Dissolved oxygen depletion due to
biological oxygen demand and/or chemical
oxygen demand (e.g., metabolic wastes) is
also a potential problem.
• Loss of a toxicant through adsorption to
exposure chambers and volatilization may
occur (Peltier and Weber, 1985).
3.5 EQUIPMENT/APPARATUS
3.5.1 Apparatus
120 larval fathead minnows — less than 30
days old
12 exposure chambers — 1 liter, glass or
plastic, labeled
12 small cups — 50 mL
graduated cylinders - 1 liter and 10 mL
mixing bucket ~ 2 liters or larger
plastic tubing - 3/8-inch outside diameter
plastic screening dilution
water — 4 L/day
test medium — 2 L/day
wide-bore pipettes — inside diameter 1.5
tunes the length of the organism
waste containers
brine shrimp nauplii
-------
3.5.2 Test Organisms
Larval fathead minnows may be cultured in-house
or obtained from an outside source. Positive
identification of the species must be made prior to
beginning the test. Fish to be used for acclimation
and toxicity tests must be healthy and have less than
5% mortality. If test medium and dilution water
are limited, use smaller test organisms. This will
also ensure that the exposure chambers are not over
loaded. Fish selected for acclimation need to be
similar in size, not more than 15 times the length of
each other. Larval fathead minnows must be fed
during the acclimation period as well as during the
test. Brine shrimp nauplii or other suitable larval
fish food may be used. Peltier and Weber (1985)
and Denny (1987) provide more detail and
information including culturing, care, handling, and
disease prevention of fathead minnows.
3.5.3 Equipment for Chemical
Analysis
Meters are needed to measure dissolved oxygen,
temperature, pH, and conductivity. Calibrate the
meters according to the manufacturer's instructions.
Measure and record alkalinity and hardness using a
standard method (American Public Health
Association, 1985).
3.6 REAGENTS
3.6.1 Dilution Water
Dilution water is moderately hard, reconstituted
deionized water unless otherwise specified. See
Horning and Weber (1985) for the preparation of
synthetic fresh water.
3.6.2 Test Medium
If the test medium is a liquid, dilutions may be
made directly for the required concentrations. If
the test medium is a liquid phase of a sediment,
pfeliminary filtration and dilutions are required.
3.7 PROCEDURES
1. Choose a range of concentrations that span
those causing zero mortality to those causing
complete mortality (indicated by a total
absence of movement, even when prodded).
The concentrations cited in Table 5: Example
3 may be adjusted to meet the criteria of the
specific situation. A geometric or logarithmic
range of concentrations may also be used
(Sprague, 1973). The example below provides
six concentrations with two 500-mL replicates.
2. Rinse all exposure chambers, except the
chamber containing 100% test medium, in
dilution water. Label the outside of the
chambers.
3. Measure 500 mL of dilution water and pour
into each control exposure chamber replicate.
Then, prepare test concentrations, working
from the lowest concentration to the highest.
4. Measure 10 mL of the test medium into a
Table 5: Example 3
Test Media Concentrations
(% test media)
0
1
10
25
50
100
Test Dilution Volumes (mL)
Diluent
1000
990
900
750
500
0
Test Media
0
10
100
250
500
1000
10
-------
beaker and dilute to 1000 mL with dilution
water. Using a graduated cyh'nder, pour 500
mL into the two exposure chambers, labelled
for 1% test concentration.
5. Repeat step 4 for all concentrations.
6. Using a pipette, randomly place one fish at a
time into a small cup until there are 10 fish in
each cup. Randomly select the cups and
carefully pour the fish into the exposure
chambers. Submerge the cup below the test
medium surface, gently tilt the cup and pour
the fish into the exposure chamber.
7. Record survival at 1 hour and then daily
thereafter. Measure and record temperature,
dissolved oxygen, pH, conductivity, alkalinity
and hardness for all test solutions after addition
of the fish.
8. Feed fish during the acclimation period and
during the toxicity test. Feed larval fish three
times daily at 4-hour intervals (e.g., 0800,1200,
and 1600). Use a freshwater-rinsed,
concentrated suspension of newly-hatched brine
shrimp. Add approximately 700-1000 nauplii
(0.1 mL) to each container (Horning and
Weber, 1985). Other food may be used if it is
suitable larval fish food.
9. Test solutions must be replaced daily. Using a
length of plastic tubing covered with netting,
siphon out the concentrations from the
exposure chambers. Leave a small amount of
test solution in the bottom of the chamber.
While siphoning, remove as much dead brine
shrimp and waste debris as possible. Mix
concentrations as done the day before and
slowly pour the new concentrations into the
exposure chambers. The temperatures of the
new test concentrations must be equal to the
temperature of the exposure chamber so that
the fish are not stressed.
10. The test is complete after the 96-hour final
mortality and chemical measurements are
recorded. Dispose of test solution in a manner
consistent with good lab practices.
3.8 CALCULATIONS
The methods used to determine the LCjo differ
depending on the results of the test. If there is no
partial mortality in any replicate (i.e. all alive or all
dead), then the Moving-Average Method may be
used to determine the LC^,. If there is partial
mortality within a replicate, then the Probit Method
should be used to calculate the LCjQ. Also the
Lowest Observable Effect Concentration (LOEC) is
recorded and the No Observable Effects
Concentration (NOEC) is recorded (Peltier and
Weber, 1985). Other methods may be used if
justified and the appropriate reference cited. See
Sprague (1973) or Peltier and Weber (1985) for
more detail on the calculations.
3.9 QUALITY ASSURANCE/
QUALITY CONTROL
Follow the guidelines in this SOP, which are
summarized in Table 6, for adequate QA/QC.
3.10 DATA VALIDATION
The following criteria provide a basis for rejecting
the results of this test:
Greater than 10% control mortality.
Greater than 10% aberrant mortality.
Temperature variation greater than 2°C.
Test medium stored more than 72 hours.
Criteria in Table 6 not met.
3.11 HEALTH AND SAFETY
When working with potentially hazardous materials,
follow U.S. EPA, OSHA, and specific health and
safety procedures.
11
-------
Table 6: Summary of Conditions for a 96-Hour Toxicity Test using Pimephales
promelas*
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
Test type
Temperature
Light Qualify
Light Intensity
Photoperiod
Test chamber size
Test solution volume
Renewal
Age of test organisms
Number/container
Replicates
Feeding
Aeration
Dilution water
Test media concentrations
Test duration
Static, daily renewal
25.0°C ± 2°C
Ambient laboratory illumination
50-100 foot candles
16 hours light, 8 hours dark
1-L container
500 mL/replicate
Daily
Less than 30 days old
10 per container
Minimum of 2
Feed 3 times daily
None unless DO concentration falls below 40% saturation,
then < 100 bubbles per minute
Moderately hard, reconstituted, deionized water, unless
otherwise specified
Minimum of 5 and 1 control
96 hours
Based on Peltier and Weber, 1985.
12
-------
4.0 24-HOUR RANGEFINDING TEST USING
LARVAL PIMEPHALES PROMELAS: SOP #2023
4.1 SCOPE AND APPLICATION
The procedure for conducting a 24-hour
rangefinding test using larval Pimephales promelas
(fathead minnows) is described below. This test is
used as a preliminary guide when testing an
effluent, leachate, or liquid phase of a sediment with
an unknown toxicity. The results of this test are
used to determine the concentration range in a
definitive toxicity test.
4.2 METHOD SUMMARY
Larval fathead minnows are exposed to various
concentrations of a test medium over a 24-hour
period. Survival and mortality data are used to
determine the concentration range to be used hi a
static or flow-through toxicity test.
4.3 SAMPLE PRESERVATION,
CONTAINERS, HANDLING, AND
STORAGE
The selected environmental medium will be sampled
utilizing the methodology detailed in ERT Standard
Operating Procedures (SOPs) #2012, Soil Sampling;
#2013, Surface Water Sampling; #2016, Sediment
Sampling, and other procedures applicable to the
medium sampled.
Once collected, the samples will be placed in
containers constructed from materials suitable for
the suspected contaminants. Because surrogate test
species will be exposed to varying concentrations of
the sample material, no chemical preservatives are
to be used. The preservation and storage protocol
is therefore limited to holding the samples on ice at
4°C for the holding tune specified by analytical
method. Prior to shipping, the laboratory
performing the toxicity tests will be notified of any
potential hazards that may be associated with the
samples.
4.4 INTERFERENCES AND
POTENTIAL PROBLEMS
• The results of a static toxicity test do not
reflect temporal changes in effluent toxicity
(Peltier and Weber, 1985). This method is
less sensitive than a flow-through toxicity
test and the sensitivity is dependent on the
accuracy of the dilutions.
• Non-target chemicals (e.g., residual
chlorine) may cause adverse effects to the
organisms giving false results.
• Dissolved oxygen depletion due to
biological oxygen demand and/or chemical
oxygen demand (e.g., metabolic wastes) is
also a potential problem.
• Loss of a toxicant through adsorption to
exposure chambers and volatilization may
occur (Peltier and Weber, 1985).
4.5 EQUIPMENT/APPARATUS
4.5.1 Apparatus
40 fathead minnows — less than 30 days old
4 small cups — 50 mL
4 exposure chambers ~ 1 liter, glass or
plastic, labeled
graduated cylinders - 1 liter and 10 mL
mixing bucket - 1 liter or larger
plastic tubing - 3/8" outside diameter
plastic screening
dilution water — 3 liters
test medium - 1.5 liters
wide-bore pipettes ~ inside diameter 1.5
times the length of the organism
waste containers
brine shrimp or other suitable food
4.5.2 Test Organisms
Test organisms may be reared in-house or received
from an outside source. Positive identification of
the test organisms must be made prior to starting
13
-------
the test. The fish to be used for a rangefinding test
must be the same age (less than 30 days old), in the
same condition, and come from the same culture as
those to be used for the definitive test. Place fish
into a holding tank and slowly drip the dilution
water into the tank over a 24-hour period. Then
leave the fish in this water for another 24 hours so
that the fish become acclimated to the dilution
water. Use populations of fish that are healthy and
have less than 5% mortality. For more detailed
information, including culturing, caring for,
handling, and disease prevention of Pimephales
promelas, see Peltier and Weber (1985) and Denny
(1987).
4.5.3 Equipment for Chemical
Analysis
Meters are needed to measure dissolved oxygen,
temperature, pH and conductivity. Calibrate the
meters according to the manufacturer's instructions.
Use a standard method to measure alkalinity and
hardness (American Public Health Association,
1985). Record all measurements on data sheets.
4.6 REAGENTS
4.6.1 Dilution Water
Dilution water is moderately hard, reconstituted
dcionizcd water unless otherwise specified. See
Horning and Weber (1985) for the preparation of
synthetic fresh water. The dilution water for a test
is the same as the water used to acclimate the fish
before the beginning of the test.
4.6.2 Test Medium
If the test medium is a liquid, dilutions may be
made directly for the required concentrations. If
the test medium is a liquid phase of a soil,
preliminary filtration and dilutions are required.
4.7 PROCEDURES
1. In order to determine the range of
concentrations to be used for a definitive
toxicity test, a preliminary rangefinding test is
conducted. Ten fish are placed into exposure
chambers with a broad range of concentrations
(0%, 1%, 10%, and 100% test media).
2. Survival and mortality (indicated by a total
absence of movement, even when prodded) are
recorded after 1 hour and 24 hours and the
results are used to determine definitive test
concentrations.
3. Replicates are not necessary for this test.
4. The concentrations cited in Table 7: Example
4 may be adjusted to meet the criteria of the
specific situation. A geometric or logarithmic
range of concentrations also may be used
(Sprague, 1973). Other ranges may be used
according to the needs of the specific situation.
5. Rinse all exposure chambers, except the
chamber containing 100% test medium, in
dilution water.
6. Measure 750 mL of dilution water and pour
into the control exposure chamber.
Table 7: Example 4
Test Media Concentrations
(% test media)
0
1
10
100
Test Dilution Volumes (mL)
Diluent
750
742.5
675.0
0.0
Test Media
0.0
7.5
75.0
750.0
14
-------
7. Measure 7.5 mL of test medium and dilute to
750 mL with dilution water. Pour this mixture
into the exposure chamber. Continue this
procedure until all the concentrations are
prepared. Always go from the lowest
concentration to the highest in order to
minimize the risk of cross-contamination.
8. Using a wide bore pipette, randomly put one
fish at a time into a small cup, placing 10 fish
into each cup. After all the fish have been
selected, pour into the exposure chambers by
gently submerging the cup below the water
surface and pouring the fish out.
9. Measure and record temperature, dissolved
oxygen, pH, conductivity, alkalinity and
hardness for each test solution after the fish
have been added to the exposure chamber,
which constitutes the beginning of the test.
4.8 CALCULATIONS
The methods used to determine the LC^ differ
depending on the results of the test. If there is no
partial mortality in any replicate (i.e. all alive or all
dead), then the Moving-Average Method may be
used to determine the LC^. If there is partial
mortality within a replicate, then the Probit Method
should be used to calculate the LC^ (Peltier and
Weber, 1985). Since the results of this test are only
preliminary, exact calculations need not be made.
An estimate of the LC50 is needed to determine the
range of concentrations to be used for the definitive
test.
Other methods to determine the LCj,, of the test
medium maybe used if justified and the appropriate
reference cited.
4.9 QUALITY ASSURANCE/
QUALITY CONTROL
Follow the guidelines in this SOP, which are
summarized in Table 8, for adequate QA/QC.
4.10 DATA VALIDATION
The following criteria provide a basis for rejecting
the results generated under this test:
• Greater than 10% control mortality.
• Criteria in Table 8 not met.
Note: Since this is only a preliminary test, the strict
guidelines used for the definitive test need not be
adhered to. However, this test should be run
according to standard laboratory guidelines.
4.11 HEALTH AND SAFETY
When working with potentially hazardous materials,
follow U.S. EPA, OSHA, and specific health and
safety procedures.
15
-------
Table 8: Summary of Test Conditions for a 24-Hour Rangefinding Toxicity Test using
Pimephales promelas*
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
Test type
Temperature
Light quality
Light intensity
Photoperiod
Test chamber size
Test solution volume
Renewal
Age of test organisms
Number/container
Feeding
Washing
Aeration
Dilution water
Test media/leachate
concentrations
Test duration
Static
25.0°C ± 2°C
Ambient laboratory illumination
50-100 foot candles
16 hours light, 8 hours dark
1-L containers
750 mL
None
Newly hatched larva (less than 24 hours old)
10 per chamber
None
N/A
None unless DO concentration falls below 40% saturation,
then < 100 bubbles per minute
Moderately hard, reconstituted, deionized water, unless
otherwise specified
Minimum of 3 and 1 control
24 hours
Based on Peltier and Weber, 1985.
16
-------
5.0 48-HOUR ACUTE TOXICITY TEST USING
DAPHNIA MAGNA OR DAPHNIA PULEX: SOP #2024
5.1 SCOPE AND APPLICATION
The procedure for conducting a 48-hour acute
toxicity test using Daphnia magpa or Daphnia pulex
is described below. This test is applicable to
leachates, effluents, and liquid phases of sediments.
5.2 METHOD SUMMARY
Larval daphnids are placed in individual containers
and exposed to various concentrations of a test
medium over a 48-hour period. Mortality is the
endpoint of the test.
5.3 SAMPLE PRESERVATION,
CONTAINERS, HANDLING, AND
STORAGE
The selected environmental medium will be sampled
utilizing the methodology detailed in ERT Standard
Operating Procedures (SOPs) #2012, Soil Sampling;
#2013, Surface Water Sampling; #2016, Sediment
Sampling, and any other procedure applicable to the
medium sampled.
Once collected, the samples will be placed in
containers constructed from materials suitable for
the suspected contaminants. Because surrogate test
species will be exposed to varying concentrations of
the sample material, no chemical preservatives are
to be used. The preservation and storage protocol
is therefore limited to holding the samples on ice at
4°C for the holding time specified by the analytical
method. Prior to shipping, the laboratory
performing the toxicity tests will be notified of any
potential hazards that may be associated with the
samples.
5.4 INTERFERENCES AND
POTENTIAL PROBLEMS
• Non-target chemicals (e.g., residual
chlorine) may cause adverse effects to the
organisms, giving false results.
• Dissolved oxygen depletion due to
biological oxygen demand and/or chemical
oxygen demand (e.g., metabolic wastes) is
also a potential problem.
• Loss of a toxicant through adsorption to
exposure chambers and volatilization may
occur (Peltier and Weber, 1985).
• The results of a static toxicity test do not
reflect temporal fluctuation in test media
toxicity (Peltier and Weber, 1985).
5.5 EQUIPMENT/APPARATUS
5.5.1 Apparatus
60 larval daphnids - acclimated for at least
24 hours to dilution water
60 exposure chambers ~ 100 mL volume,
labeled
tray to hold exposure chambers and glass
covers
wide-bore pipettes - inside diameter 1.5
tunes the length of the daphnid
graduated cylinders, 250 mL and 1 liter
pipette - 1 mL
beakers for chemical measurements, 250
mL
test medium — 1 liter
diluent ~ 3 liters
waste containers
light table — to aid in counting the
organisms
suitable food
5.5.2 Test Organisms
Test organisms may be reared in-house or obtained
from an outside source. Positive identification of
the species is required before beginning testing.
Daphnids to be used must be less than 24 hours old
and from the second to the sixth brood of healthy
adults. Populations of healthy daphnids have large
individuals, have an absence of floaters, have an
absence of ephippia, and have an absence of
parasites. Individuals are dark colored and produce
17
-------
large numbers of young (Biesinger, et al. 1987).
5.7 PROCEDURES
5.5.3 Equipment for Chemical
Analysis
Meters are needed to measure dissolved oxygen,
temperature, pH and conductivity. Calibrate the
meters according to the manufacturer's instructions.
Measure alkalinity and hardness according to a
standard method (American Public Health
Association, 1985).
5.6 REAGENTS
5.6.1 Dilution Water
Dilution water is reconstituted, deionized water.
The water type should be moderately hard unless
otherwise specified. See Horning and Weber (1985)
for the preparation of synthetic fresh water. The
dilution water for a test is the same as the water
used to culture daphnids and the water used to
acdimate daphnids before the beginning of the test.
5.6.2 Test Medium
If the test medium is a liquid, dilutions may be
made directly for the required concentrations. If
the test medium is a sediment, preliminary filtration
and dilutions are required to produce a liquid
phase.
1. Select a range of concentrations that span those
causing zero mortality to those causing
complete mortality (indicated by a total
absence of movement, even when prodded).
The concentrations cited in Table 9: Example
5 may be adjusted to meet the criteria of the
specific situation. A geometric or logarithmic
range of concentrations also may be used
(Sprague, 1973). The example provides enough
test medium for five replicates containing 50
mL each and extra for chemical analysis.
2. Rinse all exposure chambers, except the
chamber containing 100% test medium, in
dilution water. Label all chambers.
3. Mix concentrations and pour into each
exposure chamber. Work from the lowest
concentration to the highest in order to
minimize the risk of cross-contamination.
4. Measure 0.5 mL of the test medium into a
beaker and dilute to 500 mL.
5. Using a graduated cylinder, pour out 50 mL
into each exposure chamber which is labeled
for 0.1% test concentration. Pour the rest into
a beaker for chemical measurements.
6. Repeat steps 4 and 5 for all concentrations.
7. Using a wide-bore pipette, randomly select and
carefully place 10 daphnids into each exposure
Table 9: Example 5
Test Media Concentrations
(% test media)
0.0
0.1
1.0
10
50
100
Test Dilution Volumes (mL)
Diluent
500
499.5
495
450
250
0
Test Media
0.0
0.5
5.0
50
250
500
18
-------
chamber by placing the pipette tip below the
surface and gently expelling each daphnid
individually into the chamber.
8. The test begins when half of the organisms are
in the exposure chambers.
9. Measure and record mortality and survival at
one hour and then at 24 and 48 hours.
10. Measure and record temperature, dissolved
oxygen, pH, conductivity, alkalinity, and
hardness for all test solutions after the test
begins and at the completion of the test.
11. The test is complete at the end of 48 hours.
5.8 CALCULATIONS
The methods used to determine the LCj,, differ
depending on the results of the test. If there is no
partial mortality in any replicate (i.e. all alive or all
dead), then the Moving-Average Method may be
used to determine the LC^,. If there is partial
mortality within a replicate, then the Probit Method
should be used to calculate the LC^. Also the
Lowest Observable Effect Concentration (LOEC) is
recorded and the No Observable Effects
Concentration (NOEC) is recorded (Peltier and
Weber 1985). Since this is a simple acute test, only
mortality is recorded. Other methods of estimating
the LCjo may be used if justified and an accepted
reference is cited (Biesinger, et al. 1987).
5.9 QUALITY ASSURANCE/
QUALITY CONTROL
Follow the guidelines in this SOP, which are
summarized in Table 10, for adequate QA/QC.
5.10 DATA VALIDATION
The following criteria provide a basis for rejecting
the results of this test:
• Greater than 10% control mortality.
• Greater than 10% aberrant mortality in
concentrations throughout the test range.
However, there may be greater than 10%
mortality in one replicate if there is 100%
survival above that value.
• Temperature variation greater than 2°C.
• Test medium stored more than 72 hours.
• Criteria in Table 10 not met.
5.11 HEALTH AND SAFETY
When working with potentially hazardous materials,
refer to U.S. EPA, OSHA, and specific health and
safety procedures.
19
-------
Table 10: Summary of Test Conditions for a 48-Hour Acute Toxicity Test using
Daphnia magna or Daphnia pulex*
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
Test type
Temperature
Light quality
Light intensity
Photoperiod
Test chamber size
Test solution volume
Renewal
Age of test organisms
Number/container
Feeding
Aeration
Dilution water
Test media/leachate
concentrations
Test duration
Effects measured
Static, daily renewal
20.0°C ± 2°C
Ambient laboratory illumination
50-100 foot candles
16 hours light, 8 hours dark
100-mL containers
50 mL/replicate
None
Less than 24 hours old
10 per exposure chamber
Do not feed during test
None unless DO concentration falls below 40% saturation,
then <100 bubbles per minute
Moderately hard, reconstituted, deionized water, unless
otherwise specified
Minimum of 5 and 1 control
48 hours
Survival at 1, 24, and 48 hours
Based on Peltier and Weber, 1985
20
-------
6.0 7-DAY STATIC RENEWAL TOXICITY TEST
USING CERIODAPHNIA DUBIA: SOP #2025
6.1 SCOPE AND APPLICATION
The procedure for conducting a 7-day static renewal
toxicity test using Ceriodaphnia dubia is described
below. This test is applicable to effluents, leachates,
and liquid phases of sediments which require a
chronic toxicity estimate. This method uses
reproductive success as well as mortality as end
pouits for the test.
6.2 METHOD SUMMARY
Ceriodaphnia dubia are placed in individual
exposure chambers containing 15 mL of the test
medium concentration. Mortality and survival are
recorded over a 7-day period as well as the number
of broods, the brood size, and live or dead young.
These data are used to determine the Lowest
Observable Effect Concentration (LOEC), the No
Observable Effect Concentration (NOEC), the EC^
and the chronic value of the test medium.
6.4 INTERFERENCES AND
POTENTIAL PROBLEMS
• The results of a static toxicity test do not
reflect temporal changes in effluent toxicity
(Peltier and Weber, 1985). This method is
less sensitive than a flow-through toxicity
test and the sensitivity is dependent on the
accuracy of the dilutions.
• Non-target chemicals (e.g., residual
chlorine) may cause adverse effects to the
organisms giving false results.
• Dissolved oxygen depletion due to
biological oxygen demand and/or chemical
oxygen demand (e.g., metabolic wastes) is
also a potential problem.
• Loss of a toxicant through adsorption to
exposure chambers and volatilization may
occur (Peltier and Weber, 1985).
6.3 SAMPLE PRESERVATION,
CONTAINERS, HANDLING, AND
STORAGE
The selected environmental medium will be sampled
utilizing the methodology detailed in ERT Standard
Operating Procedures (SOPs) #2012, Soil Sampling;
#2013, Surface Water Sampling; #2016, Sediment
Sampling, and any other procedure applicable to the
medium sampled.
Once collected, the samples will be placed in
containers constructed from materials suitable for
the suspected contaminants. Because surrogate test
species will be exposed to varying concentrations of
the sample material, no chemical preservatives are
to be used. The preservation and storage protocol
is therefore limited to holding the samples on ice at
4°C for the holding time specified by analytical
method. Prior to shipping, the laboratory
performing the toxicity tests will be notified of any
potential hazards that may be associated with the
samples.
6.5 EQUIPMENT/APPARATUS
6.5.1 Apparatus
75 Ceriodaphnia dubia — less than 24 hours
old and released during the same 4-hour
period
60 exposure chambers/day ~ 30 mL or
larger, labeled
trays and glass covers for exposure
chambers
wide-bore pipettes — inside diameter 1.5
tunes the length of the organisms
dilution water — 1.5 L/day
test medium — 500 mL/day
graduated cylinder ~ 500 mL and 10 mL
mixing bucket - 500 mL or larger
pipettes ~ 1 mL and bulb
beakers ~ 250 mL
light table ~ to aid in counting the
organisms
suitable food
waste containers
21
-------
6.5.2 Test Organisms
Test organisms may be reared in-house or obtained
from an outside source. Positive identification of
Ceriodaphnia dttbia is required before beginning the
test (Bcrner, 1986). Ceriodaphnia dubia to be used
must be less than 24-hours old and from the second
to the sixth brood of an healthy adult. Adults to be
used should be placed into individual cups
containing dilution water 24 hours prior to the start
of the test in order to ensure less than 24-hour old
organisms.
6.5.3 Equipment for Chemical
Analysis
Meters are needed to measure dissolved oxygen,
temperature, pH and conductivity. Calibrate the
meters according to the manufacturer's instructions.
Use a standard method to measure and record
alkalinity and hardness (American Public Health
Association, 1985). Record all measurements on
data sheets.
6.6 REAGENTS
6.6.1 Dilution Water
Dilution water is moderately hard, reconstituted
delonizcd water unless otherwise specified. See
Horning and Weber (1985) for the preparation of
synthetic fresh water. The dilution water used in a
test should be the same as the water used to culture
and acclimate the test species.
6.6.2 Test Medium
If the test medium is a liquid, dilutions may be
made directly for the required concentrations. If
the test medium is a sediment, preliminary filtration
and dilutions are required to produce a liquid
phase.
6.7 PROCEDURES
1. Select a range of concentrations that span those
causing zero mortality to those causing
complete mortality (indicated by a total
absence of movement, even when prodded).
The concentrations cited in Table 11: Example
6 may be adjusted to meet the criteria of the
specific situation. A geometric or logarithmic
range of concentrations also may be used
(Sprague, 1973). The example provides enough
effluent for 10 exposure chambers per
concentration, each containing 15 mL and extra
for chemical analysis. Other ranges may be
used according to needs of the analyses.
2. Rinse all exposure chambers, except the
chamber containing 100% test medium, in
dilution water.
3. To prepare the first test solution, measure 0.30
mL of the test medium into a beaker and dilute
to 300 mL using dilution water.
4. Using a graduated cylinder, pour 15 mL into
each exposure chamber labeled for .1% test
Table 11: Example 6
Test Media Concentrations
(% test media)
0.0
0.1
1.0
10
50
100
Test Dilution Volumes (mL)
Diluent
300
299.7
297
270
150
0
Test Media
0.0
0.3
3.0
30
150
300
22
-------
concentration and pour the rest into a beaker
for chemical analyses.
5. Continue steps 3 and 4 for all concentrations.
Always work from lowest concentration to the
highest in order to minimize the risk of cross-
contamination.
6. Using a wide-bore pipette, randomly select one
acclimated Ceriodaphnia dubia (under 24-hours
old) into each cup by placing the organism
under the surface of the test medium and gently
expelling it into the test chamber.
7. Add 0.1 mL (1 drop) of a suitable food to each
exposure chamber as food.
8. Measure and record survival at 1 hour.
9. Measure and record temperature, dissolved
oxygen, pH, conductivity, alkalinity and
hardness daily of all new test solutions.
10. Measure and record dissolved oxygen daily
from both old and new test solutions and the
control. Do this prior to pouring the test
concentrations into the individual exposure
chambers.
11. On the second day, prepare new test medium
concentrations and a new set of exposure
chambers.
12. Pour new concentrations into new chambers as
done previously and use the excess for chemical
analyses.
13. Count the number of broods, the brood size,
and the number of live or dead organisms.
Ceriodaphnia dubia usually start to produce
offspring after the third day of the test and they
should have three broods by the completion of
the test. The endpoint of the test is when 60%
of the control organisms have at least three
broods and at least 90 young (an average of
nine per organism).
14. Place 0.1 mL (1 drop) of a suitable food into
the exposure chambers after the concentrations
have been renewed but before the test
organisms are transferred into the chamber.
This provides for more consistent water quality
between changes.
15. Transfer adult Ceriodaphnia dubia by carefully
removing with a wide-bore pipette and
transferring into the new exposure chamber.
16. Place a cover loosely over the exposure
chambers to prevent evaporation.
6.8 CALCULATIONS
The methods used to determine the ECso differ
depending on the results of the test. If there are no
partial effects in any replicate (i.e. all alive and
healthy or all dead), then the Moving-Average
Method may be used to determine the ECjQ. If
there are partial effects within a replicate, then the
Probit Method should be used to calculate the EC^.
Also the Lowest Observable Effect Concentration
(LOEC), the No Observable Effects Concentration
(NOEC) and the chronic value are recorded (Peltier
and Weber, 1985). Other methods of determining
the ECso may be used if justified and the
appropriate reference is cited.
6.9 QUALITY ASSURANCE/
QUALITY CONTROL
Follow the guidelines in this SOP, which are
summarized in Table 12, for adequate QA/QC.
6.10 DATA VALIDATION
The following criteria provide a basis for rejecting
the results of this test:
« Greater than 20% control mortality.
• Greater than 20% aberrant mortality in any
concentrations.
• Temperature variation greater than 2°C.
• Test medium stored more than 72 hours.
• Criteria in Table 12 not met.
• Less than 3 broods in the control group
and less than 90 young produced in the
control group (an average of 9 per
individual).
6.11 HEALTH AND SAFETY
When working with potentially hazardous materials,
follow U.S. EPA, OSHA and specific health and
safety procedures.
23
-------
Table 12: Summary of Test Conditions for 7-Day Static Renewal Toxicity Test using
Ceriodaphnia dubia*
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
Test type
Temperature
Light quality
Light intensity
Photoperiod
Test chamber size
Test solution volume
Renewal
Age of test organisms
Number/container
Feeding
Aeration
Dilution water
Test media/leachate
concentrations
Test duration
Effects measured
Static, daily renewal
25.0°C ± 2°C
Ambient laboratory illumination
50-100 foot candles
16 hours light, 8 hours dark
30-mL containers
15 mL per exposure chamber
Daily
Newly hatched larva (less than 24 hours old)
1 per chamber (10 chambers)
Feed 1 drop (0.1 mL) of suitable food per day
None unless DO concentration falls below 40% saturation,
then < 100 bubbles per minute
Moderately hard, reconstituted, deionized water, unless
otherwise specified
Minimum of 5 and 1 control
7 days
Survival and reproduction
* Based on Horning and Weber, 1985.
24
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7.0 7-DAY STATIC RENEWAL TOXICITY TEST
USING LARVAL PIMEPHALES PROMELAS: SOP #2026
7.1 SCOPE AND APPLICATION
The procedure for conducting a 7-day static renewal
toxicity test using larval Pimephales promelas
(fathead minnows) is described below. This test is
applicable to effluents, leachates, and sediments
which require a chronic toxicity estimate.
7.2 METHOD SUMMARY
Larval fathead minnows are exposed to different
concentrations of a test medium over a 7-day
period. Survival and growth results are used to
determine the No Observable Effect Concentration
(NOEC), the Lowest Observable Effect
Concentration (LOEC), the EC^, and the chronic
value (CHV) of the test medium. Test
concentrations are renewed daily.
7.3 SAMPLE PRESERVATION,
CONTAINERS, HANDLING, AND
STORAGE
The selected environmental medium will be sampled
utilizing the methodology detailed in ERT Standard
Operating Procedures (SOPs) #2012, Soil Sampling;
#2013, Surface Water Sampling; #2016, Sediment
Sampling, and any other procedure applicable to the
medium sampled.
Once collected, the samples will be placed in
containers constructed from materials suitable for
the suspected contaminants. Because surrogate test
species will be exposed to varying concentrations of
the sample material, no chemical preservatives are
to be used. The preservation and storage protocol
is therefore limited to holding the samples on ice at
4°C for the holding tune specified by the analytical
method. Prior to shipping, the laboratory
performing the toxicity tests will be notified of any
potential hazards that may be associated with the
samples.
7.4 INTERFERENCES AND
POTENTIAL PROBLEMS
• The results of a static toxicity test do not
reflect temporal changes in effluent toxicity.
This method is less sensitive than a
flow-through toxicity test and the sensitivity
is dependent on the accuracy of the
solutions (Peltier and Weber, 1985).
• Non-target chemicals (e.g., residual
chlorine) may cause adverse effects to the
organisms, giving false results.
• Dissolved oxygen depletion due to
biological oxygen demand and/or chemical
oxygen demand (e.g., metabolic wastes) i
also a potential problem.
is
Loss of a toxicant through adsorption to
exposure chambers and volatilization may
occur (Peltier and Weber, 1985).
7.5 EQUIPMENT/APPARATUS
7.5.1 Apparatus
• 120 larval fathead minnows — less than 24
hours old
• 12 exposure chambers — 1 liter, labeled
• 12 small cups ~ 50 mL
• test medium — 2 L/day
• diluent - 4.25 L/day
• graduated cylinders - 3, 1 liter
• beakers — 250 mL
• mixing buckets — 2 liters
• plastic tubing - 3/8-inch outside diameter
• plastic screening — mesh with smaller than
that of the fish
• wide-bore pipettes — inside diameter 1.5
tunes the size of the fish
• waste containers
• brine shrimp or other suitable food
25
-------
7.5.2 Test Organisms
Larval fathead minnows may be cultured in-house
or obtained from an outside source. Positive
identification of the species must be made prior to
beginning the test. Fathead minnows to be used for
the test must be healthy. Place the substrate
holding the eggs into the dilution water 24 hours
prior to the beginning of the test to ensure that the
fish to be used are less than 24-hours old. Larval
fathead minnows must be fed during the acclimation
period as well as during the test. Brine shrimp
nauplii or other suitable larval fish food may be
used. Peltier and Weber (1985) and Denny (1987)
provide more detailed information, including
culturing, caring for, handling, and preventing
disease in fathead minnows.
7.5.3 Equipment for Chemical
Analysis
Meters are needed to measure dissolved oxygen,
temperature, pH and conductivity. Calibrate the
meters according to the manufacturer's instructions.
Measure and record alkalinity and hardness using a
standard method (American Public Health
Association, 1985).
7.6 REAGENTS
7.6.1 Dilution Water
Dilution water is moderately hard, reconstituted
dcionizcd water unless otherwise specified. See
Horning and Weber (1985) for the preparation of
synthetic fresh water. Set up a laboratory or
standard dilution water control when receiving
waters are used as the dilution water.
7.6.2 Test Medium
If the test medium is a liquid, dilutions may be
made directly for the required concentrations. If
the test medium is to be a liquid phase of a soil,
preliminary filtration and dilutions are required.
7.7 PROCEDURES
1. Choose a range of concentrations that span
those causing no effect to those causing
complete mortality (indicated by a total
absence of movement, even when prodded).
The concentrations cited in Table 13: Example
7 may be adjusted to meet the criteria of the
specific situation. A geometric or logarithmic
range of concentrations also may be used
(Sprague, 1973). The example provides
enough test medium for two replicates
containing 500 mL each.
2. Rinse all exposure chambers, except the
chamber containing 100% test medium, in
dilution water.
3. Prepare the test dilutions by pouring 500 mL of
dilution water into both control chambers.
Then measure out 10 mL of the test medium
into a bucket and pour 990 mL of dilution
Table 13: Example 7
Test Media Concentrations
(% test media)
0
1
10
25
50
100
Test Dilution Volumes (mL)
Diluent
1000
990
900
750
500
0
Test Media
0
10
100
250
500
1000
26
-------
water into the bucket and mix Using a
graduated cylinder, pour 500 mL into both 1%
exposure chambers.
4. Repeat step 3 for all concentrations. Always
work from the lowest concentration to the
highest in order to minimize the risk of cross-
contamination.
5. Using a pipette, randomly place one fish at a
time into a small cup until there are 10 fish in
each cup.
6. Randomly select the cups and carefully pour
the fish into the exposure chambers by
submerging the cup below the test medium
surface, gently tilting the cup and pouring the
fish into the exposure chamber.
7. Record survival at 1 hour and then daily
thereafter.
8. Measure and record dissolved oxygen,
temperature, pH, conductivity, alkalinity and
hardness of all test solutions after the fish have
been placed into the chambers and then daily
thereafter.
9. Feed larval fish three times daily at 4-hour
intervals (e.g., 0800, 1200, and 1600). Use a
commercially prepared food suitable to larval
fish or a freshwater-rinsed concentrated
suspension of newly-hatched brine shrimp. If
using shrimp, add approximately 700-1000
nauplii (0.1 mL) to each container.
10. Prepare new dilutions daily.
11. Place plastic screening over a length of tubing
and create a siphon using the dilution water.
Carefully draw out as much of the old solution,
dead brine shrimp and waste debris as possible
from the exposure chamber without disturbing
the fish. Again, work from the lowest
concentration to the highest in order to
minimize the risk of cross-contamination.
12. Discard tubing and the waste concentrations in
a manner consistent with standard laboratory
procedures.
13. Carefully pour the new test solutions into the
exposure chambers. Steps 10 -12 are repeated
each day except for the last day of the test.
7.8 CALCULATIONS
The methods used to determine the ECa, differ
depending on the results of the test. If there are no
partial effects in any replicate (i.e. all alive and
healthy or all dead), then the Moving-Average
Method may be used to determine the ECj,,. If
there are partial effects within a replicate, then the
Probit Method should be used to calculate the ECso.
Also the Lowest Observable Effect Concentration
(LOEC), the No Observable Effect Concentration
(NOEC) and the chronic value (CHV) are recorded
(Peltier and Weber, 1985). Growth is also
measured in the larva to determine the effect of the
test medium on the life cycle. This is done by
comparing the dry weight of the fish in the various
concentrations to the dry weight of a control group
of fish raised under the same conditions.
7.9 QUALITY ASSURANCE/
QUALITY CONTROL
Follow the guidelines in this SOP, which are
summarized in Table 14, for adequate QA/QC.
7.10 DATA VALIDATION
The following criteria provide a basis for rejecting
the results of this test:
Greater than 20% control mortality.
Greater than 20% aberrant mortality.
Temperature variation greater than 2°C.
Test medium stored more than 72 hours.
Criteria in Table 14 not met.
7.11 HEALTH AND SAFETY
When working with potentially hazardous materials,
follow U.S. EPA, OSHA and specific health and
safety procedures.
27
-------
Table 14: Summary of Test Conditions for 7-Day Static Renewal Toxicity Test using
Larval Pimephales promelas*
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
Test type
Temperature
Light quality
Light intensity
Photoperiod
Test chamber size
Test solution volume
Renewal
Age of test organisms
Number/container
Replicates
Feeding
Washing
Aeration
Dilution water
Test media/leachate
concentrations
Test duration
Effects measured
Static, daily renewal
25.0°C ± 2°C
Ambient laboratory illumination
50-100 foot candles
16 hours light, 8 hours dark
1-L containers
500 mL/replicate
Daily
Newly hatched larva (less than 24 hours old)
10 per container
Minimum of 2
Feed 0.1 mL of brine shrimp nauplii 3 times per day, hi each
container
Siphon daily before solution renewal
None unless DO concentration falls below 40% saturation,
then < 100 bubbles per minute
Moderately hard, reconstituted, deionized water, unless
otherwise specified
Minimum of 5 and 1 control
7 days
Survival and growth (increase in weight)
Based on Horning and Weber, 1985
28
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8.0 96-HOUR STATIC TOXICITY TEST USING
SELENASTRUM CAPRICORNUTUM: SOP #2027
8.1 SCOPE AND APPLICATION
The procedure for conducting a 96-hour static
toxicity test using Selenastrum capricomutum is
described below. The endpoint of this test is
growth, measured by increase in cell count,
chlorophyll content, biomass, or absorbance
(turbidity). This test may be conducted on
effluents, leachates or liquid phase of sediments.
This test will also identify a test medium that is
biostimulatory (Horning and Weber, 1985).
8.2 METHOD SUMMARY
Selenastrum capricomutum is exposed to various
concentrations of a test medium over a 96-hour
period and growth is measured at the end of the
test.
8.3 SAMPLE PRESERVATION,
CONTAINERS, HANDLING, AND
STORAGE
The selected environmental medium will be sampled
utilizing the methodology detailed in ERT Standard
Operating Procedures (SOPs) #2012, Soil Sampling;
#2013, Surface Water Sampling; #2016, Sediment
Sampling, and any other procedure applicable to the
medium sampled.
Once collected, samples will be placed in containers
constructed from materials suitable for the
suspected contaminants. Because surrogate test
species will be exposed to varying concentrations of
the sample material, no chemical preservatives are
to be used. The preservation and storage protocol
is therefore limited to holding the samples on ice at
4°C for the holding time specified by the analytical
method. Prior to shipping, the laboratory
performing the toxicity tests will be notified of any
potential hazards that may be associated with the
samples.
8.4 INTERFERENCES AND
POTENTIAL PROBLEMS
• The results of a static toxicity test do not
reflect temporal changes in effluent toxicity.
• The detection limits of the toxicity of a test
medium are organism dependent (Horning
and Weber, 1985).
• Non-target chemicals (e.g., residual
chlorine) may cause adverse effects to the
organisms giving false results.
• Loss of a toxicant through adsorption to
exposure chambers and volatilization may
occur (Peltier and Weber, 1985).
• The concentrations of natural nutrients in
the test medium may affect the results
(Horning and Weber, 1985).
8.5 EQUIPMENT/APPARATUS
8.5.1 Apparatus
Selenastrum capricomutum culture
18 Erlenmeyer flasks - 250 mL
dilution water ~ 1.5 liters
test medium — 1 liter
stock nutrient solutions
centrifuge — 15 - 100 mL capacity
graduated cylinders ~ 10 mL and 100 mL
Erlenmeyer flask — 500 mL
microscope
Depending on the method used to calculate growth,
other equipment may be necessary.
8.5.2 Washing Procedure
1. Wash with warm tap water and non-phosphate
detergent.
2. Rinse with tap water.
3. Rinse with 10% HC1.
29
-------
4. Rinse with deionized water.
5. Rinse with 100% acetone.
6. Rinse with deionized water.
7. Final rinse with dilution water.
8.5.3 Test Organisms
SeJcnastnun capncomutum may be raised in-house
or received from an outside source. Positive
identification of the species is required before
beginning the test. A stock culture that is 4 to 7
days old is required for this test. Horning and
Weber (1985) provide detailed information on the
preparation of culture medium and stock culture.
8.5.4 Equipment for Chemical
Analysis
Meters are needed to measure dissolved oxygen,
temperature, pH, and conductivity. Calibrate the
meters according to the manufacturer's
specifications. Measure and record alkalinity and
hardness according to a standard method (American
Public Health Association, 1985).
8.6 REAGENTS
8.6.1 Dilution Water
Dilution water is moderately hard, reconstituted
deionized water unless otherwise specified. The
dilution water for the test is the same water used to
culture Selenastrum capricomutum. See Horning
and Weber (1985) for the preparation of synthetic
fresh water.
8.6.2 Test Medium
If the test medium is a liquid, dilutions may be
made directly for the required concentrations. If
the test medium is a liquid phase of a sediment,
preliminary filtration and dilutions are required. To
eliminate false negative results due to low nutrient
concentrations, add 1 mL of stock culture solution
(except EDTA) per liter of test medium prior to
preparing test concentrations.
8.6.3 Stock Culture Solution
The methods needed to prepare the stock culture
solution and the amount of chemicals needed to
prepare the solution are found in Horning and
Weber, 1985. One liter of test medium will provide
three replicates of 100 mL each for six
concentrations and 400 mL for chemical analyses
(Horning and Weber, 1985).
Table 15: Example 8
Test Media Concentrations
(% test media)
0
1
3
10
30
100
Test Dilution Volumes (mL)
Diluent
300
297
291
270
210
0
Test Media
0
3
9
30
90
300
30
-------
8.7 PROCEDURES
1. Maintain a stock culture of algae at 24°C ± 2°C
under continuous lighting.
2. Transfer 1-2 mL aseptically to new test
medium once a week in order to maintain an
uncontaminated and healthy culture.
3. To prepare the inoculum, follow the steps
below (Horning and Weber, 1985).
4. An inoculum is prepared from the stock
solution 2 to 3 hours prior to the beginning of
the test. Each milliliter of inoculum must
contain enough cells to provide an initial cell
density of 10,000 cells/mL hi the exposure
chamber. Therefore, each milliliter of inoculum
must contain 1 million cells if using 100 mL test
volume. Use the formula below to determine
the amount of stock solution required for the
test.
5. Volume of stock solution required (mL) = (#
of flasks) (vol. of test soln. per flask) x 10,000
cells/mL cell density in stock culture.
a. Determine the density of cells in the stock
solution.
b. Calculate the required volume of stock
solution (from the equation above).
c. Centrifuge 50% more than the calculated
value of stock solution at 1000 x g (g =
gravitational constant) for 5 minutes.
d. Decant the supernatant and resuspend in
15 mL of deionized water.
e. Repeat steps c and d.
f. Mix and determine the cell count and
dilute as necessary to obtain a cell density
of 106 cells/mL.
6. If possible, choose a range of concentrations
that will span those with no effect to that which
will cause complete mortality. The
concentrations in Table 15: Example 8 may be
adjusted to meet the specific needs of the test.
7. Measure 100 mL of dilution water into each of
the three control flasks.
8. Mix 3 mL of test medium with 297 mL of
dilution water into a mixing bucket.
9. Pour 100 mL into each 1% test medium flask.
10. Continue with these dilutions until all
concentrations are mixed.
11. Add 1 mL of test inoculum to each flask and
begin the test.
12. At 1 to 2 hours, check the cell density of the
controls to ensure sufficient test organisms.
There are no renewals of test solutions for the
duration of the test and the test is complete at
96 hours.
13. Measure and record temperature, dissolved
oxygen, pH, conductivity, alkalinity, and
hardness on all test solutions.
14. Growth is measured at the end of the test by
cell counts, chlorophyll content or turbidity
(light absorbance), or biomass. Cell counts
may be determined with an automatic particle
counter or manually under a microscope.
Chlorophyll content may be measured using in-
vivo or in-vitro fluorescence or in-vitro
spectrophotometry. Turbidity may be
measured by spectrophotometry at 750 nm.
Biomass is measured by multiplying the cell
count by the mean cell volume or by direct
gravimetric dry weight analysis. Horning and
Weber (1985) provide details of the
methodologies for these measurements.
15. At the completion of the test, samples should
be checked under a microscope to detect any
abnormal cell growth or other deviations.
16. It also may be necessary to check algal growth
on a daily basis depending on the test medium.
8.8 CALCULATIONS
The No Observable Effect Concentration (NOEC),
the Lowest Observable Effect Concentration
(LOEC), and the chronic value (CHV) are
measured and recorded at the end of 96 hours.
Dunnetts procedure or the Probit Method may be
used to calculate the NOEC and LOEC. When the
assumptions for normality and homogeneity of
variance are not met, Steel's Many - One Rank Test
may be used. Other methods may be used if
justified and the appropriate method is cited.
Calculate the percent stimulation (%S) if growth in
the concentrations exceeds the growth in the
controls.
31
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8.9 QUALITY ASSURANCE/
QUALITY CONTROL
Follow the guidelines in this SOP, which are
summarized in Table 16, for adequate QA/QC.
8.10 DATA VALIDATION
Test data is invalidated for the following reasons.
• Cell density in the controls is less than 106
cell/mL at the end of the test and the
number does not vary by more than 10%
between control replicates.
• Parameters in Table 16 are not met.
8.11 HEALTH AND SAFETY
When working with potentially hazardous materials,
follow U.S. EPA, OSHA and specific health and
safety guidelines.
Table 16: Summary of Test Conditions for a 96-Hour Static Toxicity Test using
Selenastrum capricornutum*
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
Test type
Temperature
Light intensity
Photoperiod
Exposure chamber size
Test volume
Stock culture
Cell density
Replicates
Shaking rate
Dilution water
Test duration
Effects measured
Static, non-renewal
25.0°C ± 2°C
400 ± 40 foot candles
Continuous
250-mL containers
100 mL
4-7 days old
10,000 cells per mL
3 per concentration
Twice daily by hand or 100 cpm
Reconstituted, deionized water, unless otherwise specified.
Also the same as the culture water without the EDTA
96 hours
Growth
Based on Horning and Weber, 1985
32
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9.0 10-DAY CHRONIC TOXICITY TEST USING
DAPHNIA MAGNA OR DAPHNIA PULEX: SOP #2028
9.1 SCOPE AND APPLICATION
The procedure for conducting a 10-day chronic
toxicity test using Daphnia magtia or Daphniapulex
is described below. This test is applicable to
leachates, effluents, and liquid phases of sediments.
Mortality, reproduction and growth are used to
assess the toxicity of the test medium.
9.2 METHOD SUMMARY
Larval daphnids are placed hi individual containers
and exposed to different concentrations of a test
medium over a 10-day period. Concentrations are
renewed every other day and mortality, reproduction
and growth are recorded.
chlorine) may cause adverse effects to the
organisms giving false results.
• Dissolved oxygen depletion due to
biological oxygen demand and/or chemical
oxygen demand (e.g., metabolic wastes) is
also a potential problem.
• Loss of a toxicant through adsorption to
exposure chambers and volatilization may
occur (Peltier and Weber, 1985).
• The results of a static toxicity test do not
reflect temporal fluctuation in test medium
toxicity (Peltier and Weber, 1985). Also
the effect of the toxicant is organism
dependent.
9.3 SAMPLE PRESERVATION,
CONTAINERS, HANDLING, AND
STORAGE
The selected environmental medium will be sampled
utilizing the methodology detailed in ERT Standard
Operating Procedures (SOPs) #2012, Soil Sampling;
#2013, Surface Water Sampling; #2016, Sediment
Sampling, and any other procedure applicable to
the medium sampled.
Once collected, the samples will be placed in
containers constructed from materials suitable for
the suspected contaminants. Because surrogate test
species will be exposed to varying concentrations of
the sample material, no chemical preservatives are
to be used. The preservation and storage protocol
is therefore limited to holding the samples on ice at
4°C for the holding tune specified by the analytical
method. Prior to shipping, the laboratory
performing the toxicity tests will be notified of any
potential hazards that may be associated with the
samples.
9.4 INTERFERENCES AND
POTENTIAL PROBLEMS
• Non-target chemicals (e.g., residual
9.5 EQUIPMENT/APPARATUS
9.5.1 Apparatus
60 larval daphnids ~ acclimated at least 24
hours to dilution water
60 exposure chambers — 100 mL volume,
labeled
tray to hold exposure chambers and glass
covers
wide-bore pipettes — inside diameter 1.5
times the length of the daphnid
graduated cylinders — 250 mL and 1 liter
pipette — 1 mL
beakers — 250 mL
volumetric flasks — 500 mL
test medium — 1 L/day
diluent — 3 L/day
waste containers
light table — to assist in counting the
organisms
suitable food
9.5.2 Washing Procedure
1. Wash with warm water and detergent
2. Rinse with tap water
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3. Rinse with 10% nitric acid solution
4. Rinse with deionized water
5. Rinse with 100% acetone
6. Rinse with deionized water
7. Final rinse with dilution water.
9.5.3 Test Organisms
Test organisms may be reared in-house or obtained
from an outside source. Positive identification of
the species is required before beginning the test.
Daphnids to be used must be less than 24 hours old
and from the second to the sixth brood of a healthy
adult. Populations of healthy daphnids have large
individuals, have an absence of floaters, have an
absence of ephippia, and have an absence of
parasites. Individuals are dark colored and produce
large numbers of young (Biesinger, et al. 1987).
9.5.4 Equipment for Chemical
Analysis
Meters are needed to measure dissolved oxygen,
temperature, pH and conductivity. Calibrate the
meters according to the manufacturer's instructions.
Measure alkalinity and hardness according to a
standard method (American Public Health
Association, 1985).
9.6 REAGENTS
9.6.1 Dilution Water
Dilution water is reconstituted deionized water
unless otherwise specified. See Horning and Weber
(1985) for the preparation of synthetic fresh water.
Set up a laboratory or standard dilution water
control when reconstituted deionized water is used
as the dilution water. The dilution water for a test
is the same as the water used to culture daphnids
and the water used to acclimate daphnids before the
beginning of the test.
9.6.2 Test Medium
If the test medium is a liquid, dilutions may be
made directly for the required concentrations. If
the test medium is a sediment, preliminary filtration
and dilutions are required to produce a liquid
phase.
9.7 PROCEDURES
1. Choose a range of concentrations that span
those causing zero mortality to those causing
complete mortality (indicated by a total
absence of movement, even when prodded).
The concentrations cited in Table 17: Example
9 may be adjusted to meet the criteria of the
specific situation. A geometric or logarithmic
range of concentrations also may be used
(Sprague, 1973).
Table 17: Example 9
Test Media Concentrations
(% test media)
.00
0.1
1.0
10
50
100
Test Dilution Volumes (mL)
Diluent
500.0
499.5
495.0
450.0
250.0
0
Test Media
0
0.5
5.0
50.0
250.0
500.0
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2. The example below provides enough test
medium for five replicates containing 80 mL
each and extra for chemical analysis. Other
ranges may be used according to needs of the
analyses.
3. Rinse all exposure chambers, except the
chamber containing 100% test medium, hi
dilution water before the start of the test.
4. Draw 0.5 mL of the test medium into a
volumetric flask and dilute to 500 mL. Using a
graduated cylinder, pour 80 mL into each
exposure chamber labeled for 0.1% test
concentration and pour the rest into a beaker
for chemical measurements.
5. Continue step 4 for all concentrations. Always
work from the lowest concentration to the
highest in order to minimize the risk of cross-
contamination.
6. Using a wide-bore pipette, randomly select and
carefully place one daphnid into each exposure
chamber by placing the pipette tip below the
surface and gently expelling the daphnid into
the chamber.
7. The test begins when half of the organisms are
in the exposure chambers.
8. Concentrations are renewed every other day for
the duration of the test. However, if the test
begins on a Monday, then renewals may be
done on Wednesday, Friday and the following
Monday and Wednesday.
9. Measure and record mortality and survival at 1
hour and then when test concentrations are
renewed. Count the number of live or dead
young produced by each female.
10. Measure temperature, dissolved oxygen, pH,
conductivity, alkalinity and hardness of all new
concentrations. Conduct these measurements
on old test concentrations at least three times
during the test.
11. Prepare test medium concentrations as done
previously. Pour the concentrations into new
exposure chambers, reserving extra for chemical
analyses.
12. Count the number of live or dead adults and
young, using a light table if necessary.
13. Record these results and then carefully transfer
the adult daphnid into the new concentrations.
14. Using a suitable food, feed daphnids once daily
during the test.
15. After feeding the daphnids, cover the exposure
chamber to reduce evaporation of the test
concentrations.
9.8 CALCULATIONS
The methods used to determine the ECso differ
depending on the results of the test. If there is no
partial mortality in any replicate (i.e. all alive or all
dead), then the Moving-Average Method may be
used to determine the EC^,. If there is partial
mortality within a replicate, then the Probit
Method should be used to calculate the ECX. Also
the Lowest Observable Effect Concentration
(LOEC) is recorded and the No Observable Effects
Concentration (NOEC) is recorded (Peltier and
Weber, 1985). Dunnett's many-one t procedure or
Bonferroni's t procedure (Miller, 1966) maybe used
to determine comparisons between the organisms's
response to the test medium concentrations as
compared to the control. Other methods of
estimating the response values may be used if
justified and an accepted reference is cited
(Biesinger, ejt al. 1987).
9.9 QUALITY ASSURANCE/
QUALITY CONTROL
Follow the guidelines in this SOP, which are
summarized in Table 18, for adequate QA/QC.
9.10 DATA VALIDATION
Test data is invalidated for the following reasons:
• Greater than 20% control mortality.
• Standard reference toxicant results greater
than two standard deviations from an
accepted value (American Public Health
Association, 1985).
• Greater than 20% aberrant mortality in
concentrations.
• Temperature variation greater than 2°C.
• Test medium stored more than 72 hours.
• Criteria in Table 18 not met.
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9.11 HEALTH AND SAFETY
When working with potentially hazardous materials,
follow U.S. EPA, OSHA and specific health and
safety procedures.
Table 18: Summary of Test Conditions for a 10-Day Chronic Toxicity Test using
Daphnia magna or Daphnia pulex*
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
Test type
Temperature
Light quality
Light intensity
Photoperiod
Test chamber size
Test solution volume
Renewal
Age of test organisms
Number/container
Feeding
Aeration
Dilution water
Test media/leachate
concentrations
Test duration
Effects measured
Static, renewal
25.0°C ± 2°C
Ambient laboratory illumination
50-100 foot candles
16 hours light, 8 hours dark
100-mL containers
80 mL/replicate
Every other day
Less than 24 hours old
1 per exposure chamber
Feed on day of renewal
None unless DO concentration falls below 40% saturation,
then <100 bubbles per minute
Moderately hard, reconstituted, deionized water
Minimum of 5 and 1 control
10 days
Survival, growth, and reproduction
Based on Horning and Weber, 1975.
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References
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•U.S. Government Printing Offlco: 1991 — 648-187/40581 33
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