EPA 600/3-88/029
PB88 235 510/AS
ERL-COR-496
PROTOCOLS FOR SHORT TERM TOXICITY SCREENING
OF HAZARDOUS WASTE SITES
by
Joseph C. Greene1, Cathy L. Bartels2,
William J Warren-Hicks3, Benjamin R. Parkhurst4,
Gregory L. Under2, Spencer A. Peterson1,
and William E. Milleri
1 United States Environmental Protection Agency
Corvallis Environmental Research Laboratory
Ecotoxicology Branch
Hazardous Waste Assessment Team
200 S.W. 35th Street
Corvallis, OR 97333
2Northrop Services, Incorporated
Corvallis Environmental Research Laboratory
Hazardous Waste and Water Section
200 S.W 35*h street
Corvallis, OR 97333
^Kilkelly Environmental Associates
Highway 70 West -- The Water Garden
Raleigh, NC 27622
4Western Aquatics, Inc.
P.O Box 546
203 Grand Avenue
Laramie, WY 82070
U.S. Environmental Protection Agency
Region 5, library (PL-12J)
77 West Jackson Boulevard, 12th Floor
Chicago, IL 60604-3590
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EXECUTIVE SUMMARY
This manual contains short-term methods for measuring the toxicity of chemical
contaminants in soil, sediment, surface water, and groundwater samples. The algal assay is a chronic
test, while all other tests described in the manual are acute tests. The methods are one of several
tools, including chemical analysis and field study, used to determine toxicity of hazardous waste
sites. The toxicity tests provided in this manual can be used to detect toxic materials, rank sites based
on relative short-term toxicity, and provide a cost-effective approach to monitoring the effectiveness
of site cleanup
The methods include aquatic and terrestrial tests. The algae, macroinvertebrate, and root
elongation tests are used to assess toxicity in surface water, groundwater, and sediment or soil
elutriates. The earthworm and seed germination tests are used to directly assess toxicity in soil and
sediment samples.
A critical factor in waste site evaluation is establishment of an experimental design that
satisfies the information needs for site evaluation. Components of experimental design (sampling,
statistical considerations, selection of biological tests, logistical support requirements, etc.) are
discussed in the companion document entitled, "Role of Acute Toxicity Bioassays in the Remedial
Action Process at Hazardous Waste Sites" (Athey et al. 1987)
The toxicity tests in this manual are not required by regulation. However, because toxicity
tests measure the integrated effects of complex chemical waste mixtures, they provide a reasonable
basis for assessing the toxicity of waste products independent of existing concentration criteria.
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TABLE OF CONTENTS
Section Title Page
Executive Summary ii
Glossary ix
Acknowledgments xiii
1. INTRODUCTION 1
1.1 Purpose 1
1.2 Organization of the Document 1
1.3 Background 2
1.4 Ongoing Research 4
1.5 Uses of Toxicity Tests in Identifying and Characterizing the Toxicity
of Hazardous Wastes 5
1.5.1 Site Prioritization 5
1.5.2 Waste Characterization 7
1.5.3 Site Characterization 7
1.5.4 Cleanup Standards 8
1.5.5 Site Monitoring 8
2. DATA ANALYSIS 9
2.1 Methods for Calculating Measures of Toxicity 9
2.1.1 Percent Mortality 9
2.1.2 Effect Proportion 10
2.2 Calculating the LC50 and EC50 10
2.3 Use of the Toxicity Test Results 12
3. SAMPLING CONSIDERATIONS 15
3.1 WhattoSample 15
3.2 Variability Issues 15
4. CASE STUDY 17
4.1 Background Information 17
4.2 Study Objectives 17
4.3 Study Procedures 18
4.4 Analysis and Evaluation 18
5. REFERENCES 21
Appendix A - Toxicity Test Methods 22
A.1 SCOPE AND APPLICATION 22
A.2 HEALTH AND SAFETY 23
A.2.1 General Precautions 23
A.2.2 Safety Manuals 23
in
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Section Title Page
A.3 QUALITY ASSURANCE 24
A.3.1 Introduction 24
A.3.2 Hazardous Waste Sampling and Handling 24
A.3.3 Test Organisms 24
A.3.4 Facilities, Equipment, and Test Chambers 24
A.3.5 Analytical Methods 24
A.3.6 Calibration and Standardization 24
A.3.7 Dilution Water 24
A.3.8 Test Conditions 25
A.3.9 Test Acceptability 25
A.3.10 Precision 25
A.3.11 Replication and Test Sensitivity 25
A.3.12 Quality of Test Organisms 25
A.3.13 Quality of Food for Test Organisms 26
A.3.14 Control Charts 26
A.3.15 Record Keeping 27
A.4 FACILITIES AND EQUIPMENT 28
A.4.1 General Requirements 28
A.4 1.1 Laboratory Facilities 28
A.4.1.2 Materials 28
A.4.1.3 Adhesives 28
A.4.2 Test Chambers 29
A.4.3 Cleaning 29
A.5 TEST ORGANISMS AND CULTURE METHODS 30
A.5.1 Introduction 30
A.5.2 Culture Methods for D. spp. (D. maqna and D. pulex) 30
A.5.2.1 Geographical and Seasonal Distribution 30
A.5.2.2 Life Cycle 31
A.5.2.3 Morphology and Taxonomy 31
A.5.2.4 Culturing Methods 31
A.5.2.5 Test Organisms 35
A.5.3 Culture Methods for Fathead Minnow (P. promelas) 36
A.5.3.1 Distribution 36
A.5.3.2 Life Cycle 36
A.5.3.3 Taxonomy 37
A.5.3.4 Morphology and Identification - General Characteristics 37
A.5.3.5 Hybridization 37
A.5.3.6 Culturing Methods 37
A.5.3.7 Test Organisms 40
A.5.4 Culture Methods for Brine Shrimp 40
A.5.4.1 Sources of Brine Shrimp Eggs 40
A.5.4.2 Incubation Chamber and Procedure 40
A.5.4.3 Harvesting the Nauplii 41
A.5.5 Culture Methods for Algae, S. capricornutum 41
A.5.5.1 Test Organisms 41
A.5.5.2 Stock Algal Cultures 41
A.5.5.3 Culture Medium 42
IV
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Section Title Page
A.5.6 Culture Methods for Earthworms (E. foetid a) 42
A.5.6.1 Introduction 42
A.5.6.2 Distribution 42
A.5.6.3 Life Cycle 42
A.5.6.4 Morphology and Taxonomy 45
A.5.6.5 Culture Methods 45
A.5.6.6 Test Organisms 46
A.6 DILUTION WATER 47
A.7 HAZARDOUS WASTE SAMPLING AND HANDLING 49
A.7.1 Apparatus and Equipment 49
A.7.2 Hazardous Waste Sampling, Handling, and Storage 50
A.7.2.1 Aqueous Hazardous Wastes 50
A.7.2.2 Solid Hazardous Wastes 50
A.7.2.3 Labeling Requirements 52
A.7.3 Sample Handling and Preservation 52
A.7.4 Aqueous Sample Preparation 52
A.7.5 Elutriate Preparation 52
A.7.6 Water Holding Capacity 55
A.7.7 Chemical or Physical Modification of Test Materials 55
A.8 TOXICITY TEST METHODS 56
A.8.1 Introduction 56
A.8.2 Daphnia pulex and Daphnia maqna Survival 56
A.8.2.1 Scope and Application 56
A.8.2.2 Summary of Method 56
A.8.2.3 Definitions 56
A.8.2.4 Interferences 56
A.8.2.5 Safety 56
A.8.2.6 Apparatus and Equipment 57
A.8.2.7 Reagents and Consumable Materials 58
A.8.2.8 Sample Collection, Preservation, and Handling 58
A.8.2.9 Calibration and Standardization 58
A.8.2.10 Quality Control 59
A.8.2.11 Procedure 59
A.8.2.12 Calculations 60
A.8.2.13 Precision and Accuracy 61
A.8.3 Fathead Minnow Survival (Pimphales promelas) 63
A.8.3.1 Scope and Application 63
A.8.3.2 Summary of Method 63
A.8.3.3 Definitions 63
A.8.3.4 Interferences 63
A.8.3.5 Safety 63
A.8.3.6 Apparatus and Equipment 63
A.8.3.7 Reagents and Consumable Materials 64
A.8.3.8 Sample Collection, Preservation, and Storage 65
A.8.3.9 Calibration and Standardization 65
A.8.3.10 Quality Control 65
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Section Title Page
A.8.3.11 Procedures 65
A.8.3.12 Calculations 67
A.8.3.13 Precision and Accuracy 67
A.8.4 Algal Growth (Selenastrum capricornutum) 69
A.8.4.1 Scope and Application 69
A.8.4.2 Summary of Method 69
A.8.4.3 Definitions 69
A.8.4.4 Interferences 69
A.8.4.5 Safety 69
A.8.4.6 Apparatus and Equipment 69
A.8.4.7 Reagents and Consumable Materials 71
A.8.4.8 Sample Collection. Preservation, and Handling 72
A.8.4.9 Calibration and Standardization 72
A.8.4.10 Quality Control 72
A.8.4.11 Procedures 72
A.8.4.12 Calculations 76
A.8.4.13 Precision and Accuracy 76
A.8.5 Earthworm Survival (Eisenia foetida) 78
A.8.5 1 Scope and Application 78
A.8.5.2 Summary of Method 78
A.8.5.3 Definitions 78
A.8.5.4 Interferences 78
A.8.5.5 Safety 78
A.8.5.6 Apparatus and Equipment 78
A.8.5.7 Reagents and Consumable Materials 79
A.8.5.8 Sample Collection. Preservation, and Storage 79
A.8.5.9 Calibration and Standardization 80
A.8.5.10 Quality Control 80
A.8.5.11 Procedures 80
A.8.5.12 Calculations 82
A.8.5.13 Precision and Accuracy 82
A.8.6 Lettuce Seed Germination (Lactuca sativa) 84
A.8.6.1 Scope and Application 84
A.8.6.2 Summary of Method 84
A.8.6.3 Definitions 84
A.8.6.4 Interferences 84
A.8.6.5 Safety 84
A.8.6.6 Apparatus and Equipment 84
A.8.6.7 Reagents and Consumable Materials 85
A.8.6.8 Sample Collection, Preservation, and Storage 86
A.8.6 9 Calibration and Standardization 86
A.8.6.10 Quality Control 86
A.8.6.11 Procedures 86
A.8.6.12 Calculations 88
A.8.6.13 Precision and Accuracy 88
A.8.7 Lettuce Root Elongation (Lactuca sativa) 90
A.8.7.1 Scope and Application 90
A.8.7.2 Summary of Method 90
A.8.7.3 Definitions 90
A.8.7.4 Interferences 90
VI
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Section Title Page
A.8.7.5 Safety 90
A.8.7.6 Apparatus and Equipment 90
A.8.7.7 Reagents and Consumable Materials 91
A.8.7.8 Sample Collection, Preservation.and Storage 92
A.8.7.9 Calibration and Standardization 92
A.8.7.10 Quality Control 92
A.8.7.11 Procedures 92
A.8.7.12 Calculations 94
A.8.7.13 Precision and Accuracy 94
A.9 REPORT PREPARATION 96
A.10 REFERENCES 98
VII
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LIST OF FIGURES
Figure Title Page
1 Roleof toxicity tests in the hazardous waste site evaluation process 6
A-1 Control chart 26
A-2 Waste site sample collection data sheets 53
LIST OF TABLES
Table Title Page
1 Example Use of Toxicity Test Results. Calculated LC50 or EC50 as
Percent Sample Concentration (95% Confidence Interval) 13
2 Toxicity Test Results from Samples Collected at the Railroad Tie
Treatment Plant 20
A-1 Media for D. magna and D. pulex 32
A-2 Nutrient Stock Solutions for Maintaining Algal Stock Cultures and
Test Control Cultures 43
A-3 Final Concentration of Macronutrients and Micronutrients in the
Culture Medium 44
A-4 Preparation of Synthetic Fresh Water 48
A-5 Quantities of Aqueous Hazardous Waste, Solid Hazardous Waste,
and/or Elutriate Required to Perform the Six Toxicity Tests and
Routine Chemical Analyses 51
A-6 Summary of Recommended Test Conditions for D. pulex and D. maqna
Survival Test 62
A-7 Summary of Recommended Test Conditions for Fathead Minnow
(P. promelas) Survival Test 68
A-8 Summary of Recommended Test Conditions for the Algal Growth Test
(S. capricorntum) 77
A-9 Summary of Recommended Test Conditions for the E. foetid a
Survival Test 83
A-10 Summary of Recommended Test Conditions for Lettuce Seed (L sativa)
Germination Test 89
A-11 Summary of Recommended Test Conditions for Lettuce (L sativa)
Root Elongation Test 95
VIII
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GLOSSARY
Acclimation-(l) Steady state compensatory adjustments by an organism to the alteration of
environmental conditions. Adjustments can be behavioral, physiological, or biochemical.
(2) Referring to the time period prior to the initiation of a toxicity test in which organisms are
maintained in untreated, toxicant-free dilution water or soil with physical and chemical
characteristics, e.g., temperature, pH, hardness, similar to those to be used during the toxicity test.
Acute-Involving a stimulus severe enough to rapidly induce a response. An acute effect is not
always measured in terms of lethality; it can be measured in terms of a variety of endpoints. Note
that acute means short, not mortality.
Additivity-The characteristic property of a mixture of toxicants that exhibits a cumulative toxic
effect equal to the arithmetic sum of the effects of the individual toxicants.
Alkalinity-The acid-neutralizing (proton-accepting) capacity of water; the quality and quantity of
constituents in water which result in a shift in the pH toward the alkaline side of neutrality.
Antagonism-The characteristic property of a mixture of toxicants that exhibits a less-than-additive
cumulative toxic effect.
Artificial soil-Non-toxic synthetic soil, prepared in the laboratory using a specific formulation, used
as a control and diluent medium in the earthworm and lettuce seed germination tests.
Biological oxygen demand (BOD)-The amount of oxygen necessary for the oxidative decomposition
of a material by microorganisms.
Biota-Animal and plant life.
Carcinogenic-Capable of causing cancer.
Chronic-Involving a stimulus that lingers or continues for a relatively long period of time, often one-
tenth of the life span or more. Chronic should be considered a relative term depending on the life
span of an organism. A chronic effect can be lethality, altered growth, reduced reproduction, etc.
Clitellum-Thickened saddle-like portion of certain mid-body segments in many oligochaetes and
leeches; in some species it is prominent only in sexually mature individuals; forms rings of epithelial
tissue and mucus as cocoons in which eggs are enclosed; during copulation it also gives off mucus
which envelopes the anterior ends of the two individuals.
Control--A treatment in a toxicity test that duplicates all the conditions of the exposure treatments
but contains no test material. The control is used to determine the absence of toxicity of basic test
conditions, e.g., health of test organisms, quality of dilution water.
Criteria (water quality)-An estimate of the concentration of a chemical or other constituent in water
which if not exceeded, will protect an organism, an organismal community, or a prescribed water use
or quality with an adequate degree of safety.
Dilution water-Water used to dilute the test material in an aquatic toxicity test in order to prepare
either different concentrations of a test chemical or different percentages of an aqueous sample for
the various test treatments. The water (negative) control in a test is prepared with dilution water
only.
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Effluent-A complex waste material, e.g., liquid industrial discharge or sewage, which is discharged
into the environment.
Elutriate-Solution obtained after adding water to the solid waste, shaking, and centrifuging (see
Section A.7.5 in the Appendix).
Ephippia-Special postero-dorsal part of the carapace of certain female cladocera which contains one
to several eggs usually fertilized.
Exposure-The interaction between a chemical or physical agent and a biological system.
Hardness-The concentration of ions in the water which will react with a sodium soap to precipitate
an insoluble residue. In general, hardness is a measure of the concentration of calcium and
magnesium ions in water and is frequently expressed as mg/L calcium carbonate equivalent.
Hazard-Likelihood that a chemical will cause an injury or adverse effect under conditions of its
production, use, or disposal.
Hazardous substance-A material that can pose a hazard causing deleterious effects in plants or
animals. (A hazardous substance does not in itself present a risk unless an exposure potential exists.)
lnstar--(1) Period or stage between molts of an insect during larval or nymph portion of the life
history; instars are usually numbered, the first larval instar being that stage hatching from the egg
and extending to the first ecdysis. (2) In the broad sense, the period between any two successive
molts in an arthropod, nematode, tardigrade, etc.
Leachate-Water plus solutes that has percolated through a column of soil or waste.
Lethal-Causing death by direct action. Death of organisms is the cessation of all visible signs of
biological activity.
Loading-Ratio of the animal biomass to the volume of test solution in an exposure chamber.
Macroinvertebrates-Large invertebrate organisms, sometimes arbitrarily defined as those retained
by sieves with 0.425-mm to 1.0-mm mesh screens.
Median effective concentration (ECSO)-The concentration of material to which test organisms are
exposed that is estimated to be effective in producing some sublethal response in 50% of the test
organisms. The EC50 is usually expressed as a time-dependent value, e.g., 24-h or 96-h EC50. The
sublethal response elicited from the test organisms as a result of exposure to the test material must
be clearly defined. For example, test organisms may be immobilized, lose equilibrium, or undergo
physiological or behavioral changes.
Median lethal concentration (LCSO)--The concentration of material to which test organisms are
exposed that is estimated to be lethal to 50% of the test organisms. The LC50 is usually expressed as
a time-dependent value, e.g , 24-h or 96-h LC50; the concentration estimated to be lethal to 50% of
the test organisms after 24 or 96 h of exposure. The LC50 may be derived by observation (50% of the
test organisms may be observed to be dead in one test material concentration), by interpolation
(mortality of more than 50% of the test organisms occurred at one test concentration and mortality
of fewer than 50% of the test organisms died at a lower test concentration; the LC50 is estimated by
interpolation between these two data points), or by calculation (the LC50 is statistically derived by
analysis of mortality data from all test concentrations).
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Monitoring test--A test designed to be applied on a routine basis, with some degree of control, to
ensure that the quality of water, elutriate, or hazardous waste has not exceeded some prescribed
range. In a biomonitoring test, organisms are used as "sensors" to detect changes in the quality of
water, elutriate, or hazardous waste. A monitoring test implies generation of information, on a
continuous or other regular basis.
Neonates-IMewly born or newly hatched individuals.
Parts per billion (ppb)-One unit of chemical (usually expressed as mass) per 1,000,000,000 (109) units
of the medium (e.g., water) or organism (e.g , tissue) in which it is contained. For water, the ratio
commonly used is micrograms of chemical per liter of water, 1 ug = 1 ppb; for tissues, 1 ug/kg =
1 ng/g = 1 ppb.
Parts per million (ppm)--One unit of chemical (usually expressed as mass) per 1,000,000 (106) units of
the medium (e.g., water) or organism (e.g., tissue) in which it is contained. For water, the ratio
commonly used is milligrams of chemical per liter of water, 1 mg = 1 ppm; for tissues, 1 mg/kg =
1 ug/g = 1 ppm.
Parts per thousand (ppt)--One unit of chemical (usually expressed as mass) per 1000 (103) units of the
medium (e.g., water) or organism (e.g., tissue) in which it is contained. For water, the ratio
commonly used is grams of chemical per liter of water, 1 g/L = 1 ppt; for tissues, 1 g/kg = 1 ppt. This
ratio is also used to express the salinity of seawater, where the grams of chloride per liter of water is
denoted by the symbol ppt. Full-strength seawater is approximately 35 ppt.
Parts per trillion (pptr)-One unit of material (usually expressed as mass) per 1,000,000,000,000 (1012)
units of the medium (e.g., water) or organism (e.g., tissue) in which it is contained. The ratio
commonly used is nanograms of chemical per liter of water, 1 ng/L = 1 pptr; for tissues, 1 ng/kg =
1 pptr
Percentage (%)-One unit of material (usually a liquid) per 100 units of dilution water. In tests with
industrial wastes (effluents), test concentrations are normally prepared on a volume-to-volume basis
and expressed as percent of material.
Persistence-That property of a toxicant that is a measurement of the duration of its effect. A
persistent toxicant maintains its effect after mixing, degrading slowly. A non-persistent toxicant
may have a quickly reduced effect as processes such as biodegradation, volatilization, photolysis,
etc., transform the chemical or reduce its concentration.
Phytotoxic-Toxicto plants.
Probability--A number expressing the likelihood of occurrence of a specific event, such as the ratio of
the number of outcomes that will produce a given event to the total number of possible outcomes.
Quality assurance (QA)-A program organized and designed to provide accurate and precise results.
Included are selection of proper technical methods, tests, or laboratory procedures; sample
collection and preservation; selection of limits; evaluation of data; quality control; and
qualifications and training of personnel.
Quality control (QC)-Specific actions required to provide information for the quality assurance
program. Included are standardizations, calibration, replicates, and control and check samples
suitable for statistical estimates of confidence of the data.
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Standard sample-A sample of constant and defined composition (e.g., synthetic water or artificial
soil).
Static-Describing toxicity tests in which test materials are not renewed.
Statistically significant effects-Effects (responses) in the exposed population that are different from
those in the controls at a prespecified probability level. Biological endpoints that are important for
the survival, growth, behavior, and perpetuation of a species are selected as endpoints for effect.
The endpoints differ depending on the type of toxicity test conducted and the species used. The
statistical approach also changes with the type of toxicity test conducted.
Sublethal-lnvolving a stimulus below the level that causes death.
Survival time-The time interval between initial exposure of an organism to a harmful chemical and
death.
Synergism-The characteristic property of a mixture of toxicants that exhibits a greater-than-additive
cumulative toxic effect.
Test material-A chemical, formulation, elutriate, sludge, or other agent or substance that is under
investigation in a toxicity test.
Test soil-A mixture of contaminated site soil and artificial soil to provide a medium for exposing test
organisms to solid hazardous waste.
Test solution (or test treatment) -Medium containing the material to be tested to which the test
organisms are exposed. Different test solutions contain different concentrations of the test material.
Toxicant-An agent or material capable of producing an adverse response (effect) in a biological
system, adversely impacting structure or function or producing death.
Toxic endpoints-Measurements of acute or chronic toxicity for toxic substances, including exposure
duration, concentration, and observed effects.
Toxicity—The inherent potential or capacity of a material to cause adverse effects in a living
organism.
Toxicity curve-The curve obtained by plotting the median survival times of a population of test
organisms against concentration on a logarithmic scale.
Toxicity test-The means by which the toxicity of a chemical or other test material is determined. A
toxicity test is used to measure the degree of response produced by exposure to a specific level of
stimulus or concentration of a chemical.
Water holding capacity-The quantity of water that soil retains after 24 hours following complete
saturation, generally expressed as ml/IOOg soil.
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ACKNOWLEDGMENTS
The cooperation and support of Dr. Royal Nadeau and Dr. David Charters (U.S. EPA,
Environmental Response Team, Edison, NJ) are gratefully acknowledged. The ideas and technical
comments of Dr. Peter Chapman (E.V.S. Consultants, Vancouver, British Columbia, Canada) helped
shape this document. The peer review comments of Dr. Jeffrey Giddings (Springborn Life Sciences,
Wareham, MA), Dr. Wesley Birge (University of Kentucky, Lexington, KY), Dr. Edward Neuhauser
(Niagara Power Corporation, Syracuse, NH), and Dr. William Waller (University of Texas, Dallas, TX)
helped to produce the final manuscript. Our special thanks goes to Dr. Donald Porcella, who was
responsible for the compilation of the 1983 document entitled, "Protocol for Bioassessment of
Hazardous Waste Sites," which provided the basis for testing the comparative responses of the test
organisms to waste site samples, as well as the foundation for this manuscript.
We wish to thank Mike Long, Mary Debacon, Fran Recht, Julius Nwosu, Sheila Smith, Dave
Wilborn, Mike Bollman, and Jennifer Miller of Northrop Services, Inc., Corvallis, OR, who were
responsible for performance of the toxicity bioassays covered in the test protocols We thank Al
Nebeker (U.S. EPA, Western Fish Toxicology Station, Corvallis, OR) for providing the aquatic
invertebrates, and Clarence Callahan (U.S. EPA, CERL, Corvallis, OR) and Loren Russell (Northrop
Services, Inc., Corvallis, OR) for providing the earthworms. We also thank chemists Walt Burns, Glenn
Wilson, Kevin DeWhitt (Northrop Services, Inc , Corvallis, OR), Jerry Wagner, Susan Ott (Ames
Laboratory, University of Iowa, Corvallis, OR) for their support in analytical chemistry. Special
appreciation is also extended to Marilyn Elliott, Norma Case (U.S. EPA, Corvallis, OR), who typed the
original and finished manuscripts. We also wish to thank Kiki Alexander (Northrop Services, Inc.) for
her editorial review of this protocol.
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SECTION 1
INTRODUCTION
1.1 PURPOSE
The purpose of this document is to provide toxicity tests useful for detecting and quantifying
toxicity in hazardous waste sites.
The Comprehensive Environmental Response Compensation and Liability Act (CERCLA) as
amended by the Superfund Amendment and Reauthorization Act of 1986 (SARA) requires a level of
control consistent with goals established by other legislation including the Safe Drinking Water Act
(SDWA) and the Clean Water Act (CWA). In order to meet the requirements of the Water Acts, EPA
has established acceptable limits for specific chemicals. The toxicity tests recommended in this
manual assess the short-term integrated effects of complex mixtures and provide a reasonable basis
for assessing the toxicity of waste products independent of existing concentration criteria.
1.2 ORGANIZATION OF THE DOCUMENT
This document, which is divided into two major parts, provides protocols for toxicity screening
at hazardous waste sites. The first part presents issues relating to the use of toxicity tests, including
background information, ongoing research, and application of tests (Section 1); data analysis and
calculations and uses of toxicity indices (Section 2); sampling considerations, including the choice of
media to sample and variability issues (Section 3); and finally, a hypothetical case study illustrating
the use and application of the tests (Section 4).
The second part of the document is an Appendix which provides detailed, step-by-step
protocols for using the toxicity tests Recommended protocols for toxicity tests are presented for
water flea survival (Daphnia maqna Straus and D. pulex Leydig), algal growth (Selenastrum
capricornutum Printz), fish survival (fathead minnow, Pimephales promelas Rafinesque). lettuce seed
germination (Lactuca sativa L), lettuce root elongation (Lactuca sativa). and earthworm survival
(Eisenia foetida Saviqny). This test battery of protocols can be applied to soils, sediments, elutriates
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from soils or sediments, ground water, and surface water for the purpose of measuring short-term
toxicity effects.
1.3 BACKGROUND
A high priority of the U.S. Environmental Protection Agency (EPA) is to identify, characterize,
and clean up hazardous waste sites These activities are regulated by CERCLA and SARA. Both
CERCLA and SARA address the toxic effects of hazardous wastes, and consequently, toxicity is one of
the principal characteristics used to identify and characterize hazardous waste sites.
The toxicity of wastes can be estimated using two approaches: a toxicity-based approach
and/or a chemical-specific approach. In the toxicity-based approach, toxicity tests are used to
measure toxicity directly. Toxicity is somewhat analogous to the biochemical oxygen demand (BOD)
used in waste water analyses. Both toxicity and BOD are generic measurements of complex chemical
mixtures that do not permit identification of cause-and-effect relationships. The toxicity-based
approach was developed for measuring and assisting in the regulation of the toxicity of complex
effluents discharged to surface waters (EPA 1985). It has also been used to identify and characterize
toxic wastes under regulations enforced by the Resource Conservation and Recovery Act (RCRA) of
1976 as amended (Millemann and Parkhurst 1980).
In the chemical-specific approach, chemical analyses and water quality criteria are used to
estimate toxicity. If concentrations of specific chemicals in hazardous wastes exceed criteria values,
then the concentrations are considered to be toxic. The chemical-specific approach is also used for
regulating waste water discharges under the Clean Water Act and for characterizing toxic wastes
under RCRA.
These two approaches complement each other, and depending on site-specific conditions,
either or both may be appropriate for estimating the toxicity of hazardous wastes. For complex
chemical mixtures of unknown composition, such as hazardous waste site samples, however, the
toxicity-based approach is generally considered to be the best procedure for estimating potential
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toxicity (Bergman et al. 1986, EPA 1985). Rationale for using the toxicity-based approach rather than
the chemical-specific approach includes the following:
o Toxicity tests measure the aggregate toxicity of all constituents in a complex waste mixture,
including additive, synergistic, and antagonistic effects.
o The bioavailability of toxic chemicals is measured with toxicity tests but not with chemical
analyses; therefore, chemical data may over- or underestimate the toxicities of single
chemicals.
o Chemical analyses for complex wastes (many chemicals present), especially for organics, can be
more expensive than toxicity testing.
o The specific chemicals analyzed in hazardous wastes may not include many toxic chemicals
that are actually present.
o Water quality criteria are available for relatively few chemicals potentially present in
hazardous wastes.
o It is not always clear from chemical data which compounds are causing toxicity in a complex
hazardous waste mixture.
The chemical-specific approach may be appropriate for the following cases:
o Simple wastes (few chemicals present), for which chemical analyses can be less expensive than
toxicity testing.
o Specific problem chemicals, such as carcinogens or bioaccumulative chemicals, that can be
directly measured.
o Design of treatment systems, which are more easily designed to remove specific chemicals
than to reduce a generic parameter such as toxicity.
This manual provides toxicity test methods for detecting and quantifying short-term toxicity in
solid or liquid samples collected at hazardous waste sites. These methods are cost-effective tests that
generate data useful for identifying toxic wastes, quantifying their relative toxicities, and
monitoring remediation efforts. Assessing the actual or potential site-specific effects of the wastes
-------
would require more intensive and costly toxicity tests, waste characterizations, chemical analyses,
and site-specific field assessment studies.
1.4 ONGOING RESEARCH
Ongoing research at the Corvallis Environmental Research Laboratory (CERL) has been
designed to test and evaluate the recommended toxicity test protocols on samples obtained from
hazardous waste sites nationwide. The objectives of this research are to (1) improve test precision
and accuracy, (2) increase the ability to detect toxicity, and (3) enhance the efficiency of prescribed
laboratory procedures-
Research at CERL includes evaluating problematic situations that potentially may arise when
the protocols presented here are applied under a variety of laboratory and field conditions. Specific
areas of concern include (1) test solutions with low pH or high hardness that can mask the toxicity of
other chemical components, (2) filtration of test solutions for toxicity tests other than the algal test,
and (3) addition of ethylenediaminetetraacetic acid (EDTA) to the algal test solutions to enhance
iron bioavailability.
While the recommended toxicity tests should, in general, be implemented using the protocols
presented in the Appendix, the protocols should not be considered inflexible. These protocols have
been developed as state-of-the-science methods for use in hazardous waste toxicity assessments.
Just as the science of environmental toxicology for complex mixtures is evolving, the methods
recommended in this document are expected to evolve. As new information on environmental
toxicology becomes available, new techniques undoubtedly will be developed, tested, and
evaluated. The methods and recommendations presented in this document will, as a consequence,
be revised.
Several important factors should be considered, however, prior to modification of the
recommended protocols. These factors have a strong influence on the ability of a toxicity test to
measure potential toxicity. First, altering the pH or hardness of test solutions or adding EDTA to test
solutions may alter the bioavailability and potential toxicity of many chemicals. For example, raising
-------
the pH can decrease the toxicity of many metals and can increase the toxicity of ammonia and
cyanide, and decrease the toxicity of hydrogen sulfide. In the same manner, increasing the hardness
of test solutions adds carbonates that may complex metals, thereby reducing their toxicity.
Additionally, increasing the hardness often causes increases in pH. EDTA added to test solutions can
also complex metals, reducing their toxicity. Second, filtering of test materials is required only for
the algal toxicity test (see Appendix, Section A 8) Filtration through a 0.45-um filter may remove
toxic particles or colloids, and may cause toxic chemicals present in trace amounts to be adsorbed
onto the filter. Consequently, filtration may lead to underestimation of actual toxicity.
1.5 USES OF TOXICITY TESTS IN IDENTIFYING AND CHARACTERIZING THE TOXICITY OF
HAZARDOUS WASTES
Toxicity data resulting from the battery of tests recommended in this document have
applications in the overall assessment and remediation process of hazardous waste sites (Figure 1).
Sections 1.5.1 through 1.5.5 describe these potential applications. Section 2.3 provides an example
of how toxicity data can be applied to site priontization and monitoring.
1.5.1 Site Prioritization
Over 24,000 uncontrolled hazardous waste sites have been identified by EPA (EPA 1986).
Given this large number of sites, it is important to focus available remediation resources on those
sites of greatest hazard to the environment.
Data on the toxicity of liquid or solid samples from hazardous waste sites provide a cost-
effective and rapid means for ranking the potential environmental hazards of the sites. Sites are
ranked based on the aggregate toxicity of the components of the complex sample, including
additive, synergistic, and antagonistic effects. The indices used to rank the sites are the median
lethal concentration (LC50) and the median effective concentration (EC50) (see Section 2.2). The site
with the lowest LC50 or EC50 is considered to have the highest potential for toxicity, and the site
with the highest LC50 or EC50 is considered to have the lowest potential for toxicity. Variability in
the estimate of the toxic potential of a site, caused by both field and laboratory factors, must be
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considered when comparing sites (Section 2). The use of statistically-based field sampling designs
and the application of a consistent set of toxicity test protocols will minimize this variability. The test
battery approach recommended in this document optimizes the ability to detect toxicity at a
hazardous waste site and minimizes the uncertainty in the site rankings. Differential responses
among the six toxicity tests optimize the probability of detecting short-term toxicity if it is present
and enable the sites to be prioritized according to the magnitude of potential short-term toxicity.
Although not addressed in this document, field assessments of the potentially toxic sites would be
implemented to define cause-and-effect relationships and aid in the selection of appropriate
remediation measures.
1.5.2 Waste Characterization
Both CERCLA and RCRA require identification and characterization of wastes. These
assessments are generally conducted prior to a detailed sampling program. Preliminary assessments
are based on available chemical data for the site and the documented characteristics of each
chemical. High costs associated with chemical analyses increase the importance of focusing detailed
sampling studies on areas posing the greatest hazard. The toxicity tests recommended in this
manual provide an estimate of short-term toxicity independent of chemical identification of all toxic
substances.
1.5.3 Site Characterization
One of the most important uses of toxicity tests is for characterization of environmental
conditions in and near hazardous waste sites Site characterization involves the collection of data on
hazardous substances and the assessment of potential toxicity effects at the site. Toxicity tests can
form an important part of the descriptive assessments required in the site characterization process.
Additionally, toxicity test results, combined with a properly designed field program, can be used to
assess the extent of contamination and associated potential for toxic effects.
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1.5.4 Cleanup Standards
The measured concentration of a toxic chemical at a hazardous waste site must be compared
with all relevant criteria and standards. SARA has established that many of these standards
promulgated under other Acts (e.g. SDWA, Toxic Substances Control Act [TSCA], CWA) are
mandatory for site cleanup. The criteria are relevant because they are specified by law. The lack of
effects-based criteria for these media and for many chemicals, and the disagreement over using
criteria additively in complex chemical settings, results in major difficulties in defining cleanup levels.
Toxicity test results can provide valuable information relevant to the decision process
associated with cleanup actions. The identification of potential site toxicity is an important step in
the decision process. However, cleanup actions based on detailed cause-and-effect relationships
require additional field assessment studies not covered in this report.
1.5.5 Site Monitoring
Environmental monitoring during and following remedial action is an important part of
CERCLA and RCRA assessments. Toxicity tests are a valuable monitoring tool because they provide
cost-effective, rapid assessment of potential toxicity. Length of exposure during toxicity testing
ranges from 2 (D. maqna) to 14 (earthworm) days after sample preparation. Thus, test results can be
available within approximately 4 to 16 days. Monitoring can ensure that target cleanup levels are
achieved and that no additional problems develop following site remediation or closure. If
remediation is not completely successful at a hazardous waste site, it is important to identify the
inadequacies as soon as possible to allow for implementation of additional corrective actions. As
with site ranking methods (Section 1.5.1), detailed identification of cause-and-effect relationships
requires additional field assessment studies. Protocols for field bioassessments are not included in
this report.
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SECTION 2
DATA ANALYSIS
This section presents methods for analyzing data obtained from implementation of
recommended toxicity test protocols Interpretation of the toxicity test results is dependent on the
selected methods for analysis and presentation of the data. This section will address methods for
expressing biotic response (e.g., percent mortality and inhibition), calculation of the LC50 or EC50,
and recommended methods for intersite comparisons (use of the toxicity test results).
2.1 METHODS FOR CALCULATING MEASURES OF TOXICITY
Two measures of toxicity, specific to the type of data generated, are calculated for the
recommended toxicity tests -- percent mortality and inhibition proportion. The two sections
following provide methods for calculating appropriate toxicity indices.
2.1.1 Percent Mortality
Percent mortality (defined for a specific toxicity test as [number of test units dying in a single
sample dilution/total number of test units in the dilution] X 100) is the toxicity index calculated for
four of the six toxicity tests These tests are those using D. maqna and D. pulex. fathead minnow,
earthworm, and lettuce seed germination. In the lettuce seed germination test, it is recognized that
factors other than the presence of a toxic substance can prevent seed germination. For the purpose
of calculating the toxicity index in this test, however, lack of seed germination is considered to be
equivalent to mortality
Percent mortality is calculated for each test dilution. If the total number of test units surviving
in the test dilution is equal to or greater than that of the controls, then the test solution is considered
not to exhibit short-term toxicity. For any specific toxicity test, values of the calculated percent
mortality at various dilutions are used to estimate the LC50 for the test (Section 2.2).
-------
2.1.2 Effect Proportion
The effect proportion (E) is the toxicity index calculated for the algal and lettuce root
elongation tests, and is calculated for each test dilution as:
C), E will be positive, and the test solution is considered
nontoxic. If inhibition occurs (TC), the test solution also is considered nontoxic. For either the algal or root
elongation bioassays, values of the calculated effect proportion at various dilutions are used to
calculate the EC50.
When proportional amendments of assay media are used (see Section A.8.4.11.1 of the
Appendix) in the algal growth test, effect proportions are calculated for each dilution as:
(T-IN)-P(C-IN)xmn = F
P(C-IN)
where IN = dry weight (mg/L) of inoculum used at start of test, and
P = percentage volume of algal assay media used to dilute the test samples.
P should be >_ 20% of the total test solution.
2.2 CALCULATING THE LC50 AND EC50
Data from the recommended short-term toxicity tests are used to estimate the LC50 or the
EC50. The LC50, calculated for those toxicity tests measuring mortality, is the concentration that is
10
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estimated to be lethal to 50% of the organisms within the test period. The EC50, calculated for those
toxicity tests measuring a non-lethal toxic effect, is the concentration that reduces the average
response of the test organisms by 50% within the test period. Only rarely will the concentration in
the test dilution result in exactly 50% mortality or effect of the test organisms.
Therefore, some mathematical approach is generally needed to obtain an estimate of the
LC50 or EC50 Also, because of natural variation in the sensitivity of individuals within a group of
test organisms, there is a degree of uncertainty regarding the estimated value of the LC50 or EC50.
This uncertainty is expressed as a confidence interval or range of values within which the "true" LC50
or EC50 could occur.
Unlike toxicity tests with single compounds, which usually result in a regular progression in
percent mortality or effect with increasing toxicant concentration, toxicity tests with elutriates, soils,
or complex aqueous mixtures tend to yield all-or-nothing responses. Exposures to one or more of
the higher sample concentrations (lower dilutions) result in 100% mortality of the test organisms,
whereas exposures at lower concentrations (higher dilutions) all result in 100% survival. These
results eliminate the use of some candidate methods for calculating the LC50 or EC50 at the
recommended dilutions.
Methods for calculating the LC50 or EC50 for each toxicity test include the graphical,
Litchfield-Wikoxon, binomial, Probit, Logit, moving average, moving average-angle, and Spearman-
Karber analyses (Finney 1971, Stephan 1977, Peltier 1978a,b) Although any of these methods will
provide reasonable estimates of the LC50 or EC50, those that also provide estimates of the
confidence interval are recommended for hazardous waste site rankings. Whenever possible,
methods such as the moving average-angle or Probit methods should be used because they permit
the uncertainty inherent in the estimation of the LC50 or EC50 to be calculated. When estimating
the LC50, EC50, or their estimators using parametric statistical methods such as simple linear
regression, the investigator must be careful not to bias the results by violating any of the underlying
distributional assumptions (Kennedy 1985). When the toxicity tests yield all-or-nothing results, the
Litchfield-Wikoxon, Probit, and Spearman-Karber methods cannot be used to calculate the LC50.
11
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Step-by-step methods for calculating an LC50 can be found in Peltier and Weber (1985). Computer
programs can be obtained by contacting Mr. James Dryer, U.S. EPA, Environmental Monitoring and
Support Laboratory, Cincinnati, OH, 45268. For routine analysis, use of the computer programs are
recommended for efficient and consistent results.
2.3 USE OF THE TOXICITY TEST RESULTS
Toxicity test results are appropriate for prioritizing and monitoring hazardous waste sites. The
relative index of toxicity is the LC50 or EC50 and their associated confidence intervals, which are
calculated from the data generated using laboratory procedures conducted according to the
protocols contained in the Appendix of this document. Correct interpretation of the data is
imperative for understanding and applying the toxicity test results. This section describes methods
for calculating and presenting the toxicity test results.
In general, it is recommended that screening for the purpose of assessing the relative toxicity
of hazardous waste sites be accomplished by implementing the entire battery of toxicity tests
presented in this document. For each toxicity test performed, an LC50 or EC50 can be calculated. In
most cases, the uncertainty associated with the LC50 or EC50, represented by the confidence interval,
also can be calculated (Section 2 2)
The data can be presented in a manner similar to the example provided in Table 1. In this
example, the toxicity test battery was applied to three hypothetical hazardous waste sites. The
objective of this study was to assess the relative toxicity of each of the three sites and rank the sites
with respect to their degree of toxic potential. For each test at each site, the LC50 or EC50 was
calculated using the moving average-angle method. Values of LC50 or EC50 can be compared
among sites only for toxicity tests that use the same organisms and protocols. Comparisons among
different toxicity tests is inappropriate because the types of organisms used differ, as do the
protocols, e.g., the LC50 resulting from the D magna test, conducted for 2 days, cannot be directly
compared to that for the earthworm test, conducted for 14 days. Thus, intersite comparisons must
be test-specific and are made by evaluating the magnitude of the LCSOs or ECSOs as well as their
12
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TABLE 1. EXAMPLE USE OF TOXICITY TEST RESULTS. CALCULATED LC50 or EC50 AS PERCENT
SAMPLE CONCENTRATION (95% CONFIDENCE INTERVAL)
Toxicity Test
D. maqnaa
earthworm*
fathead minnow
lettuce seed "
germination
lettuce root
elongation^
algalb
Sitel
35.5
(29.4-42.5)
6.8
(6.2-7.5)
10.5
(9.5-11.6)
45.6
(41.4-50.2)
45.6
(38-54.7)
73
(6.6-8.0)
Site 2
32.6
(27.2-39.1)
7.3
(66-8.1)
10.4
(9.5-11.4)
52.3
(47.5-57.5)
54.5
(45.4-65.4)
8.1
(73-89)
Site 3
46.9
(42.6-51.6)
15.6
(14.2-17.2)
11.2
(10.2-12.3)
75.7
(688-83.3)
868
(78.9-95.5)
14.6
(13.2-16.1)
LC50& calculated using the moving average-angle method.
ECSOs calculated using the moving average-angle method.
13
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associated uncertainty, as represented by their respective confidence intervals. For this example, a
95% confidence interval was calculated. Note that although most LC50 and EC50 mean values for
Site 2 were generally higher than for Site 1, the confidence intervals have a high degree of overlap.
Hypothesis testing, using a two-sample "t" statistic or an appropriate multiple comparison test for
example, would show no statistical differences between the LCSOs and ECSOs calculated for Site 1
and Site 2. No difference in the toxic potential of Site 1 or Site 2 can therefore be statistically
determined. The LCSOs and ECSOs calculated for Site 3, however, are higher than for either Site 1 or
Site 2. Additionally, with the exception of the fathead minnow test, the confidence intervals for the
Site 3 indices do not overlap the corresponding confidence intervals for Sites 1 and 2. Therefore,
based on the toxicity test results, Sites 1 and 2 have a higher potential for short-term toxicity than
Site 3.
Conceivably, individual toxicity tests could give inconsistent results when ranking sites. For
example, the earthworm toxicity test may rank Site 1 as more toxic than Site 2, with the D. magna
test giving opposite results. This scenario should be expected due to the different sensitivities of the
organisms in the test battery. In this case, both sites are considered to have toxic potential, possibly
resulting from different chemicals or processes. In such a case, additional field assessments (not
covered in this report) would be needed to assess the actual difference in toxic mechanisms between
the two sites.
14
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SECTION 3
SAMPLING CONSIDERATIONS
This section describes important factors for consideration when designing field sampling
protocols for toxicity screening at hazardous waste sites. A properly designed sampling program is
necessary to ensure that conclusions are based on reliable data, to maximize the information gained
from the study while minimizing the study costs, and to ensure the repeatability and precision of the
measured toxic responses. Correct interpretation of the toxicity test results is dependent upon a
properly designed and implemented sampling program.
3.1 WHAT TO SAMPLE
The choice of which media or medium to sample in the field (soil, soil water, surface water,
ground water, etc.) depends on the objectives of the study. The selected medium should maximize
the capability of detecting contamination at the hazardous waste site The media (or medium)
chosen for sampling should be (1) those into which the contaminants are known or suspected to
have been disposed, or (2) those to or from which the contaminants are likely to migrate. For
situations in which the investigator has no prior knowledge of the contaminants or their associated
spatial distribution, a general survey to obtain samples representative of the site is initially
appropriate. Resources permitting, additional surveys, the design of which can be guided using the
results of toxicity testing on the first set of samples, can aid in further investigating the potential
toxicity of the hazardous waste site. Field assessments of the hazardous waste site are necessary for
evaluating cause-and-effect relationships. Appropriate techniques for bioassessment are not
presented in this report.
3.2 VARIABILITY ISSUES
A major objective of the general surveys is to minimize variability associated with data
collection while maximizing the ability to detect site toxicity. Variability of data derived from field
15
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samples, collected during the survey of a hazardous waste site, is attributable to a number of
different factors. Sampling variability can be attributed to natural variability, variability in field
conditions when the samples are taken, sampling techniques, and sample handling in the laboratory.
Although the natural variability among samples taken at the same time and place cannot be
controlled by the investigator, some part of it may be anticipated and accounted for by a statistically-
based field sampling design.
Concentrations of contaminants usually vary both spatially and temporally in the field. Spatial
variability tends to be higher in soils and sediments than in water, because soil/sediment mixing rates
are much slower than those in water. Conversely, temporal variability in water may be greater than
in soils and sediments because transport of material is more rapid in water than in soils/sediments.
To account for spatial and temporal variability in concentrations of contaminants, statistically-
based sampling designs should be implemented for the collection of samples at hazardous waste
sites. Several types of statistical designs and the advantages and disadvantages of each for assessing
potential toxicity at hazardous waste sites can be found in Athey et al. (1987).
16
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SECTION 4
CASE STUDY
The case study presented in this section is an example selected to illustrate the use of the
toxicity tests presented in this manual. The example provided illustrates the procedures and
techniques recommended to identify and quantify short-term toxicity of hazardous waste site
samples.
4.1 BACKGROUND INFORMATION
The hazardous waste site is an abandoned railroad tie treatment plant. The site was known to
be contaminated with creosote and other wood-preserving materials. A preliminary site visit was
made to obtain background information on the history of chemical disposal, determine the
dimensions of the site, and define any special sampling problems. The site is bounded on the east
and north by a river.
Records obtained from the local department of natural resources indicate that creosote and,
occasionally, pentachlorophenol were used on the site for wood treatment. Minimal cleanup had
occurred before the site was closed. It was clear from the visit that creosote was still being emitted
from the bank into the water along some parts of the river. Piles of creosote-contaminated material,
as well as pools of black sludge, were located near ponds immediately adjacent to the storage tanks
on the south side of the site.
4.2 STUDY OBJECTIVES
The objective of the study was to identify and quantify the short-term toxicity, if any, of
contaminated water and sediments at the site. If the toxicity tests identified significant toxicity in
site samples, a more intensive field study would be designed to supply data needed for assessment of
ecological damage (methods are not addressed in this report).
17
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4.3 STUDY PROCEDURES
A bridge was chosen as the starting point for sample location. Water and sediment samples
were collected at 660 m west of the bridge (farthest downstream location), 380 m west of the bridge,
200 m west of the bridge, 120 m east of the bridge, 220 m east of the bridge (farthest upstream
location), and 3000 m upstream (the control position). In addition, sediment samples were taken
from ponds near the storage tanks The sampling points were chosen to bracket the visibly
contaminated zone. The sampling design was reviewed before the field work began by the project
manager, a statistician, the field sampling crew, and the toxicologist in charge of laboratory
analyses.
Protective clothing and masks were worn by the field sampling personnel during sample
collection.
4.4 ANALYSIS AND EVALUATION
The results of the toxicity tests are presented in Table 2. The only sites with measurable
short-term toxicity are the ponds and those sites 660 and 380 m west of the bridge. The water
sample from the 380-m west site is highly toxic, while the sediments from that location exhibit lower
toxicity. At the 660-m west site, however, the sediments are highly toxic to some organisms while
the water exhibits no measurable short-term toxicity. The results of the toxicity testing indicate at
least the potential for ecological effects from contaminated site water and sediments. From the
perspective of site prioritization, upstream sites exhibit negligible short-term toxicity, while sites at
380 m and 660 m downstream exhibit significant short-term toxicity.
Following remedial action at the site, which included sediment removal, the toxicity tests were
again used to determine if the methods for removing the short-term toxic contamination were
effective. The battery of toxicity tests was applied to sediment and water samples taken at the same
locations as the original samples The toxicity test results indicated no short-term toxicity. Periodic
toxicity testing was then implemented to monitor for the possibility of future toxic contamination.
18
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The toxicity tests cannot identify the actual causes of ecological effects at the site. A
comparison of the results of Table 2 suggests that water soluble toxic compounds in sediments from
this site may be causing significant ecological effects at substantial distances downstream. It was
recommended that ecological assessment studies should be undertaken at these downstream sites.
19
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SECTION 5
REFERENCES
Athey, L.A., J.M. Thomas, J.R. Skalski, and W.E Miller. 1987. Role of Acute Toxicity Bioassays in the
Remedial Action Process at Hazardous Waste Sites. EPA Report. EPA/600/8-87/044. Pacific Northwest
Laboratory, Richland, WA.
Bergman, H.L., R.A. Kimerle, and A.W. Maki, eds. 1986. Environmental Hazard Assessment of
Effluents. Pergamon Press, Oxford, England. 366 pp.
Environmental Protection Agency. 1985. Short-term Methods for Estimating the Chronic Toxicity of
Effluents and Receiving Waters to Freshwater Organisms. EPA/600/4-85/014, Environmental
Monitoring and Support Laboratory, Cincinnati, OH. 162 pp.
Environmental Protection Agency. 1986. National Priorities List Fact Book: June 1986. HW-7.3.
Office of Emergency and Remedial Response. Washington, DC 20460.
Finney, D.J. 1971. Probit Analysis. 3rd edition. Cambridge Press, NY. 668pp.
Kennedy, P. 1985. A Guide to Econometrics. The MIT Press, Cambridge, MA.
Millemann, R.E. and B.R. Parkhurst. 1980. Comparative toxicity of solid waste leachates to Daphnia
maqna. Environ. Internal. 4:255-260.
Peltier, W.H. and C.I. Weber. 1985. Methods for Measuring the Acute Toxicity of Effluents to
Freshwater and Marine Organisms. 3rd edition. Environmental Monitoring and Support Laboratory,
U.S. Environmental Protection Agency, Cincinnati, OH. EPA/600/4-85/013.
Peltier, W. 1978a. Methods for Measuring the Acute Toxicity of Effluents to Aquatic Life. 1st
edition. Environmental Monitoring and Support Laboratory, U.S. Environmental Protection Agency,
Cincinnati, OH. January, 1978. EPA/600/4-78/012.
Peltier, W. 1978b. Methods for Measuring the Acute Toxicity of Effluents to Aquatic Life. 2nd
edition. Environmental Monitoring and Support Laboratory, U.S. Environmental Protection Agency,
Cincinnati, OH. July, 1978. EPA/600/4-78/012.
Public Law 94-580. 1976. Resource Conservation and Recovery Act (RCRA), as amended.
Public Law 96-510. 1980. Comprehensive Environmental Response, Compensation, and Liability Act
(CERCLA), as amended.
Public Law 99-499. 1986. Superfund Amendment and Recovery Act, as amended.
Stephan, C.E. 1977. Methods for Calculating an LC50. Aquatic Toxicology and Hazard Evaluation,
ASTM STP 634 (F.L. Mayer and M.L. Hamelink, eds.), American Society for Testing and Materials,
Philadelphia, PA.
21
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APPENDIX A -- TOXICITY TEST METHODS
SECTION A.1
SCOPE AND APPLICATION
Procedures are described for obtaining information on the acute toxicity of hazardous
wastes to aquatic and terrestrial organisms. Six static acute toxicity tests are described: algal growth
(Selenastrum capricornutum). water flea survival (Daphnia pulex and D. maqna). fish survival
(fathead minnow. Pimephales promelas). earthworm survival (Eisenia foetida). seed germination
(lettuce, Lactuca sativa). and lettuce root elongation. These procedures are derived from methods in
common use for testing the acute toxicity of effluents and single chemicals (e.g., EPA 1982, ASTM
1980, Peltier and Weber 1985, Homing and Weber 1985, Edwards 1984, Thomas and Cline 1985).
Only minor modifications of these methods have been made to adapt them for tests with hazardous
wastes.
The tests can be performed with both aqueous and solid hazardous waste samples. The
algae, water flea, fish, and lettuce root elongation tests are for use with aqueous waste samples or
elutriates prepared from solid wastes. The earthworm and lettuce seed germination tests are for
testing hazardous wastes mixed with artificial soil. If desired, the earthworm and lettuce seed
germination tests could be performed with artificial soils hydrated with aqueous samples or solid
waste elutriates.
These toxicity test methods can be used to test the toxicity of other kinds of solid and
aqueous materials collected at hazardous waste sites, such as surface waters, ground waters, soils,
and sediments
Procedures are described for obtaining and culturing the test organisms, for handling and
storing hazardous wastes, and for preparing the elutriates and artificial soils required for toxicity
testing.
These toxicity test methods are state-of-the-science procedures. Because the state-of-the-
science for environmental toxicology is continually evolving, methods for performing these tests also
will evolve and improve. Therefore, these methods may be revised as better methods are developed.
Some of the methods have undergone more development, testing, and standardization than
others. For example, the aquatic tests described in this report have been in widespread use in
governmental agencies and private companies for quite some time, while the earthworm test was
only recently introduced, has not been standardized, and is much less routinely used. Consequently,
the earthworm test may undergo more changes and refinement than other tests.
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SECTION A.2
HEALTH AND SAFETY
A.2.1 GENERAL PRECAUTIONS
Collection and use of hazardous wastes for toxicity testing may involve significant risks to
personal safety and health. Personnel collecting hazardous waste samples and conducting toxicity
tests should take all safety precautions necessary for the prevention of (1) bodily injury and illness
which might result from ingest ion or invasion of infectious agents, (2) inhalation or absorption of
corrosive or toxic substances through skin contact, and (3) asphyxiation due to lack of oxygen or
presence of noxious gases.
Before sample collection and laboratory work begin, personnel should determine that all
necessary safety equipment and materials have been obtained and are in good condition. In
addition, all laboratories conducting toxicity testing on hazardous wastes should prepare a "Safety
and Health Protocol for Shipping, Receiving, Handling and Testing of Toxic Substances." This
protocol document should be consulted for laboratory-specific instructions on health and safety
procedures. To obtain an example of such a protocol, contact James C. McCarty, CERL Designated
Safety and Health Official, Environmental Research Laboratory, U.S. Environmental Protection
Agency, 200 SW 35th Street, Corvallis, OR 97333.
A.2.2 SAFETY MANUALS
For further guidance on safe practices when collecting hazardous waste samples and
conducting toxicity tests, consult general industrial safety manuals, including Walters and Jameson
(1984) and NIOSH et at. (1985).
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SECTION A.3
QUALITY ASSURANCE
A.3.1 INTRODUCTION
Quality assurance (QA) practices for hazardous waste toxicity tests consist of all aspects of the
test that affect data quality: (1) hazardous waste sampling and handling, (2) the source and
condition of the test organisms, (3) condition of equipment, (4) test conditions, (5) instrument
calibration, (6) replication, (7) use of reference toxicants, (8) record keeping, and (9) data evaluation.
The QA guidelines presented here are adapted from Horning and Weber (1985). For general
guidance on good laboratory practices related to toxicity testing, see FDA (1978), EPA (1979c, 1980s,
and 1980b), and DeWoskin (1984).
A.3.2 HAZARDOUS WASTE SAMPLING AND HANDLING
Hazardous waste samples collected for on-site and off-site testing must be preserved as
described in Section A.7, Hazardous Waste Sampling and Handling.
A.3.3 TEST ORGANISMS
The test organisms used in the procedures described in this manual are the green alga,
S. capricornutum; water fleas, D maqna and D^ pulex; the fathead minnow, P. promelas; the
earthworm, E. foetida; and lettuce, L sativa.
A.3.4 FACILITIES. EQUIPMENT. AND TEST CHAMBERS
Laboratory temperature control equipment must be adequate to maintain recommended
temperatures for test water Recommended materials must be used for fabrication of the test
equipment that comes in contact with hazardous waste materials (see Section A.4, Facilities and
Equipment).
A.3.5 ANALYTICAL METHODS
Routine chemical and physical analyses must include established QA practices outlined in
Agency methods manuals (EPA 1979a,b).
A.3.6 CALIBRATION AND STANDARDIZATION
Instruments used for routine measurements of chemical and physical parameters, such as pH,
dissolved oxygen (DO), temperature, conductivity, alkalinity, and hardness, must be calibrated and
standardized according to instrument manufacturers' procedures as indicated in the general section
on quality assurance (see EPAMethods 150.1, 360.1, 170.1,and 120.1, EPA 1979b).
Wet chemical methods used to measure hardness and alkalinity must be standardized
according to the procedures for those specific EPA methods (see EPA Methods 130.2 and 310.1, EPA
1979b).
A.3.7 DILUTION WATER
The dilution water used in the toxicity tests should be synthetic soft water, except for tests
with D. maqna. which use moderately hard water (see Sections A.6, Dilution Water, and A.8, Toxicity
Test Methods).
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A.3.8 TEST CONDITIONS
Water and air temperatures must be maintained within the limits specified for each test.
Dissolved oxygen concentrations for aquatic tests and pH of soils and solutions should be checked at
the beginning of the test and end of the test period.
A.3.9 TEST ACCEPTABILITY
The results of the reference toxicant tests are unacceptable if mean control survival is less
than 90%. The results of the definitive toxicity tests are also unacceptable if control survival is less
than 90%. The results of the algal toxicity test are unacceptable if the cell density in the controls
after 96 his less than 10&cells/mL
An individual test may be conditionally acceptable if temperature, DO, and other specified
conditions fall outside specifications, depending on the degree of the departure and the objectives
of the test (see test condition summaries in each test method section). The acceptability of the test
will depend on the best professional judgment and experience of the investigator. The deviation
from test specifications must be noted when reporting data from the test.
A.3.10 PRECISION
The ability of the laboratory personnel to obtain consistent, precise results must be
demonstrated with reference toxicants before they attempt to measure hazardous waste toxicity.
The single laboratory precision of each type of test to be used in a laboratory should be determined
by performing five or more tests with a reference toxicant. Precision can be described by the mean,
standard deviation, and relative standard deviation (percent coefficient of variation, or CV) of the
calculated end points from the replicated tests. Factors that can affect test precision include test
organism age, condition, and sensitivity; temperature control; and feeding.
A.3.11 REPLICATION AND TEST SENSITIVITY
The sensitivity of the tests depends in part on the number of replicates, the probability level
selected, and the type of statistical analysis. The minimum recommended number of replicates is
discussed in each test method section. The sensitivity of the test will increase as the number of
replicates increases
A.3.12 QUALITY OF TEST ORGANISMS
If the laboratory does not have an ongoing culturing program for test organisms and obtains
them from an outside source, the sensitivity of each batch of test organisms must be evaluated.
Evaluations are performed with a reference toxicant in a toxicity test run concurrently with the
hazardous waste toxicity tests. If the laboratory maintains breeding cultures, the sensitivity of the
offspring must be determined in a toxicity test performed with a reference toxicant at least once
each month. If preferred, this reference toxicant test may be performed concurrently with the
hazardous waste toxicity test.
A 24-h acute toxicity test is used to determine the sensitivity of fathead minnows and D. spp.
For all other test species, tests of the standard length described in each method are used to
determine the sensitivity of the test organisms.
Three reference toxicants are available from EMSL-Cincinnati to establish the precision and
validity of toxicity data generated by testing laboratories: sodium dodecylsulfate (SDS), sodium
pentachlorophenate (NaPCP), and cadmium chloride (CdCl2). The reference toxicants may be
obtained by contacting the Quality Assurance Branch, Environmental Monitoring and Support
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Laboratory, U.S. Environmental Protection Agency, Cincinnati, OH 45268; FTS 684-7325, commercial
513-569-7325. Instructions for the use and the expected toxicity values for the reference toxicants
are provided with the samples.
To ensure comparability of QA data on a national scale, all laboratories must use the same
source of reference toxicant (EMSL-Cincinnati) and the same formulation of dilution water - soft
synthetic water (moderately hard synthetic water for tests with D. magna). described in Section A.6
(Dilution Water), and algal growth medium, described in Section A.8.4 for_S. capricornutum. For the
lettuce seed germination and earthworm tests, reference toxicity tests should be performed using
100% artificial soil and test concentrations of the reference toxicant diluted in the deionized water
used to hydrate the soil.
A.3.13 QUALITY OF FOOD FOR TEST ORGANISMS
The quality of the food for fish and invertebrates is an important factor in toxicity tests.
Suitable trout chow, brine shrimp (Artemia). and other foods must be obtained as described in
Sections A.5.2-A.5.6. Limited quantities of reference Artemia cysts, information on commercial
sources of good quality Artemia cysts, and procedures for determining cyst suitability as food are
available from the Quality Assurance Branch, Environmental Monitoring and Support Laboratory,
U.S. Environmental Protection Agency, Cincinnati, OH 45268. The suitability of each new supply of
food must be determined in a side-by-side test in which the response of test organisms fed with the
new food is compared with the response of organisms fed a reference food or a previously used,
satisfactory food.
A.3.14 CONTROL CHARTS
A control chart should be prepared for each reference-toxicant-organism combination, and
successive toxicity values should be plotted and examined to determine if the results are within
prescribed limits (Figure A-1). In this technique, a running plot is maintained for the toxicity values
(X,) from successive tests with a given reference toxicant.
Upper Control Limit (X + 2S)
LC50 or | Central Tendency
EC50 I
Lower Control Limit (X - 2S)
|0 .... 5 .... 10 ... .15 .... 20 >
Toxicity Test with Reference Toxicants
Figure A-1. Control chart.
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The type of control chart illustrated (EPA 1979a) is used to evaluate the cumulative trend of
the statistics from a series of samples. The mean (X) and upper and lower control limits (1 2S) are
recalculated with each successive point, until the statistics stabilize Outliers, those values that fall
outside the upper and lower control limits, and trends of increasing or decreasing sensitivity are
readily identified. At the PQ.OS probability level, one in 20 tests would be expected to fall outside of
the control limits by chance alone.
If the toxicity value from a given test with the reference toxicant does not fall in the expected
range for the test organisms when using the standard dilution water, then the sensitivity of the
organisms and the overall credibility of the test system are suspect. In this case, the test procedure
should be examined for defects and should be repeated with a different batch of test organisms.
A.3.15 RECORD KEEPING
Proper record keeping is required. Bound notebooks should be used to maintain detailed
records of the test organisms such as species, source, age, date of receipt, and other pertinent
information relating to their history and health, and information on the calibration of equipment
and instruments, test conditions employed, and test results. Annotations should be kept current to
prevent the loss of information.
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SECTION A.4
FACILITIES AND EQUIPMENT
This section was adapted from Horning and Weber (1985).
A.4.1 GENERAL REQUIREMENTS
A.4.1.1 Laboratory Facilities
Hazardous waste toxicity tests may be performed in a fixed or mobile laboratory. Facilities
should include equipment for rearing, holding, and acclimating organisms. Temperature control can
be achieved using circulating water baths, heat exchangers, or environmental chambers. Water used
for rearing, holding, acclimating, and testing organisms may be ground water, dechlorinated tap
water (see Section A.6, Dilution Water), or synthetic water. Dechlorination can be accomplished by
aeration (allowing the water to stand in an open vessel for 24 h), carbon filtration, or the use of
sodium thiosulfate. Use of 1.0 mg (anhydrous) sodium thiosulfate/L will reduce the chlorine content
of 1.5 mg chlorine/L After dechlorination, total residual chlorine should be non-detectable. Air
used for aeration must be free of oil and fumes. Test facilities must be well ventilated and free of
fumes. During rearing, holding, acclimating, and testing, test organisms should be shielded from
external disturbances.
A.4.1.2 Materials
Materials used for exposure chambers, tubing, etc., that come in contact with hazardous
waste materials, should be carefully chosen. Tempered glass and perfluorocarbon plastics (TEFLONR)
should be used whenever possible to minimize sorption and leaching of toxic substances. These
materials may be reused following decontamination. Plastics such as polyethylene, polypropylene,
polyvinyl chloride, TYGONR, etc., may be used for test chambers or to store test materials, but
caution should be exercised in their use because they could introduce toxicants when new, or carry
over toxicants from one test to another, if reused. The use of glass carboys is discouraged for safety
reasons.
New plastic products of a type not previously used should be tested for toxicity before initial
use by exposing the test organisms in the test system where the material is used. Equipment (pumps,
valves, etc.) that cannot be discarded after each use, because of cost, must be decontaminated
according to the cleaning procedures listed in Section A.4.3, Cleaning. Fiberglass, in addition to the
previously mentioned materials, can be used for holding, acclimating, and dilution water storage
tanks, and in the water delivery system. All material should be flushed or rinsed thoroughly with the
test media before use in the test. Copper, galvanized material, rubber, brass, and lead must not
come in contact with holding, acclimation, or dilution water, or with hazardous waste and test
solutions. Some materials, such as several types of neoprene rubber (commonly used for stoppers)
may be toxic and should be tested before use
A.4.1.3 Adhesives
Silicone adhesive used to construct glass test chambers adsorbs some organochlorine and
organophosphorus pesticides, which are difficult to remove. Therefore, as little of the adhesive as
possible should be in contact with water. Extra beads of adhesive inside the containers should be
removed.
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A.4.2 TEST CHAMBERS
Test chamber size and shape vary according to the size of the test organism. Requirements
are specified in each test.
A.4.3 CLEANING
New plasticware used for sample collection or organism exposure vessels does not require
rigorous cleaning. It is sufficient to rinse the new containers once with sample before use. New
glassware, however, should be soaked overnight in acid (see below).
It is recommended that all sample containers, test vessels, pumps, tanks, and other
equipment that has come in contact with test materials be washed after use in the manner described
below to remove surface contaminants. Special cleaning requirements for glassware used in algal
toxicity tests are described in Section A.8.4.
1. Soak 15 min, and scrub with detergent in tap water, or clean in an automatic dishwasher.
2. Wash with nonphosphate detergent.
3. Rinse once with full-strength acetone to remove organic compounds.
4. Rinse twice with tap water
5. Carefully rinse once with fresh dilute (20% V:V) nitric acid or hydrochloric acid to remove
scale, metals, and bases. To prepare a 20% solution of acid, add 20 ml of concentrated acid
to 80 ml of distilled water. Neutralize with sodium carbonate.
6. Rinse well with tap water.
7. Rinse twice with deionized water.
8. Baked at400°Cfor 10 hours to remove residual organic contaminants.
All test chambers and equipment must be thoroughly rinsed with the dilution water
immediately prior to use in each test
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SECTION A.5
TEST ORGANISMS AND CULTURE METHODS
A.5.1 INTRODUCTION
The organisms used in the toxicity tests described in this manual are the fathead minnow,
P. promelas. the water fleas D. pulex and D. maqna. the green alga, S. capricornutum, the
earthworm, E. foetida. and lettuce, L sativa. These organisms are easily cultured and maintained in
the laboratory. Culturing, care, and handling procedures for D. spp.. fathead minnows, earthworms,
brine shrimp (which are required for culturing fathead minnows), and S. capricornutum are
described in Sections A.5.2-A.5.5, respectively.
Starter cultures of S. capricornutum are available from the following sources:
1. Aquatic Biology Section, Biological Methods Branch, Environmental Monitoring and Support
Laboratory, U.S. Environmental Protection Agency, Cincinnati, OH 45268.
2. American Type Culture Collection (Culture No. ATCC 22662), 12301 Parklawn Drive, Rockville,
MA 10852.
3. Culture Collection of Algae, Botany Department, University of Texas, Austin, TX 78712.
Starter cultures of the fathead minnow (P. promelas) and_D. spjx can be obtained from the
Aquatic Biology Section, Biological Methods Branch, EMSL-Cincinnati Newtown Facility,
Environmental Monitoring and Support Laboratory, U.S. Environmental Protection Agency,
Newtown, OH 45244 (Phone: FTS 778-8350; Commercial 513-527-8350). Many states have strict
regulations regarding the importation of non-native fishes. Required clearances should be obtained
from state fisheries agencies before arrangements are made for the interstate shipment of fathead
minnows.
Starter cultures of the earthworm, E. foetida. are available from Mr. Julian Stewart, Vittor &
Associates, 8100 Cottage Hill Road, Mobile, AL 36695.
Lettuce seeds (L. sativa: butter crunch variety) are available from commercial seed suppliers.
If there is any uncertainty concerning the identity of the organisms, it is advisable to have
them identified by a second party Test organisms must be destroyed after use.
A.5.2 CULTURE METHODS FOR D. SPP. (D. MA6NA AND D. PULEX)
The culture methods for D. spp. are taken from Peltier and Weber (1985).
A.5.2.1 Geographical and Seasonal Distribution
D. maqna is principally a lake dweller and is restricted to waters in northern and western
North America with hardness values > 150 mg/L (as CaCOs) (Pennak 1978). D. pulex is principally a
pond dweller, but also is found in lakes. It is found over most of the North American continent in
both hard and soft waters.
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A.5.2.2 Life Cycle
The life span of D. spp.. from the release of the egg into the brood chamber until the death
of the adult, is highly variable depending on the species and environmental conditions (Pennak
1978). The average life span of D. maqna is about 40 days at 25°C, and about 56 days at 20°C. The
average life span of D. pulex at 20°C is approximately 50 days.
In culture, a female produces a clutch of 10 to 30 eggs. The eggs hatch in the brood chamber
and the juveniles, which are already similar in form to the adults, are released in approximately two
days when the female molts (casts off her exoskeleton or carapace). D. pulex has three to four
juvenile instars, whereas D. maqna has three to five instars.
D. maqna usually has six to 22 adult instars, and D. pulex has 18 to 25. In general, the
duration of instars increases with age, but also depends on environmental conditions. Culture
conditions should assure that ephippia are not induced. A given instar generally lasts approximately
two days under favorable conditions, but when conditions are unfavorable, it may last as long as a
week. The number of young per brood is highly variable for D. spp.. depending primarily on food
availability and environmental conditions. D maqna and D. pulex may both produce as many as
30young during each adult instar. The number of young released during the adult instars of
D. pulex reaches a maximum at the tenth instar, after which there is a gradual decrease (Anderson
and Zupancic 1937). The maximum number of young produced by_D. maqna occurs at the fifth adult
instar, after which it decreases (Anderson and Jenkins 1942).
A.5.2.3 Morphology and Taxonomy
D. pulex attains a maximum length of approximately 3.5 mm, whereas D. maqna is much
larger, attaining a length of 5.0 to 6.0 mm. However, these two species can be differentiated with
certainty only by determining the size and number of spines on the postabdomenal claws, using a
dissecting or compound microscope (see Pennak 1978).
A.5.2.4 Culturinq Methods
A.5.2.4.1 Sources of Organisms-
D. spp. are available from the Environmental Monitoring and Support Laboratory-Cincinnati,
and from commercial biological supply houses Only a small number of organisms (20-30) are
needed to start a culture. D. pulex is preferred over D. maqna by some biologists because it is more
widely distributed than D. maqna and is easier to culture. However, the neonates of D. maqna are
larger and, therefore, somewhat easier to use in toxicity tests.
A.5.2.4.2 Culture Media-
Although D. spp. stock cultures can be successfully maintained in some tap waters, well
waters, and surface waters, if possible the use of synthetic water as the culture medium is
recommended because (1) it is easily prepared, (2) it is of known quality, (3) it produces predictable
results, and (4) it allows adequate growth and reproduction. Reconstituted moderately hard water
(hardness: 80 to 100 mg/L CaCOa) is recommended for D. maqna. whereas soft reconstituted water
(hardness: 40 to 48 mg/L CaCOa) is recommended for D. pulex. If D. spp. cultures cannot be
maintained in reconstituted water, prepare the culture medium from well water, surface water, or
dechlorinated tap water. Adjust the water to the desired hardness by (1) diluting with deionized or
distilled water to decrease hardness, or (2) adding reconstituted hardwater to increase hardness.
The preparation of the media (Table A-1) follows
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TABLE A-1. MEDIA FOR D. MAGNA AND D. PULEX PREPARED
FROM SYNTHETIC WATER
Concentration (mg/L)
Reagent D. maqnaa D. pulexb
NaHCO3
CaSO42H2O
MgS04
KCI
96
60
60
4
48
30
30
2
a Moderately hard reconstituted water medium.
b Soft reconstituted water medium.
The compounds are dissolved in distilled or deionized water and the media are vigorously
aerated for several hours before using. The initial pH of the media is approximately 8.0, but it will
rise as much as 0.5 unit as the D. spp. population increases. Although D. spp can survive over a wide
pH range, the optimum range is 7.0 to 8.6 (Lewis and Weber 1985). The pH of culture media should
be monitored at least twice weekly.
If synthetic water does not prove to work, culture water can be prepared using dechlorinated
tap water, well water, or a surface water which has been adjusted for hardness. The minimum
criterion for acceptable culture water is one in which healthy organisms survive for the duration of
testing without showing signs of stress.
When required, the reagents listed below can be used to increase hardness as follows:
DELTA DESIRED SAMPLE
TOTAL (DTH. mg/L) = HARDNESS - HARDNESS
HARDNESS
MULTIPLICATIVE = [(DTH. mq/L)(DESIRED VOLUME IN LITERS)]
FACTOR (MF.g) 1000mg/g
Multiply each of the following constants by the multiplicative factor, and add to the desired total
dilution or culture water volume in liters. Stir for at least 1 hour, or until dissolved.
50 mq/L Hardness 100 mq/L Hardness 200 mq/L Hardness
MgCI2-6H2O 0.593 0.593 0.593
CaSO4.2H2O 1.219 1.219 1.219
NaHCO3 1.180 1.230 1.280
KHCO3 0.120 0.147 0.236
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EXAMPLE: The deionized water hardness is 0 mg/L CaCOs; the desired hardness for the
culture water is 100 mg/L CaCOa; the total volume of culture water required is 2 L
DTH = 100-0 = 100 mg/L
MF = KlOOmq/L)(2L)l = 0.20g
1000mg/g
The salts added to 2 L of solution to obtain a total hardness of 100 mg/L CaCOa are:
MgCI2.6H2O (0.593 X 0.20 g MF) = 0.1186 g
CaSO/,.2H2O(1.2l9X0.20gMF) = 0.2438 g
NaHCO3 (1.230 X 0.20 g MF) = 0.2460 g
KHCO3 (0.147 X 0.20 g MF) = 0.0294 g
A.5.2.4.3 Feeding: Quantity and Frequency-
Food preparation and feeding are of great importance in_D. spp. culturing. D. spp. can be
cultured using algae or a prepared food consisting of a suspension of a trout chow, alfalfa, and
yeast. The latter diet is easily prepared and provides adequate nutrition for organisms used in acute
toxicity tests (Winner et al. 1977). The trout chow must conform to Fish & Wildlife Service
Specification PR(11)-78, and can be obtained through livestock feed stores. Dried yeast, such as
Fleischmann's, can be obtained at any grocery store. Dried alfalfa can be obtained at health food
stores.
The trout chow, alfalfa, and yeast food is prepared as follows:
1. Place 6.3 g of trout chow pellets, 2.6 g of dried yeast, and 0.5 g of dried alfalfa in a blender.
2. Add 500 mL of distilled or deionized water.
3. Mix at high speed for 5 min.
4. Place in a refrigerator and allow to settle for 1 h.
5. Decant the top 300 mL and save; discard the remainder.
6. Place 30- to 50-mL aliquots in small (50- to 100-mL) polyethylene bottles with screw caps and
freeze.
7. Thaw portions as needed After thawing, keep in refrigerator for a maximum of one week,
then discard.
Feed 1.5 mL prepared food per 1000 mL of medium, three times per week, i.e., Monday,
Wednesday, Friday. There may be some excess food in the medium at this rate of feeding, but if the
medium is aerated continuously and replaced each week, as discussed below, this should cause no
problems.
An alternate method of feeding D. spp. used successfully at CERL involves the use of
S. capricornutum. the green alga employed in the algal growth test (described in Section A.8.4).
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Daphnia are fed Monday through Thursday from a stock culture of S. capricornutum in a
quantity sufficient to provide a final concentration of 2 mg/L dry weight (approximately
135,000 cells/ml) in the rearing jars which contain 2 L water and 30 to 40 adult D magna. On Fridays
the quantity is doubled to allow sufficient nutrients to sustain the cultures through the weekend
without additional feeding is continuously.
Selenastrum capricornutum. cultured as a food source for Daphnia. The algae are incubated
in an environmental chamber held at 24 ±2°C with a continuous light intensity of 4304 ±430 lux.
An algal nutrient solution is prepared by adding 1.0 ml of each of the macronutrient and
micronutrient stock solutions in the order listed in Table A-2, to 900 ml of filter-sterilized deionized
water, with mixing after each addition. The final volume is brought to 1 liter with filter-sterilized
deionized water. Deionized water (not the culture medium) is filter-sterilized by passing through a
0.22 um porosity cellulose membrane filter (pre-rinsed with 100 ml deionized water) into a sterile
container.
After the algal growth medium is prepared it is autoclaved 15 minutes/L at 121°C. A final
spike of 1 ml/L BME vitamin solution (Sigma Chemical Co.) is added upon introduction of the medium
into the aspirator bottle.
D. maqna are fed, in addition to the algal cells suspensions, a fish-food and yeast solution at
the rate of 0.2 mis per 2-L jar. This nutrient solution is fed (on Tuesdays and Fridays) after the water
in the culture jars has been changed. The fish-food yeast formula is prepared by adding 12 g
fish-food (e.g., salmon or trout pellets) and 3 grams bakers yeast to 1 L deionized or distilled water.
A.5.2.4.4 Culture Temperature--
D. spp. can be cultured successfully over a wide range of temperatures, but should be
protected from sudden changes in temperature, which may cause death. The optimum temperature
is approximately 22°C, and if ambient laboratory temperatures remain in the range of 18 to 26°C,
normal growth and reproduction of D. spp. can be maintained without special temperature control
equipment.
A.5.2.4.5 Illumination-
The variations in ambient light intensities (540 to 1080 lux) and prevailing day/night cycles in
most laboratories do not seem to affect D. spp growth and reproduction significantly. However, a
minimum of 16 h of illumination should be provided each day.
A.5.2.4.6 Culture Vessels-
Culture vessels of clear glass are recommended since they allow easy observation of the
D. spp Maintain several (at least five) culture vessels, rather than only one. This will ensure backup
cultures so that, in the event of a population "crash" in one or several chambers, the entire_D. spp.
population will not be lost. If a 3-L vessel is stocked with 30 D. srjrj., it will provide approximately
300 young each week.
Initially, all culture vessels should be washed well (see Section A.4, Facilities and Equipment).
After the culture is established, clean each chamber weekly with distilled or deionized water and
wipe with a clean sponge to rid the vessel of accumulated food and dead D_. spp. (see Section
A.5.2.4.8). Once per month wash each vessel with detergent during medium replacement. Rinse
three times with tap water and then with culture medium to remove all traces of detergent.
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A.5.2.4.7 Aeration-
D. spp. can survive when the dissolved oxygen (DO) concentration is as low as 3 mg/L, but do
best when the level is above 6 mg/L. Each culture vessel should be continuously and gently aerated if
DO levels fall below 6 mg/L. This is best accomplished by using air stones. The air can come from
either an aquarium air pump or from a general laboratory compressed air supply. If the laboratory
air supply is used, first pass it through a flask full of cotton batting to filter out oil or other
contaminants.
A.5.2.4.8 Weekly Culture Media Replacement--
Careful culture maintenance is essential The medium in each stock culture vessel should be
replaced each week with fresh medium. This can be best accomplished as follows:
1. Siphon the old medium out, using plastic tubing (6 mm) covered with fine mesh netting
around the open end.
2. Retain 1/10 (300 mL) of the original medium containing theD_. spp. population.
3. Pour this old medium containing the D. spp. into a temporary holding vessel.
4. Clean the culture vessel as described above.
5. Fill the newly cleaned vessel with fresh medium. Gently transfer by pouring the contents of
the temporary holding vessel (old medium and D. spp.) into the vessel containing the fresh
medium.
If the medium is not replaced weekly, waste products will accumulate which could cause a
population crash or the production of males and/or sexual eggs.
D. spp. populations should be thinned weekly to about 8 per liter to prevent over-crowding,
which may cause a population crash or the production of males and/or ephippia. A good time to thin
the populations is during medium replacement. To transfer D. spp.. use a 15-cm disposable, jumbo
bulb pipet, or 10-mL "serum" pipet that has had the delivery end cut off and fire polished. The
diameter of the opening should be approximately 5 mm. When using a serum pipet, a pipet bulb
(such as a PropipetR) or a portable, motorized pipettor (MopetR) provides the controlled suction
needed when selectively collecting D. spp. Alternatively, Daphnia may be transferred by screening.
Liquid containing adult D. pulex and D. maqna can be poured from one container to another
without risk of air becoming trapped under their carapaces. However, the very young D. spp. are
much more susceptible to air entrapment and, for this reason, should be transferred from one
container to another using a pipet. The tip of the pipet should be kept under the surface of the
liquid when the D. spp. are released.
Each culture vessel should be covered to exclude dust and dirt and minimize evaporation.
A.5.2.5 Test Organisms
D. maqna or D. pulex. 2- to 24-h in age (neonates, or first instars), are to be used in the tests.
To obtain the necessary number of young for a test, remove adult females bearing embryos in their
brood pouches from the stock cultures 24 h preceding the initiation of the test. Place them in
400-mL beakers containing 300 mL of medium and 0.5 mL of prepared food (see Section A.5.2.4.3).
The young that are found in the beakers the following day are used for the toxicity test. Five
beakers, each containing 10 adults, usually will supply enough first instars for one toxicity test.
35
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Since the appearance of ephippia in cultures generally is indicative of unfavorable
conditions, D. spp. used for toxicity tests should not be taken from cultures that are producing
ephippia.
A.5.3 CULTURE METHODS FOR FATHEAD MINNOW (P. PROMELAS)
The culture methods for fathead minnow are adapted from Peltier and Weber (1985).
A.5.3.1 Distribution
The fathead minnow is widely distributed in North America, and is found in a wide range of
habitats. This species is most abundant in brooks, small streams, creeks, ponds, and small lakes. It is
tolerant of high temperature and turbidity, and low oxygen concentrations. The fathead minnow is
primarily omnivorous; young fish have been reported to feed on organic detritus from bottom
deposits, unicellular and filamentous algae, and planktonic organisms. Adults feed on aquatic
insects, worms, small crustaceans, and other animals.
A.5.3.2 Life Cvcle
The natural history and spawning behavior of the fathead minnow (Markus 1934, Flickinger
1973, Andrews and Flickinger 1974, and others) are well known. Male and female fathead minnows
show sexual dimorphism at maturity. The breeding males develop a conspicuous, narrow,
elongated, gray, fleshy pad of spongy tubercules on the back, anterior to the dorsal fin, and two or
three rows of strong nuptial tubercules across the snout. The sides of the body of breeding males
become almost black except for two wide vertical bars which are light in color. The females remain
quite drab.
Spawning females release a small number of eggs (usually 100 to 150) at a time. The eggs are
adhesive and attach to the under side of the spawning substrate. The females have an ovipositor
(urogenital structure) to help deposit the eggs on the under side of objects. Flickinger (1966)
indicated that the ovipositor is noticeable at least a month prior to spawning. The reported size of
the eggs varies from 1.15mm (Markus 1934) to 1.3 mm in diameter (Wynne-Edwards 1932).
Immediately after the eggs are laid, they are fertilized by the male, and the female is driven
off. Once eggs are deposited in the nest, the male becomes very aggressive and will use the large
tubercules on his snout to help drive off all intruding small fishes. In addition to fertilizing and
guarding the eggs, the male agitates the water around the eggs, which ventilates them and keeps
them free of detritus. Some males will spawn with a number of females on the same substrate, so
that the nest may contain eggs in various stages of development. The number of eggs per nest will
vary from as few as nine or 10 to as many as 12,000.
The ovaries of the females contain eggs in all stages of development. Females spawn
repeatedly as the eggs mature. A female may deposit eggs in more than one nest. Although the
average number of eggs per spawn per female is generally 100 to 150, large females may lay 400 to
500 eggs per spawn.
The hatching time depends on temperature. The average hatching time required at 25°C is
4.5 to 6 days. The newly hatched young (fry) are about 5 mm long, white in color, with large black
eyes. In warm, food-rich water, growth is rapid. Adult size is probably not reached until the second
year, and the males generally grow faster than the females. The fathead minnow is short-lived, and
rarely survives to the third year.
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A.5.3.3 Taxonomy
The specific name (P. promelas) appears to be incorrectly applied to this fish because the
fathead minnow does not fit the description originally given by Rafinesque (Lee et al. 1980). Also,
some geographic variations have been noted in the morphology of the fathead minnow.
Vandermeer (1966), in a statistical analysis of geographic variations in taxonomic characters, stated
that subspecies intergrade clinally. The American Fisheries Society (1980), however, recognizes only
one species.
A.5.3.4 Morphology and Identification •- General Characters
Fathead minnows vary greatly in many characteristics throughout their wide geographic
range. The morphology and characters for identification are taken from Trautman (1957), Clay
(1962), Hubbs and Lagler (1964), Eddy and Hodson (1961), and Scott and Crossman (1973). They are
small fish, being typically 43 mm to 102 mm, or an average of about 50 mm, in total length.
The mouth is terminal. The snout does not extend beyond the upper lip and is decidedly
oblique. Nuptial tubercules, which occur on mature males only, are large and well-developed on the
snout, and rarely extend beyond the nostrils. They occur in three main rows, with a few on the lower
jaw. In addition to nuptial tubercules, there is an elongate fleshy or spongy pad extending in a
narrow band from the nape to the dorsal fin. Wide anteriorly, the pad narrows to engulf the first
dorsal ray.
A dark spot is usually present in front of the dorsal fin in mature males. Dymond (1926) and
Trautman (1957) have described a saddle-like pattern, often associated with breeding males in which
a light area develops just behind the head and another beneath the dorsal fin. Standard lengths are
usually less than four and one-half times the body depth.
A.5.3.5 Hybridization
Trautman (1957) stated that under some conditions the fathead minnow may hybridize with
the bluntnose minnow. P. notatus (Rafinesque). He also indicated that fathead and bluntnose
minnows were competitors, and that the fathead occurred in greater population densities only
where the bluntnose was absent or comparatively few in number.
A.5.3.6 Culturinq Methods
A.5.3.6.1 Sources of Organisms-
When test fish are secured from an outside source, there is no guarantee that they will be of
the age, size, quality, or condition needed for performing bioassays. Additionally, because of the
possibility of occurrence of disease in fish brought into the laboratory, it may be necessary to give
them a prophylactic treatment for disease to prevent and/or eliminate infections. Such treatment
places a stress on the test fish and requires a quarantine period of seven days.
To avoid the problems associated with using fish of unknown condition and age, an in-house
laboratory culture facility can be developed. Such a facility can provide a continuous supply of eggs
(embryos) and/or other developmental stages of known age, which are free of disease or other
stress, for use in toxicity tests.
A.5.3.6.2 Laboratory Culture Facility--
Fathead minnows can be cultured in either a static or flow-through system. Flow-through
systems require large volumes of water and may not be feasible in some laboratories. The culture
37
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facility consists of the following components: water supply, spawning tanks, holding tanks, egg
incubation units, and rearing tanks.
1. Water Supply-Synthetic water or dechlorinated tap water can be used, but untreated well
water is preferred. If a static system is used, it is necessary to equip each aquarium with a
carbon filter system (similar to those sold for tropical fish hobbyists) to control the
accumulation of metabolic wastes in the water.
The dissolved oxygen concentration in the culture tanks should be maintained near
the 100% saturation level, using air stones if necessary. Brungs (1971), in a carefully
controlled long-term study, found that the growth of fathead minnows was reduced
significantly at all dissolved oxygen concentrations below 7.9 mg/L. Soderberg (1982)
presented an analytical approach to the reaeration of flowing water for culture systems.
Adequate procedures for culture maintenance must be followed consistently to
avoid poor water quality in the culture system. Tanks are cleaned monthly or more often as
required. They should be kept free of debris, e.g., excess food, detritus, and waste, by daily
siphoning the accumulated materials (such as dead brine shrimp nauplii or cysts) from the
bottom of the tanks with a pipet. To avoid excessive buildup of algal growth, periodically
scrape the walls of aquaria. Activated charcoal and filter pads in the aquaria filtration
systems should be changed weekly. Culture water may be maintained by preparation of
synthetic water or use of dechlorinated tap water. Distilled or deionized water is added as
needed to compensate for evaporation.
2. Spawning Tanks--The spawning unit is designed to simulate conditions in nature conducive
to spawning. Fathead minnows spawn in spring and summer, and the lab photoperiod is
maintained to mimic the natural spawning conditions. For breeding tanks, it is convenient to
use 76-L (20-gal) aquaria. Spawning tanks must be held at a temperature of 25 ± 2°C. Each
aquarium is equipped with a heater, continuous filtering unit, and spawning substrates. The
photoperiod for the spawning tanks must be rigidly controlled and maintained at 16 h light
and 8 h dark (5:00 AM to 9:00 PM is a convenient photoperiod). An illumination level of 540
to 1080 lux is adequate. The breeding fish are fed all the frozen brine shrimp and tropical
fish flake food or dry commercial fish food that they can eat twice daily (8:00 AM and 4:00
PM) during the week and once a day on weekends. If properly maintained, each breeding
tank will produce a spawn of 100 to 200 eggs approximately every four days.
It is necessary to provide a surface for the laying and fertilizing of eggs, as well as a
territory for the male. The number of substrates placed in each tank depends on the size of
the tank and the number of males. Two substrates are used for each male. The substrates
should be placed equidistant from each other and staggered from each other on the bottom
of the tanks so that the spawning territories of the males do not overlap. The recommended
spawning substrates consist of inverted 15-cm (6-in.) sections of 10-cm (4-in) diameter clay
tile or PVC cut in half longitudinally. Four substrates are used in each 76-L (20-gal) spawning
tank.
The number of fish in each aquarium, as well as the ratio of females to males,
depends on the size of the aquarium. Five to eight females and two males are placed in each
breeding tank. Spawning females must be replaced periodically (every two to three months)
with other mature, egg-laden females to ensure a continuous supply of eggs.
3. Egg Incubation Units--Once spawning conditions are right, eggs are produced. Fathead
minnows spawn in the early morning hours. They should not be disturbed except for a
morning feeding (8:00 AM) and daily examination of substrates for eggs between 9:30 AM
and 10:30 AM. In nature, the male protects, cleans, and aerates the eggs until they hatch. In
38
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the laboratory, however, it is necessary to remove the eggs from the tanks, to prevent them
from being eaten by the adults, for ease of handling when recording egg count and
hatchability.
The substrates are each lifted carefully and checked for newly spawned eggs. If eggs
are observed on a tile, remove the tile immediately from the spawning tank, stand it on end
in a small (10- to 20-L) tank, and place an active air stone at the bottom of the tank near the
egg mass. Vigorous aeration inhibits fungal growth better than weak aeration. Alternately,
the eggs can be removed from the substrate with a razor blade or with a rolling action of the
index finger, placed in a 1-L jar or beaker, and aerated with sufficient vigor to keep them in
suspension.
If fungal growth is a problem, it can be controlled for the entire incubation period by
adding 3 ml of 1% methylene blue stock solution per liter of water in the incubation vessels
(see Herwig 1979, pp. 160-161).
During the incubation period, the eggs are examined daily for viability and fungal
growth, until they hatch. Unfertilized eggs, and eggs that have become infected by fungus,
should be removed with forceps using a table top magnifier illuminator. Non-viable eggs
become milky and opaque, and are easily recognized. The non-viable eggs are very
susceptible to fungal infection, which may then spread throughout the egg mass. Fungus
should be removed quickly, and the substrates should be returned to the incubation tanks as
rapidly as possible so that viable eggs are not damaged by desiccation.
Hatching takes four to five days at an optimal temperature of 25°C. Hatching can be
delayed several (two to four) days by incubating at 10° to 15°C
4. Rearing Tanks-Newly hatched fish are transferred daily from the egg incubation tanks to
small (8-L) rearing tanks, using a large-bore pipet, until the hatch is complete. New rearing
tanks are set up on a weekly basis to separate fish by age group. A density of 150 fry per liter
is suitable for the first four weeks. The rearing tanks are allowed to follow ambient
laboratory temperatures of 20° to 23°C, but sudden, extreme variations in temperatures must
be avoided. Fathead minnow fry are fed freshly hatched brine shrimp (Artemia) nauplii
twice daily until they are four weeks old. Utilization of older (larger) brine shrimp nauplii
will result in starvation of the young fish because they are unable to ingest the larger food
organisms, (see Section A.5.4, Culture Methods for Brine Shrimp, for instructions on the
preparation of brine shrimp nauplii).
Fish older than four weeks are fed frozen brine shrimp and commercial fish starter
(#1 and #2), which is ground fish meal enriched with vitamins As the fish grow, larger pellet
sizes are used, as appropriate.
A.5.3.6.3 Disease Control-
Bacterial or fungal infections are the most common diseases encountered. However, if
normal precautions are taken, disease outbreaks will rarely, if ever, occur. If disease occurs, treat
with 0.6 ml Procaine Penicillin-G per 76-L tank. Hoffman and Mitchell (1980) have assembled a list of
some chemicals that have been used commonly for fish diseases and pests
In aquatic culture systems that use recycled water and filtration, the application of certain
antibacterial agents should be used with caution. A treatment with a single dose of antibacterial
drugs can interrupt nitrate reduction and stop nitrification for various periods of time, resulting in
changes in pH, and in ammonia, nitrite, and nitrate concentrations (Collins et al. 1976). These
changes could cause the death of the culture organisms.
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Do not transfer equipment from one tank to another without first disinfecting it. If an
outbreak of disease occurs, any equipment, such as nets, air lines, tanks, etc., that has been exposed
to diseased fish should be disinfected with sodium hypochlorite.
A.5.3.7 Test Organisms
Fish that are 1-90 days old are used in the toxicity test. It is recommended that fish 3-5
(± 12 h) old from at least three broods be used for testing. Before the fish are removed from the
holding tank, most of the water in the holding tank should be siphoned off. The fish are then
transferred to finger bowls, using a large-bore, fire-polished, glass tube (6 mm to 9 mm I.D. X 30 cm
long) equipped with a rubber bulb. It is important to note that larvae should not be handled with a
dip net. Dipping small fish such as these with a net will result in very high mortality. The same large-
bore, fire-polished, glass tube discussed above should be used to transfer the fish from the finger
bowl to the test vessels. The fish are counted as they are caught and are placed gently into the test
vessels.
A.5.4 CULTURE METHODS FOR BRINE SHRIMP
The culture methods for brine shrimp are adapted from Peltier and Weber (1985).
A.5.4.1 Sources of Brine Shrimp Eggs
Although there are many commercial sources of brine shrimp eggs, the Brazilian strain is
preferred because it has low concentrations of chemical residues. One source is Aquarium Products,
180 L Penrod Ct, Glen Burnie, MD 21061. Reference Artemia cysts are available in limited quantity
from the Artemia Reference Center, State University of Ghent, Belgium, J. Plafeousfruar 22, B 9000,
Ghent, Belgium. In the United States, contact the Environmental Research Laboratory, EPA, South
Ferry Road, Narragansett, Rl 02882, for information on reference materials and commercial sources
of good quality Artemia eggs.
A.5.4.2 Incubation Chamber and Procedure
A 2000-mL separatory funnel makes a convenient brine shrimp hatching vessel. A
satisfactory but less expensive apparatus can be prepared by cutting the bottom from a 2-L plastic
soft drink bottle and inserting a rubber stopper with a flexible tube and pinch cock. Add
approximately 1800 ml dechlorinated water and four tablespoons of non-iodized salt to the
hatching vessel and shake until the table salt (NaCI) dissolves. Alternatively, synthetic or filtered
natural seawater can be used. Add the desired quantity of eggs (usually 1.5 to 2.0 ml, dry) to the
vessel and mix well. The quantity of eggs used depends on feeding requirements. For example,
approximately 15 ml (dry) of eggs will provide enough brine shrimp nauplii to feed 1000 to 1500
newly hatched fish in four to six 8-L tanks.
After the appropriate volume of eggs is added to the hatching vessel, air is vigorously
bubbled through a 1-mL pipet which is lowered through the neck of the funnel so that the tip rests
at the bottom. Aeration will keep the eggs and newly hatched nauplii from settling on the bottom,
where the dissolved oxygen quickly would be depleted.
The eggs will hatch in 24 h at a temperature of 27°C. Hatching time varies with incubation
temperature and the geographic strain of Artemia used.
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A.5.4.3 Harvesting the Nauplii
The nauplii can be easily harvested in the following manner:
1. After 24 h at 27°C, remove the pipet supplying air and allow the nauplii to settle to the
bottom of the separatory funnel. The empty egg shells will float to the top.
2. After approximately 5 min, using the stopcock of the separatory funnel, drain the nauplii
into a 250-mL beaker.
3. After a second 5-min period and, again using the stopcock, drain any nauplii remaining in
the separatory funnel into the beaker.
4. The nauplii are further concentrated by pouring the suspension into a small cylinder that has
one end closed with #20 plankton netting.
5. The concentrate is resuspended in 50 ml of appropriate culture water, mixed well, and
dispensed with a pipet.
6. Discard the remaining contents of the hatching vessel and wash the vessel with hot soap and
water.
7. Prepare fresh salt water for each new hatch. To have a fresh supply of Artemia nauplii daily,
several hatching vessels must be set up and harvested on alternate days.
A.5.5 CULTURE METHODS FOR ALGAE, S. CAPRICORNUTUM
The culture methods for algae are adapted from Miller et al. (1978) and ASTM (1981).
A.5.5.1 Test Organisms
S. capricornutum is a unicellular coccoid green alga. Section A.5.1 provides information on
sources of "starter" cultures.
A.5.5.2 Stock Algal Cultures
Upon receipt of the "starter" culture (usually about 10 ml), a stock culture is initiated by
aseptically transferring 1 ml to a culture flask containing control algal culture medium (see Section
5.5.3). The volume of stock culture initially prepared will depend upon the number of test flasks to
be inoculated later from the stock, or other planned uses, and may range from 25 mL in a 125-mL
flask to 2 L in a 4-L flask. The remainder of the starter culture can be held in reserve for up to six
months in a refrigerator in the dark at 4°C.
Maintain the stock cultures at 24±2°C, under continuous cool-white fluorescent lighting of
4300 ± 430 lux. Shake continuously at 100 cpm.
Transfer 1 to 2 ml of stock culture weekly to 1 L of new culture medium to maintain a
continuous supply of "healthy" cells for tests. Aseptic techniques should be used in maintaining the
algal cultures, and extreme care should be exercised to avoid contamination.
To maintain unialgal culture material over a long period of time, it is advantageous to use a
semi-solid medium containing 1.0% agar. The medium is placed in sterile petri dishes, and a 1-mL
portion of a liquid algal culture is streaked onto it and incubated as described above. Place rubber
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bands around the petri dishes to reduce evaporation loss of the medium. Fresh (liquid) stock cultures
may be started at four-week intervals by transfer of cells from a single clone in a petri dish to an
appropriate volume of liquid medium.
A.5.5.3 Culture Medium
The culture medium is used to maintain the stock cultures of the test organisms. Culture
medium is used as a control in each test and as the diluent in each test. Steps for preparing the
culture medium follow:
1. Prepare five stock nutrient solutions using reagent-grade chemicals as described in Table A-2.
2. Add 1 ml of each stock solution, in the order listed in Table A-2, to approximately 900 ml of
distilled or deionized water. Mix well after the addition of each solution.
3. Dilute to 1 L, mix well, and adjust the pH to 7.0 ±0.1, using 0.1 N sodium hydroxide or
hydrochloric acid, as appropriate. The final concentration of macronutrients and
micronutrients in the culture medium is given in Table A-3.
4. Sterilize the filtration apparatus, including 0.2-um pore diameter membranes. Membranes
may be constructed of Teflon, glass fiber, or cellulose triacetate. Membranes must be
autoclaved to ensure an aseptic system.
5. Immediately filter the pH-adjusted medium through a 0.2-pm pore diameter membrane at a
vacuum of not more than 380 mm (15 in) mercury, or at a pressure of not more than one-half
atmosphere (8 psi).
6. Place the filtered medium immediately into sterile culture flasks. No further sterilization
steps are required before the inoculation of the medium
Unused sterile medium should stored at 4°C in the dark for not more than one month before
use. Storage may result in substantial loss of water by evaporation.
A.5.6 CULTURE METHODS FOR THE EARTHWORMS (EISENIA FOETIDA)
A.5.6.1 Introduction
Large numbers of E. foetida of known age and size can be easily cultured. Considerable
information has been accumulated concerning their growth (Neuhauser et al. 1980), reproduction
(Hartenstein et al. 1979), and physical requirements (Kaplan et al. 1980). This information and the
ease of culturing makes E. foetida an appropriate earthworm for testing the toxicity of solid
hazardous wastes.
A.5.6.2 Distribution
E. foetida can be found worldwide in its specific habitat, which includes manure and soils
with a high proportion of organic matter (Fender 1985).
A.5.6.3 Life Cycle
I- foetida reaches maturity in seven to eight weeks at 22 ± 2°C. It is very prolific; a single
worm produces two to five cocoons per week, each with several worms.
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TABLE A-2. NUTRIENT STOCK SOLUTIONS FOR MAINTAINING ALGAL STOCK
CULTURES AND TEST CONTROL CULTURES
Nutrient
Stock
Solution
1
2
3
4
Compound
MgCI26H2O
CaCI2 2H2O
H3BO3
MnCI24H2O
ZnCI2
FeCI36H2O
CoCI26H2O
Na2MoO42H20
CuCI22H2O
Na2EDTA2H2O
NaNO3
MgSO47H2O
K2HPO4
NaHCO3
Amount Dissolved in
500 ml Distilled Water
6.08 g
220g
92.8 mg
208 Omg
1.64mga
79.9mg
0.714 mgt>
3.63mgc
0.006 mg<*
150.0mg
12.750g
7.350 g
0.522 g
7.50 g
aZnCI2 --Weigh out 164 mg and dilute to 100 ml. Add 1 ml of this solution to Stock #1.
bCoCI2 6H2O - Weigh out 71.4 mg and dilute to 100 ml. Add 1 ml of this solution to Stock #1.
cNa2MoO4 2H2O--Weigh out 36.6 mg and dilute to 10 ml. Add 1 ml of this solution to Stock #1.
dCuCI22H2O-Weigh out 60.Omg and dilute to 1000mL. Dilute 1 ml of this solution to 10 ml.
Add 1 ml of the second dilution to Stock # 1.
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TABLE A-3. FINAL CONCENTRATION OF MACRONUTRIENTS AND
MICRONUTRIENTS IN THE CULTURE MEDIUM
Compound
NaNO3
MgCI26H2O
CaCI2 2H2O
MgSO4 7H2O
K2HPO4
NaHCO3
Compound
H3BO3
MnCI24H2O
ZnCI2
CoCI2 6H2O
CuCI22H2O
Na2MoO42H2O
FeCI3 6H20
Na2EDTA2H2O
Macronutrient
Concentrations (mg/L)
Element
25.5
12.2
4.41
14.7
1.04
15.0
Micronutrient
N
Mg
Ca
S
P
K
Na
C
Concentrations (ug/L)
4.20
2.90
1.20
1.91
0.186
0.469
11.0
2.14
Element
185
416
3.27
1.43
0.012
7.26
160
300
B
Mn
Zn
Co
Cu
Mo
Fe
—
32.5
115
1.57
0.354
0.004
288
33.1
-
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A.5.6.4 Morphology and Taxonomy
Classification of the earthworm Eisenia foetid a is controversial. Some authorities recognize
two subspecies, foetida and andrei (Edwards 1984), while others recognize the subspecies as distinct
species (Fender, 1985). Identification with the naked eye is not 100% accurate. There are no studies
which indicate a sensitivity difference between andrei and foetida: conventional toxicological
literature does not differentiate between the two. For these reasons, this manual simply refer to
E. foetida as the designated name of the species used in the test. CERL has used andrei as its test
organism for the last two years. In any case, results of the tests should include an identification of
the organism used.
A.5.6.5 Culture Methods
A.5.6.5.1 Sources of Organisms-
Starter cultures of E. foetida can be obtained from Mr. Julian Stewart, Vittor & Associates,
8100 Cottage Hill Road, Mobile, AL 36695.
A.5.6.5.2 Culture Vessels-
The earthworms can be grown in any number of suitable containers including glass,
polyethylene, and wood. The size of the container affects the ability to handle and move the
earthworms as required by the laboratory protocol. Excessive container weight may make moving,
feeding, and harvesting procedures difficult. The containers should be covered with a glass lid
containing air holes.
A.5.6.5.3 Culture Media-
The earthworms are grown in peat moss bedding. The bedding pH is adjusted between 5
and 8 by adding up to three percent by weight of CaCOa. When using peat, care must be taken to
avoid over-watering. Peat tends to become water logged with time and anaerobic conditions
develop. Indications of anaerobic conditions occur after two to three months and include (1) a
change in color between the bottom bedding material and the upper one to two inches of material,
and (2) development of a strong odor.
A.5.6.5.4 Culture Conditions-
E. foetida stock cultures are maintained in a growth chamber in the dark at the same
temperature as used for testing. The chambers must be capable of maintaining a temperature of
22 ± 2°C.
A.5.6.5.5 Feeding-
A mixture of alfalfa pellets is used as the maintenance organic food substrate for the
earthworms. The mixture is saturated with water and aged for two weeks in a sealed container. The
aged food is sprinkled on the surface of the culture tray. Alfalfa pellets can be obtained from
agricultural feed and supply stores.
A.5.6.5.6 Culture Maintenance-
Crowding of adult worms in the growth container must be avoided. Crowding decreases the
growth rate and reproduction efficiency. Splitting the bedding material in half every three to
four months with pH-adjusted peat moss will prevent overcrowding (Neuhauser et al 1980).
45
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When the bedding appears dry on the surface, water should be added. However, saturation
can cause the earthworms to crawl out of the rearing container. Therefore, water should be added
at a rate that keeps the material wet, but avoids standing water in the bottom of the rearing pan.
Water use will depend on the ambient temperature and relative humidity. Watering additions can,
however, easily be adjusted to the particular conditions of the rearing room.
A pH range of 5 to 8 is best for optimum growing conditions. Earthworms subjected to
moisture and pH conditions outside the optimum range will leave the container or die.
A.5.6.5.7 Production Level-
At least four culture containers should be maintained to ensure a sufficient number of
earthworms on a continuing basis.
A.5.6.6 Test Organisms
Adult E^ foetida (at least two months old with a clitellum) are used for the toxicity test. The
earthworms should weigh 300 to 500 mg and be from the same culture container. From 150 to
300 earthworms may be required for each test.
46
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SECTION A.6
DILUTION WATER
This section is adapted from Homing and Weber (1985).
Recommended dilution water is synthetic soft water, except for tests with D. maqna. which
require moderately hard water (see Section A.5.2) and for algal tests, which use algal growth
medium. Soft dilution water can also be made by diluting well water or dechlorinated tap water
(see below) with distilled or deionized water to obtain soft water with the same pH, hardness, and
alkalinity as the synthetic soft water. The dilution water is considered acceptable if D. spp. show
adequate survival, growth, and reproduction when cultured in the water. If possible, water should
conform to ASTM specification D 1193, Type III (1981). Type III grade reagent water shall be
prepared by distillation, ion exchange, reverse osmosis, or a combination thereof, followed by
polishing with a 0.45 um membrane filter.
Dechlorinated water should be used as dilution water only as a last resort, because it is
usually difficult to completely remove all the residual chlorine or chlorinated organics, which may be
very toxic to the test organisms. Sodium thiosulfate is recommended for dechlorination (1.0 mg
anhydrous sodium thiosulfate/L will reduce chlorine concentrations of 1.5 mg/L). After
dechlorination, total residual chlorine must be non-detectable.
If it is necessary to pass the dilution water through a deionizer to remove unacceptably high
concentrations of copper, lead, zinc, fluoride, or other toxic substances before use, it must be
reconstituted to restore the calcium and magnesium removed by the deionization process.
To prepare synthetic fresh water, use the reagents listed in Table A-4. For example, to
prepare 20 L of soft synthetic water, follow these steps:
1. Place 19 L of distilled or deionized water in a properly cleaned plastic carboy.
2. Add sufficient MgSO4, NaHC03, and KCI to the plastic carboy, and stir well.
3. Add sufficient CaSO4 2H2O to 1 L of distilled or deionized water in a separate flask, place on a
magnetic stirrer until the calcium sulfate has dissolved, and add to the plastic carboy and stir
well.
4. Aerate vigorously for 24 h (with air filtered through cotton to remove oil) to dissolve the
added chemicals and stabilize the medium.
The measured pH, hardness, and alkalinity of the aerated water will approximate that
indicated under "Final Water Quality" in Table A-4.
47
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TABLE A-4. PREPARATION OF SYNTHETIC FRESH WATER*
Reaqent Added (mq/L)b
Water
Type
Very
Soft
Soft*
Moderately
Hard*
Hard
Very
Hard
NaHCO3
12.0
48.0
96.0
192.0
384.0
CaSO4.2H2O MgSO4
7.5 7.5
30.0 30.0
60.0 60.0
120.0 120.0
240.0 240.0
KCI
0.5
2.0
4.0
8.0
16.0
pHc
6.4-6.8
7.2-7.6
7.4-7.8
7.6-8.0
8.0-8.4
Final Water
Hardness^
10-13
40-48
80-100
160-180
280-320
Quality
Alkalinity
10-13
30-35
60-70
110-120
225-245
aTaken in part from Marking and Dawson (1973).
bAdd reagent-grade chemicals to distilled or deionized water.
(Approximate equilibrium pH after 24 h of aeration.
^Expressed as mg CaCOa/L.
eRecommended dilution for tests using species other than D. maqna.
fRecommended for tests using D. maqna.
48
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SECTION A.7
HAZARDOUS WASTE SAMPLING AND HANDLING
In this section, procedures are described for sampling and handling hazardous wastes and for
making the elutriates required for testing the toxicity of solid hazardous waste samples to aquatic
and terrestrial test organisms.
A.7.1 APPARATUS AND EQUIPMENT
1. Sample Containers
a. 3-gal plastic pails with lids and handles
Cardinal Plastics #384-P, or equivalent
Akron, OH
(206) 562-9600
b. 5-gal steel paint cans with crimp lids
Freund Can Company #1260-4450, or equivalent
Chicago, IL
1-800-621-2808
c. 2.5-gal cubitainer
VWR Scientific #243000-155, or equivalent
P.O. Box 7900
San Francisco, CA
(415)468-7150
d. Plastic Trash Bags, 1 x 2'
VWR Scientific # 11215-392, or equivalent
e. ORM-E Labels
Labelmaster, Chicago, IL 60646
(312)478-0900
2. Flint-glass jars (1/2-gal or 2-L) with Teflon-lined lids for preparing elutriates.
3. Top-loading balance, 1- to 2000-g capacity
4. Crystallizing dishes, 100-mm diameter
5. Drying oven
6. End-over-end shaker
Rota-Tox rotary extractor
Associated Design and Manufacturing Co.
Alexandria, VA
7. Glass beakers, 250 mL
8. Qualitative crepe filter paper, 185-mm diameter, coarse porosity
9. Glass chemical funnel, 100-mm (top inside diameter) x 95-mm (stem length)
49
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10. 10-mLpipets
11. 500-mL glass Erlenmeyer flasks
12. Aluminum foil
13. Refrigerated centrifuge with 1-L or more capacity.
A.7.2 HAZARDOUS WASTE SAMPLING. HANDLING. AND STORAGE
Proper collection, packaging, and shipping of hazardous waste site samples is critical. Proper
sampling and shipping ensures sample integrity, safety in handling, and an adequate data base for
sample processing and future sampling requirements. The following sections outline the minimum
requirements for sampling and shipping hazardous wastes.
A.7.2.1 Aqueous Hazardous Wastes
From 3.7 to 7 L of hazardous waste solution are required to perform all six toxicity tests and
to conduct routine chemical analyses (Table A-5). Except for the fathead minnow test, which
requires the greatest volume of solution, the volumes in Table A-5 are greater than those actually
required and make some allowance (100%) for wastage and repetition of tests.
All sample containers should be rinsed with sample water before being filled with sample.
Fill the 2.5-gal cubitainers with the aqueous sample. Seal the screw cap with tape. Place the
cubitainer into two plastic bags. Seal the bags with tape and place the sealed sample into a 3-gal
plastic pail, which is then sealed. Place the plastic pail into a 5-gal metal paint can and crimp the lid.
Complete the information on tape or label, which identifies the packager and date, using an
indelible pen. Affix strips of tape over the crimped edges in at least two places.
Laboratory chain-of-custody procedures must always be followed when handling hazardous
wastes.
A.7.2.2 Solid Hazardous Wastes
From 3.6 to 4.5 kg of solid hazardous waste is required to perform all six toxicity tests and to
conduct routine chemical analyses (Table A-5). Except for the fathead minnow test, the volumes in
Table A-5 are more than those actually required and make some allowance (100%) for waste and
repetition of tests.
Line the 3-gal plastic pail with two plastic bags. Completely fill plastic bags inside the pail
with waste material, then seal the plastic bags with tape. Secure lid on pail with tape and insert the
pail into a plastic outer bag. Seal the outer bag with tape and insert soil or sediment sample into the
5-gal metal paint can. Crimp lid on can.
Complete the information on tape or label, which identifies the packager and date, using an
indelible pen. Affix strips of tape over the crimped edges in at least two places.
Laboratory chain-of-custody procedures must always be followed when handling hazardous
wastes.
50
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TABLE A-5. QUANTITIES OF AQUEOUS HAZARDOUS WASTE. SOLID HAZARDOUS WASTE,
AND/OR ELUTRIATE REQUIRED TO PERFORM THE SIX TOXICITY TESTS AND ROUTINE
CHEMICAL ANALYSES
Aqueous or Solid Hazardous Waste
Aqueous Waste or Solid
Elutriate Required Sample Required
Test (mL) (g. dry weight)
2 SEP..
Fathead Minnow
<. 5-d old
> 5-d old
Algae
Lettuce root elongation
Earthworm
Lettuce seed germination
Chemical Analyses
Totals
Fathead minnows
< 5-d old
> 5-d old
600
1200
4500
600
240
none
none
1000
3640
6940
150
300
1125
150
60
1500
1200
250
3610
4435
51
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A.7.2.3 Labeling Requirements
All containers will be identified according to the labeling requirements discussed below. A
data sheet (Figure A-2) must be filled out for each sample with as much detail as possible.
U.S. Department of Transportation (DOT) regulations require that environmental samples
collected from hazardous materials disposal sites are labeled as Other Regulated Materials. "E" Class
(ORM-E). This label will be affixed to the lid and the bucket of all collected environmental samples.
This label identifies the sample as being potentially hazardous, flammable, corrosive, poisonous, etc.,
but containing less than a reportable quantity of the sample. All such labels should be clearly
identifiable (white on black or vice versa) and affixed with permanent ink or paint. A highlighted
border of at least 1 in. is required. If sample contents are known, or if reportable quantities of
various substances (corrosive, poison, etc.) are contained or anticipated in the sample, then labeling
must comply with DOT CFR-49 specifications. These specifications are found in Section 172 of the
DOT Hazardous Materials Shipping and Handling Regulations. These regulations can be found at
the office of any carrier authorized to haul hazardous materials.
A.7.3 SAMPLE HANDLING AND PRESERVATION
Aeration during collection and transfer of solid and aqueous hazardous wastes should be
minimized to reduce the loss of volatile chemicals. The time elapsed from collection of a sample to
the initiation of the toxicity tests should not exceed 72 h. Sample toxicity may be affected when held
for longer than 72 h. Samples must be chilled after collection and maintained at 4°C until used for
testing, unless toxicity tests are initiated within 24 h of sample collection.
Samples collected for on-site tests should be used within 24 h. Samples collected for off-site
toxicity testing are to be chilled to 4°C when collected, shipped on ice to the central laboratory, and
transferred to a refrigerator (4°C) until start of test. Every effort must be made to initiate the test
with a sample on the day of its arrival in the laboratory.
A.7.4 AQUEOUS SAMPLE PREPARATION
The aqueous hazardous wastes must be filtered through a (30-um) plankton net to remove
indigenous organisms that may attack the test organisms. Waters used in algal toxicity tests must be
filtered through a 0.45-pm pore diameter filter before use. It may be necessary to first coarse-filter
the waste water through a nylon sieve having 2-mm holes to remove debris and/or break up large,
floating or suspended solids.
The dissolved oxygen (DO) concentration in the dilution water should be near saturation
before use. Aeration will bring the DO and other gases into equilibrium with air, minimize oxygen
demand, and stabilize the pH.
If the dilution water and waste water must be warmed to bring them to the prescribed test
temperature, supersaturation of dissolved gases may become a problem. To prevent this problem,
the waste and dilution waters are heated to 25°C and checked for DO with a probe. If the DO
exceeds 100% saturation, the solutions are aerated vigorously with an air stone (usually 1 to 2 min)
until the DO is lowered to 100% saturation.
A.7.5 ELUTRIATE PREPARATION
From 3.7 to 7 L of elutriate are required to perform all six toxicity tests and to conduct
routine chemical analyses (Table A-5). The volume of elutriate prepared must be sufficient for all
test concentrations and for chemical analyses. Except for the fathead minnow test, the volumes in
Table A-5 are more than those actually required and make some allowance (100%) for wastage and
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HMAT Sample Data Sheets
1. Date of Collection
2. Name of Sample Site
3. Location of Site
4. Site Sequential Sample Number
5. Type of Sample Soil 6. Quantity Processed Soil
Water Water
Groundwater Groundwater
Other Other
7. Septh of Sample (m) (cm)
8. Date Received at CERL
9. Chemical Constituents
Known
Suspected
10. Collected by: Name
Address
Phone
11. Notes
Figure A-2. Waste site sample collection data sheets.
S3
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repetition of tests. Standard procedures for effluents and surface waters do not recommend
filtering the elutriate. During the last 2 years, in cooperation with EPA's Environmental Response
Team, CERL has filtered elutriate, surface waters, and ground waters. This was done for the specific
purpose of assessing solute toxicity. If samples are filtered, then interpretation of results should
recognize the potential for altered toxicity (see Section 1.4 and Section 7.7). If the sample is not
filtered and suspended solids remaining in the waste water cause a physical stress to the D. spp and
fathead minnows, then sample filtration is required to remove the solids (see Sections A.8.2.11.8 and
A.8.3.11.6).
The elutriates are prepared by adding deionized water to the waste equal to four times the
dry weight of the sample. For the following calculation assume 1 mL of water weighs 1 g. Since most
wastes will already contain some water, this residual water must be factored into the calculation of
the amount of deionized water to be added. Waste samples should not be dried before preparing
elutriates.
First, to determine the moisture content of the sample, place 125 g of sample into a clean
crystallizing dish and weigh.
The combined weight of the dish and sample equals the initial wet weight Dry the sample at
100 ± 5°C for 24 h. Cool in a desiccator and reweigh the dish and sample. The combined weight (to
nearest gram) of the dish and dried sample equals the final dry weight. Save the dried soil in an air-
tight container for use in determining the sample water holding capacity. The moisture fraction of
the sample is calculated as follows:
Moisture
Fraction = initial wet weight (g) - final dry weight (g)
(MF) subsample weight (125 g)
Each 2-L jar will hold about 1500 ml of water plus 375 g of dry sample (375 x 4 = 1500 mL).
Three to five jars will be required to prepare enough elutriate for all tests. Calculate the volume of
deionized water to be added to each jar as follows:
Water
Added to = [1500 ml] - [moisture fraction x 375 g dry sample]
Elutriate Jar
(mL)
Calculate the total wet weight of sample to add to the elutriate jar:
Wet
Sample = [375 g dry sample] + [moisture fraction x 375 g dry sample]
Weight
(9)
Add the correct amount of wet sample to each elutriate jar and then add the correct amount
of deionized water.
Mix the jar contents (end-over-end) in the dark at 20 ± 2°C for 48 h.
Centrifuge the suspension at 10,000 rpm for 10 min at 4°C. Combine and thoroughly mix the
elutriates from each elutriate jar Do not concentrate the elutriates. It is best to use the elutriates for
toxicity testing immediately after preparation. If they are not used immediately, store them at 4°C in
the dark and use them within 72 h. Refer to Section A.7.4 for sample filtration requirements.
54
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A.7.6 WATER HOLDING CAPACITY
The earthworm and lettuce seed germination toxicity tests require that the moisture content
of the test soils be determined. Water holding capacities of both the artificial and site soil must be
known so appropriate moisture adjustments can be made.
Place 100 g of dried sample (as measured by the procedure in Section A.7.5) into a 250-mL
glass beaker. Add 100 ml of deionized water to the sample. Mix thoroughly with a glass stir rod to
ensure that all sample particles are wetted and that a slurry of sample and water exists.
Next, fold a circle of 185-mm diameter, coarse porosity, qualitative crepe filter paper into
quarters and place in a 100-mm (top inside diameter) by 95-mm (stem length) glass funnel. The
folded filter paper should be level with the top of the glass funnel. Slowly add 9 ml of deionized
water, using a pi pet, to the filter paper to wet the entire surface. Measure the combined weight of
the funnel and hydrated filter paper. Add the weight of the dried soil (100 g) to the weight of the
funnel and hydrated filter paper to obtain the initial weight.
Place the funnel in a 500-mL glass Erlenmeyer flask. Slowly pour the slurry of soil and water
onto the hydrated filter paper held in the funnel. Rinse any soil remaining on the beaker and stir rod
into the funnel with deionized water. Use the minimum volume of water necessary to ensure that all
of the solid material has been washed onto the filter. Cover the funnel tightly with aluminum foil
and allow it to drain for 3 h at room temperature.
Weigh the funnel, hydrated filter paper, and soil to obtain the final weight. The water
holding capacity, expressed as mL water/100 g soil, equals the difference between the final and
initial weights of the funnel, filter, and sample
The water holding capacity of the artificial soils used in the lettuce seed germination and
earthworm test must also be determined, as above. If the recipe is not changed, the water holding
capacity of the artificial soil should not change.
A.7.7 CHEMICAL OR PHYSICAL MODIFICATION OF TEST MATERIALS
For the purpose of detecting and quantifying, toxicity tests should be run as described in the
appendix. However, some modifications or adjustments may be desirable to answer site specific
lexicological questions. If such adjustments or modifications are made, they may modify the
toxicities of the solutions.
For example, increasing test solution (or soil) pH, which will also increase the alkalinity and
hardness (if a calcium or magnesium compound is used), will significantly affect the toxicities of most
metals by increasing complexation with carbonates and bicarbonates and decreasing metal
solubilities and toxicities. Increasing the calcium concentration will generally decrease metal uptake
and toxicity. Increasing the pH will also (1) increase the toxicities of ammonia and cyanide,
(2) decrease the toxicity of hydrogen sulfide, (3) alter the toxicities of polar organic chemicals and
(4) reduce the toxicities of acids. Decreasing the pH will generally have opposite effects on toxicity as
those for increasing the pH.
Filtering will remove particulate matter, some of which may contribute to the toxicity of the
entire mixture, plus some toxic chemicals in solution may be adsorbed by the filter. Thus, filtering
may reduce the toxicities of some test solutions.
Modifying the chemical and physical properties of the test solutions may lead to
underestimating the toxicities of some solutions or lead to identifying some toxic solutions as non-
toxic (false negatives). If such modifications are made, it would be desirable to run parallel tests on
unaltered test materials, so that the effects of these modifications can be quantified. In any case,
such modifications should only be done with consideration of their potential effects on the toxicities
of the test materials.
55
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SECTION A.8
TOXICITY TEST METHODS
A.8.1 INTRODUCTION
In this section, methods for the six toxicity tests are described. Section A.8.2 presents the
methods for the D. pulex and D. maqna toxicitv test; Section A.8.3, the fathead minnow survival test;
Section A.8.4, the algal growth test; Section A.8.5, the earthworm survival test; Section A.8.6, the
lettuce seed germination test; and Section A.8.7, the lettuce root elongation test. For each toxicity
test, the scope and application, apparatus and equipment, reagents and consumable materials, and
procedures, as well as other useful information, are provided.
A.8.2 DAPHNIA PULEX AND DAPHNIA MAGNA TOXICITY TEST
A.8.2.1 Scope and Application
This method (adapted from Peltier and Weber [1985], Horning and Weber [1985], and ASTM
[1980]) measures the acute toxicity of hazardous waste solutions to the cladocerans, D. pulex and
D. maqna. during a 48-hour static exposure. The responses measured include the synergistic,
antagonistic, and additive effects of all the chemical, physical, and biological components that
adversely affect the physiological and biochemical functions of the test organisms. This method
should be performed by, or under the supervision of, professionals experienced in aquatic toxicity
testing.
Detection limits of the toxicity of a hazardous waste solution or pure substance are organism
dependent.
A.8.2.2 Summary of Method
The test species, D. pulex or D. maqna, is exposed in a static system for 48 h to different
concentrations of hazardous waste solutions. Test results are based on survival.
A.8.2.3 Definitions
For definitions of key terms, refer to the Glossary.
A.8.2.4 Interferences
Toxic substances may be introduced by contaminants in dilution water, glassware, sample
hardware, and testing equipment (see Section A.4, Facilities and Equipment). Adverse effects of low
dissolved oxygen (DO) concentrations or high concentrations of suspended and/or dissolved solids
may mask the presence of toxic substances. Pathogenic organisms in the dilution water and test
solution may affect test organism survival and confound test results.
Improper hazardous waste handling and elutriate preparation may also adversely affect test
results (see Section A.7, Hazardous Waste Sampling and Handling).
A.8.2.5 Safety
For a discussion on safety, see Section A.2, Health and Safety.
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A.8.2.6 Apparatus and Equipment
1. Laboratory D. spp. culture unit - See Section A.5.2. This test requires at least 180 2- to
25-hour-old D. spp. neonates.
2. Sample containers - for sample shipment and storage, see Section A.7, Hazardous Waste
Sampling and Handling.
3. Environmental chamber, incubator, or equivalent facility with temperature control
(20 ± 2°C).
4. Water purification system - Millipore Milli-Q or equivalent that produces Type II ASTM
water.
5. Test vessels - 100-mL borosilicate glass beakers. Three beakers are required for each test
concentration. The beakers should be covered with glass covers during the test.
6. Racks for test vessels - Plexiglass racks drilled to hold 10 test vessels each.
7. Dissecting microscope -- for examining organisms in the test chambers.
8. Light box - for illuminating organisms during examination.
9. Volumetric flasks and graduated cylinders -- Class A, borosilicate glass or non-toxic plastic
labware, 10-to 1000-mL. for culture work and preparation of test solutions.
10. Volumetric pipets--Class A, 1-to 100-mL.
11. Serological pipets- 1-to 10-mL, graduated.
12. Pipet bulbs and fillers- PropipetR or equivalent.
13. Disposable polyethylene pipets, droppers, and glass tubing with fire-polished edges, 5 mm ID
-- for transferring organisms.
14. Wash bottles -- for rinsing small glassware and instrument electrodes and probes.
15. Glass or electronic thermometers - for measuring water temperatures.
16. Bulb-thermograph or electronic-chart type thermometers -- for continuously recording
temperature.
17. National Bureau of Standards certified thermometer--see EPA Method 170.1, EPA 1979b.
18. pH, DO, and specific conductivity meters - for routine physical and chemical measurements.
Unless the test is being conducted to specifically measure the effect of one of the above
parameters, a portable, field-grade instrument is acceptable.
19. Miscellaneous apparatus and equipment, and transfer containers should be constructed of
materials as indicated in Section A.4, Facilities and Equipment.
57
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A.8.2.7 Reagents and Consumable Materials
1. Reagent water -- defined as activated-carbon-filtered distilled or deionized water that does
not contain substances toxic to the test organisms. A water purification system may be used
to generate reagent water (see number 4 above).
2. Dilution water -- see Section A.6, Dilution Water. Soft dilution water (40 to 48 mg/L as
CaCOa) is recommended for tests with D. pulex, and moderately hard water (80 to 100 mg/L
as CaCOa) is recommended for tests with D. maqna.
3. Hazardous waste solutions - see Section A.7, Hazardous Waste Sampling and Handling.
4. Reagents for hardness and alkalinity tests (see EPA Methods 130.2 and 310.1, EPA 1979b).
5. pH buffers 4, 7, and 10 (or as per instructions of instrument manufacturer) for standards and
calibration check (see EPA Method 150.1, EPA 1979b).
6. Membranes and filling solutions for DO probe (see EPA Method 360.1, EPA 1979b), and
reagents for modified Winkler analysis.
7. Laboratory quality assurance samples and standards for the above methods.
8. Specific conductivity standards (see EPA Method 120.1, EPA 1979b).
9. Reference toxicant solutions (see Section A.3, Quality Assurance).
10. Test organisms -- D. pulex or D. maqna 2- to 24-hour-old neonates. (See information on
culturing methods in Section A.5.2). D. maqna or D. pulex can be used as the test species if
the lowest concentration of test solution has a hardness value _>80 mg/L as CaCOa. Only
D. pulex should be used as the test species if the lowest concentration of test solution has a
hardness value <80 mg/L as CaCOa. Use of D. maqna in soft water solutions may lead to
mortality caused by osmotic stress.
a. The test organism (species being used) cultures should be started at least two weeks
before the brood animals are needed, to provide an adequate supply of neonates for
the test. Only a few individuals are needed to start a culture because of their
prolificacy.
b. D. spp. may be shipped or otherwise transported in polyethylene bottles. A low
density population (20-30 animals) will live as long as one week in a 1-L bottle filled
three-fourths full with culture medium containing the trout chow diet (see Section
A.5.2). Animals received from an outside source should be transferred to new culture
medium gradually, over a period of 1 to 2 days, to avoid mass mortality.
c. Culture - See Section A.5.2 for D. spp. culture methods.
A.8.2.8 Sample Collection. Preservation, and Handling
For a discussion on sample collection, preservation, and handling, see Section A.7, Hazardous
Waste Sampling and Handling.
A.8.2.9 Calibration and Standardization
For a discussion on calibration and standardization, see Section A.3, Quality Assurance.
58
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A.8.2.10 Quality Control
For a discussion on quality control, see Section A.3, Quality Assurance.
A.8.2.11 Procedure
A.8.2.11.1 Test Solutions-
One of two dilution factors, 0.3 or 0.5, is commonly used. A dilution factor of approximately
0.3 allows testing between 100% and 1%, using only five concentrations (100%, 30%, 10%, 3%, and
1 %). This series of dilutions minimizes the level of effort, but, because of the wide interval between
test concentrations, provides poor test precision ( ± 300%). A dilution factor of 0.5 provides greater
precision (±100%), but requires several additional dilutions to span the same range of
concentrations. Improvements in precision decline rapidly as the dilution factor is increased beyond
0.5. A dilution factor of 0.5 is generally preferred.
The volume of hazardous waste solution required for three replicates per concentration,
each containing 50 ml of test solution, is approximately 300 ml. Enough test solution
(approximately 700 ml) should be prepared at each concentration to provide 400 ml additional
volume for chemical analyses.
A.8.2.11.2 Hardness of Test Solutions-
Measure the hardness of the lowest concentration of hazardous waste solution to be tested.
The hardness of the aqueous hazardous waste or elutriate must be measured before the test is
initiated to determine if D. maqna or D. pulex is the appropriate species for the test.
D. maqna or D. pulex can be used as the test species if the lowest concentration of test
solution has a hardness valueJ>.80 mg/L as CaCOs. Only D. pulex should be used as the test species if
the lowest concentration of test solution has a hardness value <80 mg/L as CaCOa.
A.8.2.11.3 Obtaining Neonates for the Test-
This test method requires 2- to 24-hour-old neonates to begin the test. (See Section A.5.2,
D. spp. Culture Methods.)
A.8.2.11.4 Start of the Test-
Measure the DO concentration of the aqueous sample or elutriate and dilution water. If the
DO in the test solution and/or dilution water is low (<60% saturation), aerate before preparing the
test solutions. Although aeration may be necessary, a loss of toxicity may occur due to volatilization
or chemical interactions.
The test should begin as soon as possible, preferably within 24 h of sample collection. In no
case should the test be started more than 72 h after sample collection. Just prior to testing, the
temperature of the sample should be adjusted to 20 ± 2°C and maintained at that temperature until
portions are added to the dilution water.
Begin the test by randomly placing one neonate in each test beaker until 10 neonates are in
each beaker. Because of their small size and difficulty in handling, the test chambers are usually
placed in racks, 10 to a rack. The position of the test chambers is randomized in the racks at the
beginning of the test, and on following days, the positions of the racks are randomized.
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A.8.2.11.5 Light, Photoperiod, and Temperature-
The light quality and intensity should be at ambient laboratory levels, approximately 540 to
1080 lux, with a photoperiod of 16 h of light and 8 h of darkness. The test water temperature should
be maintained at 20 ± 2°C.
A.8.2.11.6 Feeding--
The D. spp. are not fed during the test.
A.8.2.11.7 Routine Chemical and Physical Analyses-
At a minimum the following measurements are made:
o DO, pH, conductivity, alkalinity and hardness are measured at the beginning and end of the
test in a control beaker and in one test beaker at each test concentration.
o Temperature is measured at the beginning of each 24-h exposure period in a control beaker
and at each test concentration.
A.8.2.11.8 Observations During the Test-
Protect the D. spp. from unnecessary disturbance during the test. Make sure the D. SPP.
remain immersed during the performance of the above operations.
The D. spp. are best counted with the naked eye. The organisms are more easily seen if
viewed against a black background. If counts are made without the aid of a stereo microscope, place
the test vessels on a black strip of tape on a light box.
The numbers of live and dead D. spp in each test chamber are recorded every 24 ± 2 h, and
the dead D. spp. are discarded. Death is indicated by lack of movement of the body or appendages
when gently prodded. Unusual behavior such as lethargy, floating on the surface, and unusual
phenomena, such as the presence of surface films, precipitates, or organisms caught in paniculate
matter, should be noted and recorded.
A.8.2.11.9 Termination of the Test--
The test is terminated after 48 h of exposure, when the numbers of live and dead D. spp. are
recorded.
A.8.2.11.10 Acceptability of Test Results--
For the test results to be acceptable, mean survival in the controls must be at least 90%.
A.8.2.11.11 Summary of Test Conditions--
A summary of test conditions is listed in Table A-6.
A.8.2.12 Calculations
See Section 2 of the main text for data analysis methods.
The effect measured during the toxicity tests using D. spp. is death.
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Report the LC50 and its 95% confidence limits. The LC50 is an estimate of the median lethal
concentration.
A.8.2.13 Precision and Accuracy
Section A.3, Quality Assurance, describes precision of the test; the accuracy of toxicity tests
cannot be determined.
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TABLE A-6. SUMMARY OF RECOMMENDED TEST CONDITIONS FOR D. PULEX
AND D. MAGNA TOXICITY TEST
1. Test type:
2. Temperature (°C):
3. Light quality:
4. Light intensity:
5. Photoperiod:
6. Test vessel size:
7. Test solution volume:
8. Age of test organisms:
9. Number of test
organisms per chamber:
10. Number of replicate
chambers per dilution:
11. Feeding regime:
12. Aeration:
13. Dilution water:
14. Site sample hardness:
Static
20 ± 2°C
Ambient laboratory light
540 to 1080 lux (ambient laboratory levels)
16 h light, 8 h dark
100 mL
50 mL
2-24h
10
Do not feed
None
D. pulex - 40 to 48 mg/L CaC03
D. maqna 80 to 100 m
<80 mg/L CaCO3 D. maqna
>80 mg/L CaCOa D. pulex
15.
16.
17.
18.
Dilution factor:
Test duration:
pH range:
Effect measured:
0.5
48 h
>:6-<.
Death
10
If pH is outside this range, results may reflect pH toxicity. Adjustments of pH to either 6 or 10 may
result in altered toxicity to other constituents (see Section 1.4 and Section A.7.7).
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A.8.3 FATHEAD MINNOW TOXICITY TEST (PIMEPHALES PROMELAS)
A.8.3.1 Scope and Application
This method (adapted from Peltier and Weber [1985], Horning and Weber [1985], and ASTM
[1980]) estimates the acute toxicity of hazardous waste solutions to the fathead minnow
(P. promelas). using fish 3 to 5 days old in a static test. The responses measured include the
synergistic, antagonistic, and additive effects of all the chemical, physical, and biological
components which adversely affect the physiological and biochemical functions of the test
organisms. This method should be performed by, or under the supervision of, professionals
experienced in aquatic toxicity testing.
Detection limits of the toxicity of a hazardous waste solution or pure substance are organism
dependent.
A.8.3.2 Summary of Method
Fathead minnows 3 to 5 days old are exposed in a static system for 48 h to different
concentrations of hazardous waste solutions. Test results are based on the survival of the fish.
A.8.3.3 Definitions
For the definitions of key terms, refer to the Glossary.
A.8.3.4 interferences
Toxic substances may be introduced by contaminants in or on dilution water, glassware,
sample hardware, and testing equipment (see Section A.4, Facilities and Equipment). Adverse effects
of low dissolved oxygen (DO) concentration or high concentrations of suspended and/or dissolved
solids may mask the presence of toxic substances Improper hazardous waste handling and elutriate
preparation may adversely affect test results (see Section A.7, Hazardous Waste Sampling and
Handling).
Pathogenic organisms in solutions may affect test organism survival, and also confound test
results.
To avoid further interferences, the fish should not be fed during the test.
A.8.3.5 Safety
For a discussion on safety, see Section A.2, Health and Safety.
A.8.3.6 Apparatus and Equipment
1. Fathead minnow and brine shrimp culture units -- see Sections A.5.3 and A.5.4. This test
requires 150-300 fish, one to 90 days old. It is preferable to obtain fish from an in-house
fathead minnow culture unit. Fish 3-5 days old (±12 h) are strongly recommended for
testing. If fish less than five days old are used, smaller test vessels (150-mL) with a smaller
volume (100 ml) of test solution can be used. Using fish less than five days old would reduce
the volume of test solution required from 4500 ml to 1200 ml. If it is not feasible to culture
fish in-house, fish can be shipped in well-oxygenated water in insulated containers. All fish
used in a test should be ± 1 days in age and should come from a pool of fish consisting of at
least three separate spawnings.
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2. Sample containers - for sample shipment and storage (see Section 4, Facilities and
Equipment).
3. Environmental chamber or equivalent facility with temperature control (20 ± 2°C).
4. Water purification system -- Mi Hi pore Milli-Q or equivalent.
5. Test chambers - borosilicate glass or non-toxic disposable plastic labware. If fish S 5 days old
are used, 150-mL beakers are appropriate. If older fish are used, 1-L beakers are appropriate.
At least three beakers are required for each concentration and control. To avoid potential
contamination from the air, the chambers should be covered during the test.
6. Volumetric flasks and graduated cylinders - Class A, borosilicate glass or non-toxic plastic
labware, 10- to 1000-mL for making test solutions.
7. Volumetric pipets- Class A, 1-to 100-mL.
8. Serological pipets - 1-to 10-mL, graduated.
9. Pipet bulbs and fillers -- PropipetR or equivalent.
10. Droppers and glass tubing with fire-polished edges, 4 mm ID - - for transferring small fish.
11. Small dip nets for transferring larger fish.
12. Wash bottles -- for washing embryos from substrates and containers and for rinsing small
glassware and instrument electrodes and probes.
13. Glass or electronic thermometers - for measuring water temperatures.
14. Bulb-thermograph or electronic-chart type thermometers - for continuously recording
temperature.
15. National Bureau of Standards certified thermometer (see EPA Method 170.1, EPA 1979b).
16. pH, DO, and specific conductivity meters -- for routine physical and chemical measurements.
Unless the test is being conducted to specifically measure the effect of one of the above
parameters, a portable, field-grade instrument is acceptable.
17. Miscellaneous apparatus and equipment -- transfer containers, pumps, and automatic
dilution devices should be constructed of materials as indicated in Section A.4, Facilities and
Equipment.
A.8.3.7 Reagents and Consumable Materials
1. Reagent water - defined as activated-carbon-filtered distilled or deionized water that does
not contain substances toxic to the test organisms. A water purification system may be used
to generate reagent water (see number 4 above).
2. Aqueous hazardous wastes or a hazardous waste elutriate - see Section A.7, Hazardous
Waste Sampling and Handling.
3. Dilution water -- see Section A.6, Dilution Water.
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4. Reagents for hardness and alkalinity tests (see EPA Methods 130.2 and 310.1, EPA 1979b).
5. pH buffers 4, 7, and 10 (or as per instructions of instrument manufacturer) for standards and
calibration check (see EPA Method 150.1, EPA 1979b).
6. Membranes and filling solutions for DO probe (see EPA Method 360.1, EPA 1979b), or
reagents for modified Winkler analysis.
7. Laboratory quality assurance samples and standards for the above methods.
8. Specific conductivity standards (see EPA Method 120.1, EPA 1979b).
9. Reference toxicant solutions (see Section A.3, Quality Assurance).
10. Test organisms -- Fathead minnows one to 90 days old. Fish three to five days old ( ± 12 h) are
strongly recommended, (see Section A.5.3, Culture of Fathead Minnows).
A.8.3.8 Sample Collection. Preservation, and Storage
For a discussion on collecting, preserving, and storing samples, see Section A.7, Hazardous
Waste Sampling and Handling.
A.8.3.9 Calibration and Standardization
For a discussion on calibration and standardization, see Section A.3, Quality Assurance.
A.8.3.10 Quality Control
For a discussion on quality control, see Section A.3, Quality Assurance.
A. 8.3. 11 Procedures
A.8.3.1 1 .1 Test Solutions-
One of two dilution factors, 0.3 or 0.5, is commonly used. A dilution factor of approximately
0.3 allows testing between 100% and 1%, using only five concentrations (100%, 30%, 10%, 3%, and
1 %). This series of dilutions minimizes the level of effort, but, because of the wide interval between
test concentrations, provides poor test precision ( ± 300%). A dilution factor of 0.5 provides greater
precision (± 100%), but requires several additional dilutions to span the same range of
concentrations. Improvements in precision decline rapidly as the dilution factor is increased beyond
0.5. A dilution factor of 0.5 is generally preferred
If the test solution is known or suspected to be highly toxic, a lower range of concentrations
should be used, beginning at 10%. If a high rate of mortality is observed during the first 1 to 2 h of
the test, additional dilutions at the lower range of concentrations can be added.
For fish _<^5 days old, the total volume of solution required for three replicates per
concentration, each containing 100 ml of test solution, is approximately 600 ml. For older fish, the
volume of solution required for three replicates per concentration, each containing 750 ml of test
solution, is approximately 4500 ml. Enough test solution at each concentration should be prepared
to provide 400 ml additional volume for chemical analyses.
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A.8.3.11.2 Start of the Test--
Measure the DO concentration of the aqueous sample or elutriate and dilution water. If the
DO in the test solution and/or dilution water is low (60% saturation), aerate before preparing the
test solutions. Although aeration may be necessary, a loss of toxicity may occur due to volatilization
or chemical interactions.
Tests should begin as soon as possible, preferably within 24 h of sample collection. If the
persistence of sample toxicity is not known, the maximum holding time should not exceed 36 h. In
no case should the test be started more than 72 h after sample collection. Just prior to testing, the
temperature of the sample should be adjusted to 20 ± 2°C and maintained at that temperature until
portions are added to the dilution water.
Regardless of the size or age of fish used in a test, the loading of fish per chamber must not
exceed 0.8 g/L. Weigh a subsample of fish to be used in the test to determine their average weight
and the appropriate loading rate.
The test is initiated by placing fish, one or two at a time, into each test chamber, until each
chamber contains up to 10 fish, for a total of at least 30 fish for each concentration.
Randomize the position of test chambers at the beginning of the test. The amount of water
added to the chambers when transferring the fish to the compartments should be kept to a
minimum to avoid unnecessary dilution of the test concentrations.
A.8.3.11.3 Light, Photoperiod. and Temperature--
The light quality and intensity should be at ambient laboratory levels, which is approximately
540 to 1080 lux, with a photoperiod of 16 h of light and 8 h of darkness. The water temperature in
the test chambers should be maintained at 20 ± 2°C.
A.8.3.11.4 Feeding--
As mentioned previously, the fish are not fed during the test.
A.8.3.11.5 Routine Chemical and Physical Analyses-
At a minimum, the following measurements are made:
o DO and temperature are measured at the beginning of each 24-h exposure period and at the
end of the exposure period in a control beaker and in one test beaker at each test
concentration.
o Temperature, pH, conductivity, alkalinity, and hardness are measured at the beginning and
end of the test in a control beaker and at each test concentration.
A.8.3.11.6 Observations During the Test--
Protect the fish from unnecessary disturbance during the test. Make sure the fish remain
immersed during the performance of the above operations.
The numbers of live and dead fish in each test chamber are counted and recorded 24 ±2 h
after the test is initiated, and the dead fish are discarded. Death is indicated by lack of movement of
the body or appendages when gently prodded. Unusual behavior, such as lethargy, floating on the
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surface, and unusual phenomena, such as the presence of surface films, precipitates, and organisms
caught in participate matter, should be noted and recorded.
A.8.3.11.7 Termination of the Test--
The test is terminated after 48 h of exposure. At termination, the numbers of live and dead
fish in each test chamber are counted.
A.8.3.11.8 Acceptability of Test Results-
For the test results to be acceptable, mean survival in the controls must be at least 90%.
A.8.3.11.9 Summary of Test Conditions-
A summary of test conditions is listed in Table A-7.
A.8.3.12 Calculations
The effect measured during the toxicity tests using fathead minnow is death.
See Section 2 of the main text for data analysis methods.
Report the LC50 and its 95% confidence limits. The LC50 is an estimate of the median lethal
concentration.
A.8.3.13 Precision and Accuracy
Section A.3, Quality Assurance, describes precision of the test; the accuracy of toxicity tests
cannot be determined.
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TABLE A-7. SUMMARY OF RECOMMENDED TEST CONDITIONS FOR FATHEAD
MINNOW (P. PROMELAS) TOXICITY TEST
1. Test type:
2. Temperature(°C):
3. Light quality:
4. Light intensity:
5. Photoperiod:
6. Test chamber size:
7. Test solution volume:
8. Age of test organisms:
9. Number of test
organisms per chamber:
10. Number of replicate
chambers per dilution:
11. Feeding regime:
12. Aeration:
13. Dilution water:
14. Test concentrations:
15. Dilution factor:
16. Test duration:
17. Effect measured:
Static
20 ± 2°C
Ambient laboratory light
540-1080 lux (ambient laboratory levels)
16 h light, 8 h dark
150-mLor1-L
100or750mL/replicate
3-5 days
10 fish/chamber; not to exceed
0.8 g/L. Minimum of 30 fish/test concentration.
Minimum of 3
Do not feed
If the DO concentration falls below 40% saturation,
gently aerate solutions.
Same as culture water
At least 5 and a control
0.5
48 h
Death
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A.8.4 ALGAL GROWTH (SELENASTRUM CAPRICORNUTUM)
A.8.4.1 Scope and Application
Unicellular algae are important producers of oxygen and form the basis of the food web in
aquatic ecosystems. Algal species and communities are sensitive to environmental changes and their
growth may be inhibited or stimulated by the presence of pollutants.
The method (Porcella 1983, Horning and Weber 1985) measure the toxicity of hazardous
waste solutions to the fresh water alga, S. capricornutum. during a four-day, static exposure. The
responses measured include the synergistic, antagonistic, and additive effects of all the chemical,
physical, and biological components that adversely affect the physiological and biochemical
functions of the test organisms.
Detection limits of the toxicity of a hazardous waste solution or pure substance are organism
dependent.
A.8.4.2 Summary of Method
A Selenastrum population is exposed in a static system to a series of concentrations of
hazardous waste solutions for 96 h. The response of the population is measured in terms of changes
in cell density (cell counts per ml), biomass, or chlorophyll content.
A.8.4.3 Definitions
For the definitions of key terms, refer to the Glossary.
A.8.4.4 Interferences
Toxic substances may be introduced by contaminants in or on dilution water, glassware,
sample hardware, and testing equipment (see Section A.4, Facilities and Equipment). In addition,
high concentrations of suspended and/or dissolved solids may mask the presence of toxic substances.
Improper hazardous waste sampling and elutriate preparation may also adversely affect test results
(see Section A.7, Hazardous Waste Sampling and Handling). The EDTA present in the stock culture
medium used for dilution water may chelate some toxic metals potentially present in hazardous
waste solutions. This may lead to the underestimation of the toxicities of these solutions.
Pathogenic and/or predatory organisms in the dilution water and test solutions may affect test
organism survival, thereby confounding test results. The amount of natural nutrients present in the
test solutions or dilution water may also affect test results. The pH of test solutions should fall
between 5 to 10; values outside this range can inhibit growth and confound test results (Miller et al.
1978).
A.8.4.5 Safety
For a discussion on safety, see Section A.2, Safety and Health.
A.8.4.6 Apparatus and Equipment
1. Laboratory Selenastrum culture unit -- see Section A.5 5. To test solution toxicity, sufficient
numbers of log-phase- growth organisms must be available.
2. Sample containers - for sample shipment and storage, see Section A.7, Hazardous Waste
Sampling and Handling.
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3. Environmental chamber, incubator, or equivalent facility-- with cool-white fluorescent
illumination (4300 ± 430 lux) and temperature control (24 ± 2°C).
4. Mechanical shaker -- Capable of providing orbital motion at the rate of 100 cycles per minute
(cpm).
5. Light meter--with a range of 0-11,000 lux.
6. Water purification system - Millipore Milli-Q or equivalent.
7. Balance, analytical - capable of accurately weighing 0.0001 g.
8. Reference weights, Class S -- for checking performance of balance.
9. Glass or electronic thermometers -- for measuring water temperatures.
10. Bulb-thermometer or electronic-chart type thermometers ~ for continuously recording
temperature.
11. National Bureau of Standards certified thermometer (see EPA Method 170.1, EPA 1979b).
12. Meters: pH and specific conductivity - for routine physical and chemical measurements.
Unless the test is being conducted to specifically measure the effect of one of the above
parameters, a portable, field-grade instrument is acceptable.
13. Fluorometer (optional) -- Equipped with chlorophyll detection light source, filters, and
photomultiplier tube (Turner Model 110 or equivalent).
14. Cuvettes for spectrophotometer - 1- to 5-cm light path.
15. Electronic particle counter (optional) -- Coulter Counter, ZBI, or equivalent, with mean cell
(particle) volume determination.
16. Microscope - with 10X, 45X, and 100X objective lenses, 10X ocular lenses, mechanical stage,
substage condenser, and light source (inverted or conventional microscope).
17. Counting chamber -- Sedgwick-Rafter, Palmer-Maloney, or hemocytometer.
18. Centrifuge-with swing-out buckets having a capacity of 15 to 100 ml.
19. Centrifuge tubes-- 15-to 100-mL, screw-cap.
20. Filtering apparatus -- for membrane and/or glass fiber filters.
21. Volumetric flasks and graduated cylinders -- Class A, 10-to 1000-mL, borosilicate glass, for
culture work and preparation of test solutions.
22. Volumetric pipets- Class A, 1-to 100-mL.
23. Serological pipets-- 1-to 10-mL, graduated.
24. Pipet bulbs and filters -- PropipetR or equivalent.
25. Wash bottles - for rinsing small glassware, instrument electrodes, and probes.
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26. Culture flasks--1- to 4-L borosilicate, Erlenmeyer flasks.
27. Test flasks- 125- or 250-mL borosilicate, Erlenmeyer flasks.
28. Preparation of glassware - prepare all graduated cylinders, test flasks, bottles, volumetric
flasks, centrifuge tubes, and vials used in algal bioassays as follows:
a. Wash with non-phosphate detergent solution, preferably heated to 50°C or hotter.
Brush the inside of flasks with a stiff-bristle brush to loosen any attached material.
The use of a commercial laboratory glassware washer or heavy-duty kitchen
dishwasher is highly recommended.
b. Rinse thoroughly with tap water and drain well.
c. All new test flasks and all flasks that, through use, may become contaminated with
toxic organic substances, must be rinsed with acetone or heat-treated before use. To
thermally degrade organics, place glassware in a high temperature oven at 400°C for
30 min. After cooling, proceed with the next step.
d. If acetone is used in Step c, rinse thoroughly with tap water. If the heat treatment is
used, go directly to Step e.
e. Carefully rinse with a 10% solution (by volume) of reagent-grade hydrochloric acid
(HCI); fill vials and centrifuge tubes with the 10% HCI solution and allow to stand a
few minutes; fill all larger containers to about one-tenth capacity with HCI solution
and swirl so that the entire surface is bathed.
f. Rinse with tap water and drain well.
g. To neutralize any residual acid, rinse with a saturated solution of Na2CC>3.
h. Rinse five times with tap water and then five times with deionized or distilled water.
i. Dry in an oven, cover the mouth of each vessel with aluminum foil or other closure, as
appropriate, before storing.
29. Use of sterile, disposable pipets will eliminate the need for pipet washing and minimize the
possibility of contaminating the cultures with toxic substances.
A.8.4.7 Reagents and Consumable Materials
1. Reagent water - defined as carbon-filtered, distilled or deionized water that does not
contain substances toxic to the test organisms. A water purification system may be used to
generate reagent water (see number 7 above).
2. Test solution and dilution water — see Section A.6, Dilution Water, and Section A.7,
Hazardous Waste Sampling and Handling. The dilution water is algal medium (see Section
A.5.5).
3. Reagents for hardness and alkalinity tests (see EPA Methods 130.2 and 310.1, EPA 1979b).
4. pH buffers 4, 7, 8 and 10 (or as per instructions of instrument manufacturer) for standards
and calibration check (see EPA Method 150.1, EPA 1979b)
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5. Laboratory quality assurance samples and standards for the above methods.
6. Specific conductivity standards (see EPA Method 120.1, EPA 1979b).
7. Reference toxicant solutions (see Section A.3, Quality Assurance).
8. Acetone -- pesticide quality or equivalent.
9. Dilute hydrochloric (or nitric) acid -- carefully add 10 ml of concentrated HCI (or HNOa) to
90 ml of reagent water.
10. Test Organisms -- log-phase-growth S. capricornutum. See Section A.5.5 for culturing
methods.
A.8.4.8 Sample Collection. Preservation, and Handling
For a discussion on collecting, preserving, and handling, see Section A.7, Hazardous Waste
Sampling and Handling.
A.8.4.9 Calibration and Standardization
For a discussion on calibration and standardization, see Section A.3, Quality Assurance.
A.8.4.10 Quality Control
For a discussion on quality control, see Section A.3, Quality Assurance.
A.8.4.11 Procedures
A.8.4.11.1 Test Solutions--
One of two dilution factors, 0.3 or 0.5, is commonly used. A dilution factor of approximately
0.3 allows testing between 100% and 1%, using only five concentrations (100%, 30%, 10%, 3%, and
1 %). This series of dilutions minimizes the level of effort, but, because of the wide interval between
test concentrations, provides poor test precision (± 300%). A dilution factor of 0.5 provides greater
precision (±100%), but requires several additional dilutions to span the same range of
concentrations. Improvements in precision decline rapidly as the dilution factor is increased beyond
0.5. A dilution factor of 0.5 is generally preferred.
If the test solution is known or suspected to be highly toxic, a lower range of concentrations
should be used (such as 10%, 3%, 1%, 0.3%, and 0 1%).
The volume of test solution required for the test is 0.5 or 1 L depending on whether 125 ml
or 250 ml beakers respectively are used. To reduce the volume of hazardous water solution
required, it is recommended that the 125 ml flask with 50 ml of test solution be used. Prepare
enough test solution at each test concentration (approximately 550 ml or 700 ml) to provide 50 ml
or 100 ml of test solution for each of three replicate test chambers and 400 ml for chemical analyses.
Dilution water consists of stock culture medium.
Test solutions may be toxic and/or nutrient poor. "Poor" growth in an algal toxicity test,
therefore, may be due to toxicity or nutrient limitation, or both. To eliminate false negative results
due to low nutrient concentrations, 1 ml of each stock nutrient solution is added per liter of test
solution before preparing the test dilutions. Thus, all test treatments and controls will contain at
least the basic amount of nutrients.
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The amount of nutrients present in the test dilutions may effect test results (Section A.8.4.4),
however, ASTM is still considering the proportional amendment method (Porcella 1983). In the
proportional amendment method, full strength algal assay medium (AAM) is used to dilute site
samples for the test series. Dilution of the site sample with AAM should insure adequate nutrients
for the algae. An 80 percent test solution (20 percent AAM), however, is the maximum permissible
test concentration which ensures the presence of adequate nutrients. AAM nutrients are not spiked
into the test solution prior to its dilution with algal assay medium. The different concentrations of
AAM in the test series is accounted for in the formula for calculating percent effect (Section 2.1.2).
If the growth of the algae in the test solutions is to be measured with an electronic particle
counter, the test solution and dilution water must be filtered through a GF/A, GF/C, or equivalent
pore diameter filter, and checked for "background" particle count before it is used in the test. Note:
Filtration may strip volatile substances from the solution, decreasing its toxicity.
If samples contain volatile substances, the test sample should be added below the surface of
the dilution water towards the bottom of the test container through an appropriate delivery tube.
A.8.4.11.2 Preparation of Inoculum-
The inoculum is prepared no more than 2 to 3 h prior to the beginning of the test, using
S. capricornutum harvested from a four- to seven-day stock culture. Each milliliter of inoculum must
contain enough cells to provide an initial cell density of 10,000 cells/ml in the test flasks. Assuming
the use of 125-mL flasks, each containing 50 ml of test solution, the inoculum must contain 500,000
cells/mL. Assuming the use of 250-mL flasks, each containing 100 ml of test solution, the inoculum
must contain 1,000,000 cells/mL. The following steps provide an example of how to estimate the
volume of stock culture required to prepare the inoculum:
If the four- to seven-day stock culture used as the source of the inoculum has a cell density of
2,000,000 cells/mL. a test employing 25 flasks, each containing 100 ml of test medium and
inoculated with a total of 1,000,000 cells, would require 25,000,000 cells or 12.5 ml of stock
solution (25,000,000/2,000,000) to provide sufficient inoculum. It is advisable to use a volume
20% to 50% in excess of the minimum volume required to cover accidental loss in transfer
and handling.
1. Determine the density of cells (cells/mL) in the stock culture (for this example, assume
2,000,000 per ml).
2. Calculate the required volume of stock cultures as follows:
Vol. of test
= No. of test flasks X solution/flask X 10" cells/mL
Cell density (cells/mL) in stock culture
= 25 flasks X 100 ml/flask X 10^ cells/ml
2x106cells/mL
= 12.5 mL stock culture
[Note: cell density is determined according to procedures described below.]
3. Add 0.5 ml of stock culture to each flask.
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4. Mix well and determine the cell density in the makeup water. Some cells will be lost
in the concentration process. Dilute the cell concentrate as needed to obtain a cell
density of 1,000,000 cells/mL, and check the cell density in the final inoculum.
A.8.4.11.3 Start of the Test--
Tests should begin as soon as possible, preferably within 24 h of test solution preparation. If
the persistence of test solution sample toxicity is not known, the maximum holding time should not
exceed 36 h. In no case should the test be started more than 72 h after test solution preparation.
Just prior to testing, the temperature of the sample should be adjusted to 24 ± 2°C and maintained at
that temperature until the sample is diluted for testing.
The test begins when the algae are added to the test flasks. Mix the inoculum well, and add
1 ml to the test solution in each flask.
A.8.4.11.4 Light. Photoperiod, and Temperature--
Test flasks are incubated under continuous illumination at 4300 ±430 lux at 24±2°C, and
should be shaken continuously at 100 cpm on a mechanical shaker. Flask positions in the incubator
should be randomly rotated each day to minimize possible spatial differences in illumination and
temperature on growth rate. This step may be omitted if it can be verified that test specifications are
met at all positions.
A.8.4.11.5 Routine Chemical and Physical Analyses-
At a minimum, the following measurements are made:
o Alkalinity, hardness, pH, and conductivity are measured in extra control solutions and extra
test solutions (see Section A.8.7.11.1) at the beginning of the test and in a control flask and
one test flask at each test concentration at the end of the test.
o Temperature is measured at the beginning of each 24-h exposure period in a control flask
and at each test concentration.
A.8.4.11.6 Observations During the Test-
Toxic substances in the test solutions may degrade or volatilize rapidly, and the inhibition in
algal growth may be detectable only during the first one to two days in the test. It is recommended,
therefore, to determine the algal growth response daily, as described below.
A.8.4.11.7 Termination of the Test-
The test is terminated 96 h after initiation. It is recommended the algal growth in each flask
is measured by cell counts. If there interferences on the particle counter, then the chlorophyll
content can be measured to determine the reliability of the particle counter. Regardless of the
method used to monitor growth, the algae in the test solutions should be checked under the
microscope to detect abnormalities in cell size or shape.
1. Cell counts-Several types of automatic electronic and optical particle counters are available
for use in the rapid determination of cell density (cells/mL) and mean cell volume (MCV) in
um3/cell. The Coulter Counter is widely used and is discussed in detail by Miller etal. (1978).
74
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If biomass data are desired for algal growth potential measurements, a Model ZBI or
ZB Coulter Counter is used. However, the instrument must be calibrated with a reference
sample of cells of known volume.
When the Coulter Counter is used, an aliquot (usually 1 ml) of the test culture is
suspended in a 1% sodium chloride electrolyte (such as lsotonR), in a ratio of 1 ml of test
culture to 9 ml (or to 19mL)of 0.22-um filtered saline solution (dilution of 10:1 or 20:1). The
resulting dilution is counted using an aperture tube with a 100-um diameter aperture. Each
cell (particle) passing through the aperture causes a voltage drop proportional to its volume.
Depending on the model, the instrument stores the information on the number of particles
and the volume of each, and calculates the mean cell volume.
The following procedure is used:
a. Mix the algal culture in the flask thoroughly by swirling the contents of the flask
approximately six times in a clockwise direction, and then six times in the reverse
direction; repeat this the two-step process at least once.
b. At the end of the mixing process, stop the motion of the liquid in the flask with a
strong brief reverse mixing action, and quickly remove 1 ml of cell culture from the
flask with a sterile pipet.
c. Place the aliquot in a counting beaker, and add 9 ml (or 19 ml) of electrolyte
solution (such as Coulter lsotonR)
d. Determine the cell density (and MCV, if desired).
As alternative methods, cell counts may be determined using a Sedgwick-Rafter,
Palmer-Maloney, hemocytometer, or inverted microscope. For details on microscope
counting methods, see APHA (1985) and Weber (1973). Whenever feasible, 400 cells per
replicate are counted to obtain ± 10% precision at the 95% confidence level. This method
has the advantage of allowing for the direct examination of the condition of the cells.
2. Chlorophyll Content-Chlorophyll may be measured m vivo fluorometrically, or in vitro either
fluorometrically or spectrophotometrically. in vivo fluorometric measurements are
recommended because of the simplicity and sensitivity of the technique and rapidity with
which the measurements can be made (Rehnberg et al. 1982).
The measurements are made as follows:
a. Adjust the "blank" reading of the fluorometer using the filtrate from an equivalent
dilution of test solution filtered through a 0.45-um particle retention filter.
b. Mix the contents of the test culture flask by swirling successively in opposite
directions (at least three times), and remove 4 mL of culture from the flask with a
sterile point.
c. Place the aliquot in a small disposable vial and record the fluorescence as soon as the
reading stabilizes. (Do not allow the sample to stand in the instrument more than
1 min.).
d. Discard the sample.
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A.8.4.11.8 Summary of Test Conditions-
A summary of test conditions is listed in Table A-8.
A.8.4.11.9 Acceptability of Test Results-
The test results are acceptable if the algal cell density in the control flasks exceeds
106 cells/mL at the end of the test and does not vary more than 10% among replicates.
A.8.4.12 Calculations
The effect measured during the toxicity tests is inhibition of growth. If the growth in any
treatment is greater than the growth of controls, it is considered non-toxic.
See Section 2 in the main text for data analysis methods.
Report the EC50 and its 95% confidence limits. The EC50 is an estimate of the median
effective concentration.
A.8.4.13 Precision and Accuracy
Section A.3, Quality Assurance, describes precision of the test; the accuracy of toxicity tests
cannot be determined.
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TABLE A-8. SUMMARY OF RECOMMENDED TEST CONDITIONS FOR THE ALGAL
GROWTH TEST (S. CAPRICORNUTUM)
1. Test type:
2. Temperature(°C):
3. Light quality:
4. Light intensity:
5. Photoperiod:
6. Test flask size:
7. Test solution volume:
8. pH range:
9. Age of stock culture
used for inoculum:
10. Initial cell density:
11. Number of replicate
chambers per dilution:
12. Shaking rate:
13. Dilution water:
14. Dilution factor:
15. Test duration:
16. Effect measured:
Static
24 ± 2°C
"Cool white" fluorescent light
4300 ±430 lux
Continuous illumination
125mLor250mL
SOmLor 100mL
>.5but
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A.8.5 EARTHWORM SURVIVAL (EISENIA FOETIDA)
A.8.5.1 Scope and Application
This method (modified from the methods described by Edwards [1984] and Goats and
Edwards [1982]) estimates the acute toxicity of solid hazardous wastes to the earthworm (£. foetida)
in a 14-d static test. The responses measured include the synergistic, antagonistic, and additive
effects of all the chemical, physical, and biological components that adversely affect the
physiological and biochemical functions of the test organisms. The method uses soil as the exposure
medium because the exposure conditions closely resemble natural conditions. The method could
also be used to test aqueous samples by using 100% artificial soil and by using the aqueous samples
in place of distilled or deionized water to hydrate the test soils. This method should be performed
by, or under the supervision of, professionals experienced in environmental toxicity testing.
Detection limits of the toxicity of an hazardous waste solution or pure substance are
organism dependent.
A.8.5.2 Summary of Method
Earthworms (E_. foetida) are exposed in a static system for 14 days to different concentrations
of solid hazardous wastes mixed with artificial soil. Test results are based on survival of the worms.
A.8.5.3 Definitions
For definitions of key terms, refer to the Glossary.
A.8.5.4 Interferences
Toxic substances may be introduced by contaminants in water, glassware, sample hardware,
artificial soil, and testing equipment (see Section A.4, Facilities and Equipment). Low dissolved
oxygen (DO) concentrations or saturation of soils with water may mask the presence of toxic
substances.
Pathogenic organisms in test materials may also affect test organism survival, and confound
test results.
Improper hazardous waste sampling and handling may adversely affect test results (see
Section A.7, Hazardous Waste Sampling and Handling).
A.8.5.5 Safety
For a discussion on safety, see Section A.2, Health and Safety.
A.8.5.6 Apparatus and Equipment
1. Earthworm culture units -- see Section A.5.6. This test requires 150-300 earthworms >60
days old weighing 300 to 500 mg. It is preferable to obtain the earthworms from an in-house
culture unit.
2. Sample containers -- for sample shipment and storage (see Section A.7, Hazardous Waste
Sampling and Handling).
3. Environmental chamber or equivalent facility with temperature control (22 ± 2°C).
78
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4. Water purification system -- Millipore Milii-Q or equivalent.
5. Balance, analytical - capable of accurately weighing earthworms to 0.001 g.
6. Balance, top-loading -- capable of weighing soil samples to 1.0 g.
7. Reference weights, Class S - for checking performance of balance. Weights should bracket
the expected weights of the weighing pans and the expected weights of the pans plus worms
or soil.
8. Test chambers -- standard 1-pint canning jars with screw-top lids and rings.
9. Volumetric flasks and graduated cylinders -- Class A, borosilicate glass or non-toxic plastic
labware, 10-to 1000-mL
10. Volumetric pipets-Class A, 1-to 100-mL.
11. Serological pipets-- 1-to 10-mL, graduated.
12. Pipet bulbs and fillers-- PropipetR or equivalent.
13. Bulb-thermograph or electronic-chart type thermometers-for continuously recording
temperature.
14. National Bureau of Standards certified thermometer (see EPA Method 170.1, EPA 1979b).
A.8.5.7 Reagents and Consumable Materials
1. Reagent water -- defined as activated-carbon-filtered, distilled or deionized water that does
not contain substances toxic to the test organisms. A water purification system may be used
to generate reagent water (see number 4 above).
2. Solid hazardous waste sample -- see Section A.7, Hazardous Waste Sampling and Handling.
3. Artificial soil consisting of (by weight) 10% 2.36-mm-screened sphagnum peat, 20% colloidal
kaolinite clay, and 70% grade 70 silica sand. The artificial soil must be well mixed after it is
prepared. Peat, sand, and clay can be obtained from local suppliers.
4. pH buffers 4, 7, and 10 (or as per instructions of instrument manufacturer) for standards and
calibration check (see EPA Method 150.1, EPA 1979b).
5. Laboratory quality assurance samples and standards for the above methods.
6. Reference toxicant (see Section A.3, Quality Assurance).
7. Test organisms -- earthworms (E. foetida): see Section A.5.6 for culture methods for
earthworms. The species taxonomy should be verified using appropriate systematic keys
(Fender 1985). Adult E.. foetida (at least two months old with a clitellum) used for each test
should weigh 300 to 500 mg and be from the same culture container.
A.8.5.8 Sample Collection. Preservation, and Storage
For a discussion on collecting, preserving, and storing samples, see Section A.7, Hazard Waste
Sampling and Handling.
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A.8.5.9 Calibration and Standardization
For a discussion on calibration and standardization, see Section A3, Quality Assurance.
A.8.5.10 Quality Control
For a discussion on quality control, see Section A.3, Quality Assurance.
A.8.5.11 Procedures
A.8.5.11.1 Test Soils-
One of two dilution factors, 0.3 or 0.5, is commonly used. A dilution factor of approximately
0.3 allows testing between 100% and 1 %, using only five concentrations (100%, 30%, 10%, 3%, and
1 %). This series of dilutions minimizes the level of effort, but, because of the wide interval between
test concentrations, provides poor test precision {± 300%). A dilution factor of 0.5 provides greater
precision (±100%), but requires several additional dilutions to span the same range of
concentrations. Improvements in precision decline rapidly as the dilution factor is increased beyond
0.5. A dilution factor of 0.5 is generally preferred.
If the hazardous waste is known or suspected to be highly toxic, a lower range of
concentrations should be used (such as 10%, 5%, 2.5%, 1.25%, and 0.63%). If a high rate of
mortality is observed during the first 1 to 2 h of the test, additional dilutions at the lower range of
concentrations should be added.
The volume of hazardous waste required for three replicates per concentration, each
containing 200 g of test soil, is approximately 1200 g. Prepare enough test soil at each concentration
to provide sufficient soil for chemical analyses.
A.8.5.11.2 Glassware Preparation-
Drill or punch a 1/16-in. hole into the center of each canning jar lid. Clean all jars using the
procedures outlined in Section A.4, Facilities and Equipment. The rinse cycle in some dishwashers is
colder than the wash cycle; this creates a thermal shock, which may crack the soft glass jars. The
cleaned jars should be dried at 50°C in a forced air oven and stored with lids (without holes) on in a
dust-free environment until needed. The lids should be examined after each use because the seal
may deteriorate during repeated washing. Replace the lids and screw rings as needed.
A.8.5.11.3 Start of the test-
Tests should begin as soon as possible, preferably within 24 h of test soil collection. If the
persistence of sample toxicity is not known, the maximum holding time should not exceed 36 h. In
no case should the test be started more than 72 h after test soil collection. Just prior to testing, the
temperature of the test soils should be adjusted to 20 ± 2°C.
Homogenize the solid waste material using a blender. Mix the homogenized solid waste
material with artificial soil to prepare 700 g each of a geometric series of test soil concentrations,
e.g., 100, 50, 25, 12.5, 6.25, 3.13% dry weight/dry weight, plus controls (100% artificial soil). To
ensure even distribution of the test soil mixture, the total amount for each concentration is mixed
together in a blender before dividing into replicates.
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After mixing, test soils need to be hydrated with deionized water to create a moist, but not
saturated, testing environment. The earthworm test soils are to be hydrated to 75% of water
holding capacity. Hydration water required to achieve the desired hyd ration is calculated as follows:
Hydration Water to be added (mL/IOOg) = THWts - EHWts
THWts (total test soil hydration water desired, ml_/100 g) =
PHYD x [(PAS X WHCas) + (PWS x WHCwS)],
EHWts (existing test soil hydration water, mL/IOOg) =
[(PAS x MFas) + (PWS x MFW$)] x 100,
where PHYD = proportion of hydration required (e.g., 0.75);
PAS = proportion of artificial soil in test soil
(e.g., 0.50);
WHCas = water holding capacity of the artificial soil
in mL/IOOg;
PWS = proportion of waste sample in the test soil;
WHCWS = the water holding capacity of the waste sample
inmL/100g;
MFas = moisture fraction of the artificial soil; and
MFWS = moisture fraction of the waste sample.
Ten earthworms are placed into each of the three replicate jars each containing 200 g (dry
weight) of test soil. (Randomize the position of test chambers at the beginning of the test.) Adult
earthworms should be handled with a spatula.
The earthworms should be placed on the surface of the test soil in a pint jar, capped, and
secured. The tests are incubated to give a soil temperature of 20 ± 2°C under continuous light. The
test jars must not be shaded.
A.8.5.11.4 Light, Photoperiod, and Temperature--
Lighting should be at continuous ambient laboratory levels, which is approximately (540 to
1080 lux), with no shading.
A.8.5.11.5 Feeding-
To avoid further interferences, the worms are not fed during the test.
A.8.5.11.6 Routine Chemical and Physical Analyses-
At a minimum, the following measurements are made:
o The pH of the test soils should be measured at the beginning and end of the test.
o The temperature of the environmental control chamber should be continuously monitored.
o The TOC of the test soils should be measured in one test jar at all test concentrations and in
the control.
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A.8.5.11.7 Observations During the Test-
Mortality is assessed by emptying the test soil onto a tray and sorting the worms from the
soil. Dead worms are discarded. (E. foetida are considered dead when they do not respond to a
gentle touch to their anterior.) Live worms are placed back into their test jars and placed on the
surface of the soil. The numbers of live and dead worms in each test chamber are recorded at 14 days
and the dead worms are discarded.
Decay of dead worms can be rapid and, if all 10 worms per container are not found, it can be
assumed that the worms decayed beyond recognition.
A.8.5.11.8 Termination of the Test--
The test is terminated after 14 days of exposure. At termination, the number of live and
dead worms in each test chamber are counted.
A.8.5.11.9 Acceptability of Test Results-
For the test results to be acceptable, mean survival in the controls must be at least 90%.
A.8.5.11.10 Summary of Test Conditions--
A summary of test conditions is listed in Table A-9.
A.8.5.12 Calculations
The effect measured during the toxicity tests using E. foetida is death.
See Section 2 in the main text for a description of data analysis methods. Both 7-d and 14-d
LCSOs and 95% confidence intervals should be calculated.
A.8.5.13 Precision and Accuracy
Section A.3, Quality Assurance, describes test precision; the accuracy of toxicity tests cannot
be determined.
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TABLE A-9. SUMMARY OF RECOMMENDED TEST CONDITIONS FOR E. FOETIDA
SURVIVAL TEST
1. Test type:
2. Soil temperature (°C):
3. Light quality:
4. Light intensity:
5. Photoperiod:
6. Test vessel type and size:
7. Test soil mass:
8. TestsoilpH*
9. Artificial soil (% weight):
10. Test soil moisture content:
11. Renewal of test materials:
12. Age of test organisms:
13. Number of test organisms
per chamber:
14. Number of replicate chambers
per dilution:
15. Feeding regime:
16. Dilution factor:
17. Test duration:
18. Effect measured:
Static
20 ± 2°C
Ambient laboratory light
540-1080 lux
Continuous illumination
1-pint glass canning jars with rings and lids 1/8 inch air hole
200 g
>.4but <.10
10% 2.36 mm-screened sphagnum peat, 20% colloidal
kaolinite clay, and 70%-grade 70 silica sand
75% of water holding capacity
None
> 60 days
10
Do not feed
0.5
14 days
Death
* If pH is outside this range, results may reflect phtoxicity. Adjustments of ph to 4.0 or 10.0 may
result in altered toxicity of other constituents. See Sec 1.4. and A 7.7.
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A.8.6 LETTUCE SEED GERMINATION (LACTUCA SATIVA)
A.8.6.1 Scope and Application
This method (modified from the method described by Thomas and Cline [1985]) estimates
the acute toxicity of solid hazardous wastes to lettuce (L sativa) in a 120-h static test. The responses
measured include the synergistic, antagonistic, and additive effects of all the chemical, physical, and
biological components that adversely affect the physiological and biochemical functions of the test
organisms. The method uses soil as the exposure medium because the exposure conditions closely
resemble natural conditions. The method could also be used to test aqueous samples by using 100%
artificial soil and using the aqueous samples in place of distilled or deionized water to hydrate the
test soils. This method should be performed by, or under the supervision of, professionals
experienced in environmental toxicity testing.
Detection limits of the toxicity of a hazardous waste solution or pure substance are organism
dependent.
A.8.6.2 Summary of Method
Lettuce seeds (L. sativa) are exposed in a static system for 120 h to different concentrations of
solid hazardous wastes mixed with artificial soil. Test results are based on the successful germination
of the seeds.
A.8.6.3 Definitions
For definitions of key terms, refer to the Glossary.
A.8.6.4 Interferences
Toxic substances may be introduced by contaminants in water, glassware, sample hardware,
artificial soil, and testing equipment (see Section A.4, Facilities and Equipment).
Improper hazardous waste sampling and handling may adversely affect test results.
Pathogenic organisms in test materials may affect test organism survival, and also confound
test results.
A.8.6.5 Safety
For a discussion on safety, see Section A.2, Health and Safety.
A.8.6.6 Apparatus and Equipment
1. Wire mesh screens for sizing seeds (fractions of an inch): 1/6 x 1/28, 1/6 x 1/30, 1/6 x 1/32, 1/6 x
1/3, A.T. Ferrell and Company, Saginaw, Ml 48601 or Seedburo Equipment Company,
Chicago, IL 60607.
2. Forceps
3. pH meter
4. Storage bottles
5. 12" x 12" polyethylene resealable bags (e.g., ZiplocR)
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6. An illuminated magnifier
7. Sample containers -- for sample shipment and storage (see Section A.7, Hazardous Waste
Sampling and Handling).
8. Controlled environmental chamber capable of maintaining a uniform temperature of
24 ± 2°C and 4300 ± 430 lux of light operating on a clock timer to control diurnal cycling.
9. Water purification system - Millipore Milli-Q or equivalent.
10. Balance, top loading - capable of weighing soil samples to 0.1 g.
11. Reference weights, Class S - for checking performance of balance. Weights should bracket
the expected weights of the weighing pans and the expected weights of the pans plus
samples.
12. Test chambers - the bottom halves of plastic petri dishes, 150-mm wide by 15-mm high
placed in 12" x 12" polyethylene resealable bags.
13. Volumetric flasks and graduated cylinders -- Class A, borosilicate glass or non-toxic plastic
labware, 10-to 1000-mL.
14. Volumetric pipets-Class A, 1-to 100-mL.
15. Serological pipets- 1-to 10-mL, graduated.
16. Pipet bulbs and fillers - PropipetR or equivalent.
17. Bulb-thermograph or electronic-chart type thermometers - for continuously recording
temperature.
18. National Bureau of Standards certified thermometer (see EPA Method 170.1. EPA 1979b).
A.8.6.7 Reagents and Consumable Materials
1. Reagent water - defined as activated-carbon-filtered, distilled or deionized water that does
not contain substances toxic to the test organisms. A water purification system may be used
to generate reagent water (see number 9 above).
2. Solid hazardous waste sample
3. Artificial soil - the artificial soil used in the lettuce seed germination test is commercially
available, 20-mesh, washed silica sand.
4. Cover sand — commercially available 16-mesh sand, which has been passed through a 20-
mesh sieve to remove fines.
5. pH buffers 4, 7, and 10 (or as per instructions of instrument manufacturer) for standards and
calibration check (see EPA Method 150.1, EPA 1979b).
6. Laboratory quality assurance samples and standards for the above methods.
7. Reference toxicant (see Section A.3, Quality Assurance).
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8. Test organisms -- lettuce (butter crunch variety), Lactuca sativa L. The seeds used in this test
are available from commercial seed companies. Seed from one lot should be purchased in
amounts adequate for one year's testing. Information on seed lot, year, or growing season
in which they were collected and germination percentage should be provided by the seed
source. Only untreated (not treated with fungicide, repellents, etc.) seeds are acceptable for
use in this toxicity test.
A.8.6.8 Sample Collection. Preservation, and Storage
For a discussion on collecting, preserving, and storing samples, see Section A.7, Hazardous
Waste Sampling and Handling.
A.8.6.9 Calibration and Standardization
For a discussion on calibration and standardization, see Section A.3, Quality Assurance.
A.8.6.10 Quality Control
For a discussion on quality control, see Section A.3, Quality Assurance.
A.8.6.11 Procedures
A.8.6.11.1 Test Soils--
One of two dilution factors, 0.3 or 0.5, is commonly used. A dilution factor of approximately
0.3 allows testing between 100% and 1%, using only five concentrations (100%, 30%, 10%, 3%, and
1 %). This series of dilutions minimizes the level of effort, but, because of the wide interval between
test concentrations, provides poor test precision (± 300%). A dilution factor of 0.5 provides greater
precision (± 100%), but requires several additional dilutions to span the same range of
concentrations. Improvements in precision decline rapidly as the dilution factor is increased beyond
0.5. A dilution factor of 0.5 is generally preferred.
A.8.6.11.2 Glassware Preparation--
All glassware used in preparing the soil samples must be thoroughly washed as described in
Section A.4, Facilities and Equipment.
A.8.6.11.3 Start of the Test-
Sample preparation should begin as soon as possible, preferably within 24 h of sample
collection. If the persistence of sample toxicity is not known, the maximum holding time should not
exceed 36 h. Just prior to testing, the temperature of the test soils should be adjusted to 24 ± 2°C.
Homogenize the solid waste material using a blender. Mix the homogenized solid waste
material with artificial soil to prepare 400 g each of a geometric series of test soil concentrations, i.e.,
100, 50, 25, 12.5, 6.25, 3.13% dry weight hazardous waste/dry weight artificial soil, plus controls
(100% artificial soil). To ensure even distribution of the test soil mixture, the total amount for each
concentration is mixed together in a blender before dividing into replicates.
After purchase, size grading of seeds is carried out on the entire seed lot. Small samples, 100-
150, are sized at a time.
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In addition, the seed lot must be inspected. Trash, empty hulls, and damaged seed are
removed.
Nest the four wire mesh sizing screens, with the one containing the largest holes on top and
those with successively smaller holes in sequence below. A blank or bottom pan is used to collect the
fraction that passes through all screens.
Pour the seeds onto the top screen, then shake the whole set of nested screens (by hand or
with a vibrator) until all the seed remains on one screen or reaches the bottom pan. The separated
fractions are set aside and retained. This procedure is repeated until all the seed in the lot is sized.
The size class containing the most seed is selected and used for the duration of the tests. The seeds in
each size class are divided into small lots, placed in separate envelopes or sacks, and stored in
airtight, waterproof containers in a refrigerator at 4°C.
Place 100 g of each concentration of air-dried control or test soil in each of three replicate
150-mm plastic petri dishes. Randomize the position of the test containers. Place 40 seeds in each
dish. Space the seeds at least 0.5 inch from the edge of the petri dish to ensure that they will be
covered with test soil. Press the seeds into the test soil with the bottom of a clean beaker. Pour 90 g
of cover sand on top of the hydrated test soil. Level the cover sand with a ruler or small piece of
cardboard. Place petri dish cover over the bottom to prevent water loss and spillage prior to
incubation.
Hydrate the test soils with deionized water to create a moist, but not saturated, testing
environment. The lettuce seed germination test soils are to be hydrated to 85% of water holding
capacity. Hydration water required to achieve the desired hydration is calculated as follows.
Hydration Water to be added (mL/100g) = THWts - EHWts
THWts (total test soil hydration water desired, mL/100 g) =
PHYD x [(PAS X WHCas) + (PWS x WHCWS)],
EHWts (existing test soil hydration water, mL/100 g) =
[(PAS x MFas) -(- (PWS x MFWS)] x 100,
where PHYD = proportion of hydration required (e.g., 0.85);
PAS = proportion of artificial soil in test soil (e.g., 0.50);
WHCas = water holding capacity of the artificial soil in mL/100 g;
PWS = proportion of waste sample in the test soil;
WHCWS = the water holding capacity of the waste sample in mL/100 g;
MFas = moisture fraction of the artificial soil; and
MFWS = moisture fraction of the waste sample.
If MFas = 0, which would be the case if the artificial soils are prepared from dry ingredients,
then EHWts = PWS x MFWS x 100.
Place each covered petri dish into a resealable polyethylene bag, centered over the bottom
of the bag. Raise the sides of the bag into a vertical position over the dish. Remove the dish cover
and seal (airtight) the bag, leaving as much air space as possible. Randomly distribute the bags in an
environmental chamber.
A.8.6.11.4 Light, Photoperiod, and Temperature-
Incubate at 24±2°C in the dark for 48 h, followed by 16 h of light and 8 h dark until
termination of the test at the end of 120 h. Light intensity should be 4300 ± 430 lux.
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A.8.6.11.5 Routine Chemical and Physical Analyses-
At a minimum, the following measurements are made:
o The pH of the test soils should be measured at the beginning and end of the test (Black 1965,
1982).
o Soil temperature is measured at the beginning of each 24-h exposure period in one replicate
of each test concentration and in the control.
A.8.6.11.6 Observations During the Test-
No observations are made during the test.
A.8.6.11.7 Termination of the Test-
After 120 h, the number of germinated seeds in each dish is determined by counting each
seedling that protrudes above the soil surface.
A.8.6.11.8 Acceptability of Test Results-
For the test results to be acceptable, mean germination in the controls must be at least 90%.
A.8.6.11.9 Summary of Test Conditions--
A summary of test conditions is listed in Table A-10.
A.8.6.12 Calculations
The effect measured during the toxicity tests using lettuce (L sativa) seeds is germination.
See Section 2 in the main text for a description of data analysis methods.
Report the LC50 and its 95% confidence limits. The LC50 is an estimate of the median lexhal
concentration.
A.8.6.13 Precision and Accuracy
Section A.3, Quality Assurance, describes test precision; the accuracy of toxicity tests cannot
be determined
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TABLE A-10. SUMMARY OF RECOMMENDED TEST CONDITIONS FOR LETTUCE
SEED (L. SATIVA) GERMINATION TEST
1. Test type:
2. Temperature (°C):
3. Light quality:
4. Light intensity:
5. Photoperiod:
Test vessel type
and size:
Static
24 ± 2°C
Fluorescent
4300 ±430 lux
Initial 48 h dark, followed by 16 h of light and 8 h of dark until
termination of the test.
The bottom halves of plastic petri
dishes, 150-mm wide by 15-mm high, placed in 12" x 12"
polyethylene resealable bags.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
Test soil mass:
Test soil moisture
content:
Artificial soil:
Test soil pH:
Renewal of test
materials:
Age of test organisms:
Number of test organisms
per chamber:
Number of replicate
chambers per dilution:
Dilution factor:
Test duration:
Effect measured:
100 g
85% of water holding capacity
20-mesh, washed silica sand
J>4but <_10
None
seeds
40
3
0.5
120 h
Germination
If pH is outside this range, results may reflect pH toxicity. Adjustment of pH to 4.0 or 10.0 may
result in altered toxicity to other constituents. See Sec 1.4 and A.7.7.
89
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A.8.7 LETTUCE ROOT ELONGATION (LACTUCA SATIVA)
A.8.7.1 Scope and Application
This method (as modified from the method described by Porcella [1983] and Ratsch [1983])
estimates the acute toxicity of aqueous hazardous wastes and hazardous waste elutriates to lettuce
seedlings (L sativa) in a 120-h static test. The responses measured include the synergistic,
antagonistic, and additive effects of all the chemical, physical, and biological components that
adversely affect the physiological and biochemical functions of the test organisms. This method
should be performed by, or under the supervision of, professionals experienced in environmental
toxicity testing.
Detection limits of the toxicity of a hazardous waste solution or pure substance are organism
dependent.
A.8.7.2 Summary of Method
Lettuce seeds (L sativa) are exposed to different concentrations of hazardous waste solutions
on wet filter paper for 120 h in the dark. Test results are based on the percent inhibition of seedling
lettuce root elongation compared to controls.
A.8.7.3 Definitions
For definitions of key terms, refer to the Glossary.
A.8.7.4 Interferences
Toxic substances may be introduced by contaminants in water, glassware, sample hardware,
artificial soil, and testing equipment (see Section A.4, Facilities and Equipment).
Improper hazardous waste sampling and handling may adversely affect test results.
Pathogenic organisms in test materials may affect test organism survival and growth, and
also confound test results.
A.8.7.5 Safety
For a discussion on safety, see Section A.2, Health and Safety.
A.8.7.6 Apparatus and Equipment
1. Wire mesh screens for sizing seeds (fractions of an inch): 1/6 x 1/28, 1/6 x 1/30, 1/6 x 1/32, 1/6 x
1/3, AT. Ferrell and Company, Saginaw, Ml 48601 or Seedburo Equipment Company,
Chicago, IL 60607.
2. Forceps.
3. pH meter.
4. Storage bottles for the hazardous waste solutions (see Section A.7, Hazardous Waste
Sampling and Handling).
5. 33-gal, black plastic garbage bags.
6. Metric ruler.
90
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7. Whatman filter paper, grade 3, 9-cm.
8. An illuminated magnifier.
9. A glass plate - for measuring root lengths.
10. Sample containers - for sample shipment and storage (see Section A.7, Hazardous Waste
Sampling and Handling).
11. Controlled environmental chamber capable of maintaining a uniform temperature of
24 ± 2°C
12. Water purification system -- Millipore Milli-Q or equivalents.
13. Balance, top loading-capable of weighing soil samples to 0.1 g.
14. Reference weights, Class S -- for checking performance of balance. Weights should bracket
the expected weights of the weighing pans and the expected weights of the pans plus
samples.
15. Test chambers - glass petri dishes, 100-mm wide by 15-mm with covers.
16. Volumetric flasks and graduated cylinders - Class A, borosilicate glass or non-toxic plastic
labware, 10-to 1000-mL
17. Volumetric pipets-Class A, 1-to100-mL.
18. Serological pipets - 1- to 10-mL, graduated.
19. Pipet bulbs and fillers -- PropipetR or equivalent.
20. Bulb-thermograph or electronic-chart type thermometers -- for continuously recording
temperature.
21. pH, DO, and specific conductivity meters -- for routine physical and chemical measurements.
Portable, field-grade instruments are acceptable.
22. National Bureau of Standards certified thermometer (see EPA Method 170.1, EPA 1979b).
A.8.7.7 Reagents and Consumable Materials
1. Reagent water -- defined as activated-carbon-filtered, distilled or deionized water that does
not contain substances toxic to the test organisms. A water purification system may be used
to generate reagent water (see number 12 above).
2. Aqueous hazardous waste sample or a hazardous waste elutriate (see Section A.7, Hazardous
Waste Sampling and Handling).
3. Dilution water -- Deionized water.
4. Reagents for hardness and alkalinity tests (see EPA Methods 130 2 and 310.1, EPA 1979b).
91
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5. pH buffers 4, 7, and 10 (or as per instructions of instrument manufacturer) for standards and
calibration check (see EPA Method 150.1, EPA 1979b).
6. Membranes and filling solutions for the dissolved oxygen probe (see EPA Method 360.1, EPA
1979b), or reagents for modified Winkler analysis.
7. Laboratory quality assurance samples and standards for the above methods.
8. Reference toxicant (see Section A.3, Quality Assurance).
9. Test organisms - lettuce (butter crunch variety), Lactuca sativa L. The seeds used in this test
are available from commercial seed companies. Seed from one lot should be purchased in
amounts adequate for one year's testing. Information on seed lot, year, or growing season
in which they are collected and germination percentage should be provided by the source of
seed. Only untreated (not treated with fungicide, repellents, etc.) seeds are acceptable for
use in this toxicity test.
A.8.7.8 Sample Collection. Preservation, and Storage
For a discussion on collecting, preserving, and storing samples, see Section A.7, Hazard Waste
Sampling and Handling.
A.8.7.9 Calibration and Standardization
For a discussion on calibration and standardization, see Section A.3, Quality Assurance.
A.8.7.10 Quality Control
For a discussion on quality control, see Section A.3, Quality Assurance.
A.8.7.11 Procedures
A.8.7.11.1 Test Solutions--
One of two dilution factors, 0.3 or 0.5, is commonly used. A dilution factor of approximately
0.3 allows testing between 100% and 1%, using only five concentrations (100%, 30%, 10%, 3%, and
1 %). This series of dilutions minimizes the level of effort, but, because of the wide interval between
test concentrations, provides poor test precision (± 300%). A dilution factor of 0.5 provides greater
precision (±100%), but requires several additional dilutions to span the same range of
concentrations. Improvements in precision decline rapidly as the dilution factor is increased beyond
0.5. A dilution factor of 0.5 is generally preferred
Twenty milliliters of solution is required for each test concentration. The minimum volume
of hazardous waste solution required to run the test with three replicates per test concentration is
about 240 ml. Prepare enough test solution (approximately 500 ml) for the highest and lowest
concentrations to provide 300 ml additional volume for routine chemical analyses. (See Section
A.8.7.11.4)
A.8.7.11.2 Start of the Test-
Sample preparation should begin as soon as possible, preferably within 24 h of sample
collection. If the persistence of sample toxicity is not known, the maximum holding time should not
exceed 36 h Just prior to testing, the temperature of the test solutions should be adjusted to
24 ± 2°C.
92
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After purchase, size grading of seeds is carried out on the entire seed lot. Small samples, 100-
150 grams, are sized at a time.
In addition, the seed lot must be inspected. Trash, empty hulls, and damaged seeds are
removed.
Nest the four wire mesh sizing screens, with the one containing the largest holes on top and
screens with successively smaller holes in sequence below. A blank or bottom pan is used to collect
the fraction that passes through all screens.
Pour the seeds onto the top screen, then shake the whole set of nested screens (by hand or
with a vibrator) until all the seed remains on one screen or reaches the bottom pan. The separated
fractions are set aside and saved. This procedure is repeated until all the seed in the lot is sized. The
size class containing the most seed is selected and used for the duration of the tests. The seeds in
each size class are divided into small lots, placed in separate envelopes or sacks, and stored in
airtight, waterproof containers in a refrigerator at 4°C.
Place a sheet of Whatman No. 3 filter paper in each of three replicate 100-mm plastic petri
dishes. Prepare 20 ml of each test concentration using deionized water to dilute the hazardous
waste solutions to the appropriate test concentrations.
Each replicate petri dish containing filter paper receives 4 ml of test solution. (The
remaining 8 ml may be saved for chemical analysis.) Place five seeds, equally spaced, in a circle on
the filter paper, equidistant from the edge to the center. Place the lid on each petri dish.
Set the petri dishes in layers in a black 33-gal plastic garbage bag lining a cardboard box.
(Randomize the position of test containers at the beginning of the test.) Place moist laboratory
paper towels between layers to keep the humidity level elevated and to prevent drying of the filter
paper. Tape the garbage bag and close the box to seal the system. The box is placed in a controlled
environment chamber and incubated for 120 h at 24 ± 2°C.
A.8.7.11.3 Light, Photoperiod, and Temperature-
Incubate at 24 ± 2°C in the dark.
A.8.7.11.4 Routine Chemical and Physical Analyses--
At a minimum, the following measurements are made:
o The pH, alkalinity, and hardness of the test solutions should be measured at the beginning of
the test.
o Temperature is measured at the beginning of each 24-h exposure period.
A.8.7.11.5 Observations During the Test-
No observations are taken during the test.
A.8.7.11.6 Termination of the Test-
Measurement of root length is made at 120 h from the start of dark incubation. It is
important to measure each plate as nearly as possible to 120 h (not to exceed ± 30 min.).
93
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Remove the seeds from the filter paper and place on the glass work surface. Measure the
distance from the transition point between the hypocotyl and root to the tip of the root. At the
transition between the hypocotyl and the primary root, the axis may be slightly swollen, contain a
slight crook, or change noticeably in size. The lengths of all roots are measured to the nearest
millimeter and entered on the data sheet. For additional descriptions and photographs helpful in
making root measurements, see USDA (1952) and Wellington (1961).
A.8.7.11.7 Acceptability of Test Results-
For the test results to be acceptable, mean germination in the controls must be at least 90%.
A.8.7.11.8 Summary of Test Conditions-
A summary of test conditions is listed in Table A-11.
A.8.7.12 Calculations
The effect measured during the toxicity tests using lettuce (L sativa) root elongation is
percent inhibition of lettuce root elongation compared to controls.
See Section 2 in the main text for data analysis methods.
Report the EC50 and its 95% confidence limits. The EC50 is an estimate of the median
effective concentration.
A.8.7.13 Precision and Accuracy
Section A.3, Quality Assurance, describes test precision; the accuracy of toxicity tests cannot
be determined.
94
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TABLE A-11. SUMMARY OF RECOMMENDED TEST CONDITIONS FOR LETTUCE
(L. SATIVA) ROOT ELONGATION TEST
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Test type:
Temperature (°C):
Light quality:
Light intensity:
Photoperiod:
Test vessel type and
size:
Test solution volume:
Test Solution pH:
Dilution water:
Renewal of test
Static
24 ± 2°C
Dark
Dark
Dark
Glass petri dishes, 100-mm wide by
15-mm high
4mL
^4but
-------
SECTION A.9
REPORT PREPARATION
The general format and content recommended for reports follows.
1. INTRODUCTION
1.1 Sample number
1.2 Toxicity testing objectives or requirements
1.3 Hazardous waste site location
1.4 Contractor (if toxicity testing is contracted)
1.4.1 Name of firm
1.4.2 Phone number
1.4.3 Address
2. SITE CHARACTERISTICS
2.1 Types of hazardous wastes known to be present
2.2 Geology
2.3 Hydrology
2.4 Descri pti on of waste treatment
2.5 Volume of waste present
3. SOURCE OF HAZARDOUS WASTE, ELUTRIATE, DILUTION WATER, AND ARTIFICIAL SOIL
3.1 Samples
3.1.1 Sampling point
3.1.2 Collection dates and times
3.1 3 Sample collection method
3.1.4 Physical and chemical data
3.2 Elutriate Preparation
3.3 Dilution Water
3.3 1 Source
3.3.2 Collection date and time
3.3.3 Pretreatment
3.3.4 Physical and chemical characteristics
3.4 Artificial Soil
3.4.1 Composition
3.4.2 Pretreatment
3.4.3 Physical and chemical characteristics
4. TEST METHODS
4.1 Toxicity test method used
4.2 Endpoint(s) of test
4.3 Deviations from reference method, if any, and the reason(s)
4.4 Date and time test started
4.5 Date and time test terminated
4.6 Type of test chambers
4.7 Volume of solution or hazardous waste used/chamber
4.8 Number of organisms/test chamber
4.9 Number of replicate test chambers/treatment
96
-------
4.10 Acclimation of test organisms (mean and range)
4.11 Test temperature (mean and range)
TEST ORGAN ISMS
5.1 Scientific name (and variety, if any)
5.2 Age
5.3 Life stage
5.4 Mean length and weight (where applicable)
5.5 Source
5.6 Diseases and treatment (where applicable)
QUALITY ASSURANCE
6.1 Standard toxicant used and source
6.2 Date and time of most recent test
6.3 Dilution water used in test
6.4 Results (LC50 or EC50 and confidence limits)
6.5 Physical and chemical methods used
RESULTS
7.1 Provide raw biological data in tabular form, including daily records of affected organisms
in each concentration (including controls)
7.2 Provide table of LCSOs or ECSOs
7.3 Indicate statistical methods to calculate endpoints
7.4 Provide summary table of physical and chemical data
7.5 Tabulate QA data
97
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SECTION A.10
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* U.S. GOVERNMENT PRINTING OFFICE: 1989- 648-163/8706
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