THE Coastal Resource Coordinator’s 

Bioassessment Manual

Report No. HAZMAT 93-1 (revised)

Prepared by 

Hazardous Materials Response and Assessment Division 

Office of Ocean Resources Conservation and Assessment 

National Oceanic and Atmospheric Administration 
Purpose and Use of This Guidance

This manual and any internal procedures adopted for its implementation are intended solely 

as guidance. They do not constitute rulemaking by any agency and may not be relied upon to 

create a right or benefit, substantive or procedural, enforceable by law or in equity, by any 

person. Any agency or person may take action at variance with this manual or its internal 

implementing procedures. 

This document should be cited as: MacDonald, D.A., M.B. Matta, L.J. Field, C. Cairncross, 

and M.D. Munn. 2003. The coastal resource coordinator’s bioassessment manual. Report 

No. HAZMAT 93-1 (revised). Seattle, WA. National Oceanic and Atmospheric 

Administration. 160 pp + appendices. 
THE Coastal Resource Coordinator’s

BIOASSESSMENT MANUAL

Donald A. MacDonald 

Mary B. Matta 

L. Jay Field 

Charles Cairncross 

and 

Mark D. Munn*

(E.V.S. Consultants, Inc. 

Seattle, Washington) 

December 1992 – Revised July 2003 

Report No. HAZMAT 93-1 

NOAA / Hazardous Materials Response and Assessment Division 

7600 Sand Point Way NE 

Seattle, Washington 98115 

*Current address: U.S. Geological Survey 

Water Resources Division 

 Tacoma, Washington 
TABLE OF CONTENTS 

LIST OF TABLES AND FIGURES................................................................ vi

PREFACE.......................................................................................................... ix

CHAPTER 1 BIOASSESSMENT: AN OVERVIEW.......................... 1-1

CHAPTER 2 THE ROLE OF BIOASSESSMENT IN THE 

REMEDIAL PROCESS 

 INTRODUCTION ........................................................ 2-1 

 PRELIMINARY ASSESSMENT .................................... 2-1 

 REMEDIAL INVESTIGATION ...................................... 2-2 

FEASIBILITY STUDY: BIOASSESSMENT 

AS A TOOL TO ESTABLISH SITE CLEANUP 

TARGET LEVELS. ..................................................... 2-4 

REMEDIAL DESIGN/REMEDIAL ACTION: 

BIOASSESSMENT AS A TOOL TO 

EVALUATE THE SUCCESS OF REMEDIAL 

ACTIONS................................................................... 2-6 

 REFERENCES............................................................ 2-7 

CHAPTER 3 TOXICITY TESTS

 INTRODUCTION ........................................................ 3-1 

Objectives of Toxicity Testing ................................................ 3-1

Advantages of Toxicity Testing ............................................. 3-2

Disadvantages of Toxicity Testing ........................................ 3-3

 TEST SELECTION.......................................................................... 3-4

Testing Water, Soil, or Sediment............................................ 3-5

Selecting a Test Organism ...................................................... 3-7

Acute or Chronic Testing........................................................ 3-9

 Endpoints .................................................................................. 3-9

Sediment Test Conditions: Static or Flow-Through.........3-10

 Cost Effectiveness ..................................................................3-10

SAMPLING DESIGN CONSIDERATIONS................... 3-11 

 i July 2003
INTERPRETATION OF RESULTS..............................3-12 

Comparison with Control Samples .....................................3-12

 EC50s and LC50s .....................................................................3-13

Other Factors Influencing Toxicity Tests............................3-13

Determining the Cause of Toxicity......................................3-14

SUMMARY................................................................3-16 

 REFERENCES ..........................................................3-16

CHAPTER 4 BIOACCUMULATION 

 INTRODUCTION ......................................................................... 4-1

Advantages of Bioaccumulation Studies.............................. 4-2

Disadvantages of Bioaccumulation Studies ......................... 4-2

 TEST ORGANISMS ....................................................................... 4-4

Type of Organism .................................................................... 4-4

Test Organism Selection.......................................................... 4-7

 SUMMARY..................................................................4-9 

 REFERENCES ............................................................4-9 

CHAPTER 5 BIOMARKERS

 INTRODUCTION .......................................................................... 5-1

Objectives and Purpose........................................................... 5-4

Advantages of Biomarkers ..................................................... 5-4

Disadvantages of Biomarkers................................................. 5-5

SELECTING A TEST...................................................5-6 

TYPES OF BIOMARKERS ...........................................5-7

Enzyme and Protein Systems ................................................. 5-7

 Detoxification Enzymes .......................................................... 5-7

 Binding Proteins....................................................................... 5-9

Contaminant Metabolites in Bile .........................................5-11

 Genetic Disorders...................................................................5-11

Immune System Responses ..................................................5-12

 Histopathology.......................................................................5-13

 Pathology.................................................................................5-14

 Growth.....................................................................................5-16

 Reproduction ..........................................................................5-16

 DATA INTERPRETATION.........................................5-18 

July 2003 ii
 SUMMARY ............................................................... 5-18 

 REFERENCES.......................................................... 5-19 

CHAPTER 6 BENTHIC COMMUNITY STUDIES 

 INTRODUCTION ........................................................ 6-1 

 STUDY OBJECTIVES ................................................. 6-2 

 SAMPLING METHODS ............................................... 6-3 

Quantitative vs non-quantitative sampling ......................... 6-3

 Sampling Devices..................................................................... 6-5

 DATA ANALYSIS..................................................... 6-10 

 Ecological Analysis ................................................................6-11

 Statistical approaches ............................................................6-13

EPA’S RAPID BIOASSESSMENT PROTOCOLS......... 6-13 

 SUMMARY ...................................................................................6-15

 Study Objective ......................................................................6-15

 Sampling Methods .................................................................6-15

 Sampling Devices...................................................................6-15

 Data Analysis..........................................................................6-16

 REFERENCES.......................................................... 6-16 

CHAPTER 7 STUDY DESIGN AND STATISTICAL ANALYSIS 

 INTRODUCTION ........................................................ 7-1 

 STUDY DESIGN ......................................................... 7-2 

 Question Formulation ............................................................. 7-2

 Reconnaissance Survey ........................................................... 7-5

 Station selection........................................................................ 7-6

Random versus nonrandom designs .................................... 7-8

 Sample Replication .................................................................. 7-9

STATISTICAL ANALYSIS......................................... 7-11 

Hypothesis Formulation and Testing .................................7-12

 Randomization .......................................................................7-16

Correlation and Regression Analysis..................................7-17

SUMMARY....................................................................................7-17

 REFERENCES ...............................................................................7-19

 iii July 2003
CHAPTER 8 SUMMARY OF RECOMMENDED BIOASSAY 

PROTOCOLS 

 INTRODUCTION.........................................................8-1 

 SOIL...........................................................................8-1 

Lettuce Seed (Latuca sativa) Germination Bioassay 

(Thomas and Cline, 1985) ....................................................... 8-1

 Earthworm (Eisenia foetida) Lethality Bioassay 

(Porcella 1983).........................................................................8-10

 WATER COLUMN ......................................................................8-10

 Selenastrum capricornutum Algal Lethality Bioassay 

(Porcella, 1983) 8-10

 Water flea Daphnia magna EC50 Bioassay 

(Porcella, 1983) 8-11

 Ceriodaphnia dubia Survival and Reproduction 

Bioassay (EPA, 1985) 8-11

 Fathead Minnow Pimephales promelas Larval 

Survival and Growth Bioassay (EPA, 1985) 8-12

SEDIMENTS ..........................................................8-13 

 Daphnia magna Lethality and Partial Life Cycle 

Bioassay (Nebeker et al., 1984).............................................8-13

ASTM Designation: E 1367 - 92. 1996. Standard Guide 

for Conducting 10-Day Static Sediment Toxicity Tests 

with Marine and Estuarine Amphipods. [Rhepoxynius 

 abronius, Eohaustorius estuarius, Ampelisca abdita, 

 Grandidierella japonica] .........................................................8-13

ASTM Designation: E 1706 - 95b (Replaces 1383-90). 1996. 

Standard Guide for Conducting Sediment Toxicity Tests 

with Freshwater Invertebrates. [Hyalella azteca, 

Chironomus tentans, Chironomus riparius]. ...........................8-17 

 ASTM Designation: E 1611 - 94. 1996. Standard guide 

for conducting sediment toxicity tests with marine and 

estuarine polychaetous annelids. (Neanthes 

 arenaceodentata, Neanthes virens ).........................................8-20 

July 2003 iv
 Protocols From Recommended Guidelines 

For Conducting Laboratory Bioassays On 

Puget Sound Sediments (Puget Sound Estuary 

Program, 1991)........................................................................8-21

TEST SELECTION............................................... 8-31 

 REFERENCES.......................................................... 8-37 

CHAPTER 9 AQUATIC PHYSICO-CHEMICAL PARAMETERS 

 DEFINITION ............................................................... 9-1 

 TEMPERATURE......................................................... 9-1 

SALINITY................................................................... 9-2 

SEDIMENT GRAIN SIZE ............................................. 9-4 

TOTAL SOLIDS/MOISTURE CONTENT ...................... 9-5 

TOTAL VOLATILE SOLIDS AND TOTAL ORGANIC 

CARBON ................................................................... 9-6 

OIL AND GREASE ..................................................... 9-9 

 TOTAL SULFIDE ..................................................... 9-10 

ACID VOLATILE SULFIDES ..................................... 9-11 

 pH ........................................................................... 9-12 

EH (ELECTRODE POTENTIAL) ................................ 9-13 

SUMMARY....................................................................................9-14

ADDITIONAL READING.............................................................9-15

 REFERENCES ...............................................................................9-15

APPENDIX A GLOSSARY ............................................... A-1 

APPENDIX B PROTOCOLS ............................................ B-1 

APPENDIX C TOXICITY TEST SENSITIVITY COMPARISON 

TABLE ...................................................... C-1 

 v July 2003
LIST OF TABLES AND FIGURES 

TABLES 

Table 3-1 Advantages and disadvantages of toxicity tests. ....................................3-3

Table 3-2 Steps in toxicity test selection.....................................................................3-5

Table 4-1 Advantages and disadvantages of bioaccumulation studies. ...............4-3

Table 4-2 Advantages and disadvantages of the various types of organisms 

used in bioaccumulation studies. ..............................................................4-5

Table 4-3 Organism selection criteria for bioaccumulation studies, adapted 

from Phillips, 1977. ......................................................................................4-8

Table 5-1 Advantages and disadvantages of using biomarkers at 

waste sites. ....................................................................................................5-6

Table 5-2 Sensitivity of biomarkers to specific contaminant groups.....................5-7

Table 5-3 Biomarker tests currently applied to specific groups of 

organisms. .....................................................................................................5-8

Table 6-1 Stream-net samplers used in assessing benthic communities in 

freshwater streams.......................................................................................6-6

Table 6-2 Dominant grab samplers used in assessing benthic 

communities. ...............................................................................................6-8

Table 6-3 Advantages and disadvantages of artificial substrates. ......................6-10

Table 7-1 Examples of parametric and nonparametric tests for the 

comparison of samples adapted from Elliott (1977). ...........................7-14

Table 7-2 Principals of environmental study design from Green (1979). ..........7-18

Table 8-1 Bioassay tests by habitat and media..........................................................8-2

Table 8-2 Releative sensitivities of selected test organisms and 

endpoints....................................................................................................8-32

July 2003 vi
Table 8-3 Rating of selection criteria for freshwater sediment toxicity testing 

organisms. ..................................................................................................8-36

FIGURES 

Figure 1-1 Assessment categories and example methods for addressing 

various levels of concern at a hazardous waste site. ............................ 1-3

Figure 3-1 TIE strategy to evaluate contributions of contaminant groups 

(Ankley et al., 1991). ................................................................................3-15

Figure 5-1 Exposure-effects pathway and potential indigenous organism 

measures of response and adverse effects.............................................. 5-2

 vii July 2003
July 2003 viii
PREFACE 

This manual has been designed for the use of NOAA's Coastal Resource Coordinators as an 

introduction to and a general guide for using bioassessment techniques for evaluating 

conditions at hazardous waste sites. It is meant to be a ready reference to help them make 

suggestions for or evaluate proposed ecological work plans for these sites. It is not meant to 

be a cookbook for work plan development. More detailed information on the use and 

applicability of these techniques is available from the CRC technical support staff in Seattle. 

The manual has been designed in loose-leaf form so that it can be readily modified as 

changes occur in the rapidly developing field of bioassessment or gaps in the information 

are identified. An early planned modification will be an expansion of the discussion on 

laboratory bioaccumulation methodologies. The users should be aware that this guide is 

not all inclusive of the currently available bioassessment methodologies but does include 

those methodologies that have been most commonly used at sites of concern to NOAA. 

At this time I would like to thank the people, who in addition to the authors, made this 

manual possible. Reviewers Edward R. Long, Rebecca Hoff, Nancy Beckvar, Peter Knight, 

Sean Morrison, Diane Wehner, Karen Wurst, Waynon Johnson, and Chris Mebane for their 

critical comments on earlier drafts of the manual; NOAA Hazardous Materials Response 

and Assessment Division librarian, John Kaperick for helping to obtain pertinent 

documents; technical editor Charlene Swartzell for making the final version readable; 

graphic artist Virginia Curl for cover graphics, and CRC Branch Manager, Alyce Fritz, for 

her support and guidance. 

Donald A. MacDonald 

November 20, 1992 

 ix July 2003
July 2003 x
HAZMAT 93-1–Introduction

CHAPTER 1 

BIOASSESSMENT: AN OVERVIEW 

DEFINITION 

Bioassessment is the characterization of environmental conditions through the use of 

biological organisms. The major objectives of this document are to provide some general 

guidelines on: the application of bioassessment procedures to the different stages of the 

hazardous waste site remedial process, the design of bioassessment studies, the use of 

specific bioassessment methodologies, and the concurrent physico-chemical measurements 

needed. In addition, a summary of recommended toxicity testing protocols, most of which 

represent the present state-of-the-art, is provided for use in specific situations. The 

bioassessment methodologies discussed in this document are generally restricted to those 

applicable to aquatic environments since this is the environment of concern to the National 

Oceanic and Atmospheric Administration (NOAA). 

There are three levels of questions concerning contamination of the environment near a 

hazardous waste site: 

1. Are contaminants present? 

2. Are contaminants bioavailable? 

3. Are contaminants causing or have the potential to cause bioeffects? 

While chemical analyses are an important first step in the characterization of hazardous 

waste sites, by themselves they yield little information on impacts or potential impacts to 

biological resources. The presence of a contaminant does not mean it is bioavailable, and 

the fact that it is bioavailable does not necessarily mean it is capable of causing bioeffects. In 

the context of this document, bioeffect means an effect on a biological organism, 

population, or community that is detrimental to the health of the organism, population, or 

community. Determining the potential for causing bioeffects is a necessary component in 

the determination of the environmental impact of a hazardous waste site. 

Bioassessment methodologies use well-defined tests with biological organisms to determine 

biological sensitivities to contaminated soil, sediment, or water samples from hazardous 

waste sites. These methodologies can be grouped into four general categories: toxicity tests, 

bioaccumulation, biomarkers (biochemical effects, physiological effects, incidence of 

disease), and community studies. Individual bioassessment methodologies are incapable of 

proving cause and effect at hazardous waste sites. However, an integrated approach using 

 1-1 July 2003 
HAZMAT 93-1–Introduction

a number of assessment methodologies, both biological and chemical, can provide a 

preponderance of evidence linking observed bioeffects to a hazardous waste site. Chemical 

analysis of environmental media indicates if contaminants are present in the environment. 

Bioaccumulation studies indicate if the contaminants are bioavailable; this is especially 

important for chemicals that are not acutely toxic. Toxicity tests indicate if contaminated 

media are capable of causing bioeffects, in particular, acute toxicity. Community studies 

and biomarkers indicate if indigenous organisms, in the vicinity of the waste site, are 

experiencing bioeffects. Taken singly the data generated from each of these methods gives 

some information about conditions in the vicinity of the waste site, but, taken as part of a 

properly integrated bioassessment study, the same data can strongly suggest that the 

hazardous waste site is the cause of the observed bioeffects (Figure 1-1). 

July 2003 1-2 
HAZMAT 93-1–Introduction

Figure 1-1. Assessment categories and example methods for addressing 

various levels of concern at a hazardous waste site. 

 1-3 July 2003 
HAZMAT 93-1–Introduction

July 2003 1-4 
HAZMAT 93-1 Role of Bioassessment

CHAPTER 2 

THE ROLE OF BIOASSESSMENT IN THE REMEDIAL 

PROCESS 

INTRODUCTION 

The selection of the appropriate bioassessment procedure(s) is dependent on the objectives 

of the particular stage in the remedial process. The amount of information required to make 

necessary decisions regarding further activities at the site tends to increase as the overall 

assessment of a site proceeds from the preliminary screening to the endangerment 

assessment and selection of remedial action. Regardless of the stage in the remedial 

process, it is of primary importance to clearly establish the intended application of 

bioassessment data prior to such data collection, including the specific questions that are 

being addressed, the proposed decision criteria, and appropriate sampling strategy and 

statistical design. If properly planned, data collected at each stage can be used to guide the 

development of work plans for subsequent stages. The early incorporation of an overall 

sampling design that adequately addresses the potential risks to natural resources reduces 

the possibility of additional sampling later that might delay or hinder the remedial process. 

The role of bioassessment approaches during the different stages of a site remediation is 

discussed in more detail in this chapter. 

PRELIMINARY ASSESSMENT 

The data needs for the preliminary assessment are usually directed toward broad-scale 

screening for evidence of release of contaminants to the environment and the presence or 

absence of toxicity. Bioassessment procedures, particularly bioaccumulation studies and 

toxicity tests, can play an important role in site screening. 

The bioaccumulation of chemical contaminants in tissues of resident organisms effectively 

demonstrates the bioavailability of contaminants associated with the site. For contaminants 

known to bioaccumulate to a greater extent in higher trophic level organisms (e.g., PCBs, 

mercury), determining the degree of contamination in a representative higher trophic level 

organism may provide a worst-case evaluation appropriate for the preliminary assessment. 

Combined with information on the site history, visual observations of site characteristics 

and chemical analyses, toxicity tests can provide qualitative information on the distribution 

2-1 July 2003 
HAZMAT 93-1 Role of Bioassessment

of toxic substances and the relative toxicity of the site. Toxicity tests can also provide an 

indication of the relationship between toxicity and chemical concentrations that may be 

particularly useful in defining areas where contamination is of concern. For this purpose, 

the use of two toxicity tests is recommended. As resources permit, additional acute toxicity 

tests could be employed to broaden the range of toxic substances that can be detected. This 

is especially important at sites with complex mixtures of contaminants or where the site 

history indicates the possible presence of substances for which analyses are not normally 

done (e.g., pharmaceutical or dye manufacturing). At sites where the presence of 

substances known to be especially toxic to a specific organism or type of organism is 

suspected, additional toxicity tests for those substances should be included. For example, 

since fish are particularly sensitive to the pesticides endosulfan, rotenone, and toxaphene, 

inclusion of an appropriate laboratory fish toxicity test would be recommended if the 

presence of one or more of these substances was suspected. 

REMEDIAL INVESTIGATION 

During the remedial investigation (RI) stage, bioassessment tools provide important 

information for the ecological risk assessment. They can be useful in characterizations of 

both the exposure and ecological effects. Chemical information obtained during the 

preliminary assessment can be used to help guide the site characterization during the RI 

phase of the study. Chemical analyses should provide information on the types of 

substances present and the possible ranges of their concentrations. Using basic information 

available for many compounds on the hazardous substance list regarding their toxicity to 

different organisms, chemical information can indicate which bioassessment approaches 

may be most fruitful. At sites where only one, or a few closely related substances of concern 

are predominant, it may be possible to select a bioassessment technique known to be 

sensitive to those substances. This is important when it is necessary to limit the number of 

different tests run on each sample in order to increase the total number of samples that can 

be tested. For example, acute Crustacea toxicity tests may alone be an adequate 

bioassessment option in situations where trace metals are the primary concern, since 

Crustacea used in common toxicity tests are reasonably sensitive to toxicity from most 

metals. This would allow an increase in the spatial or temporal coverage of sampling, while 

maintaining confidence that the toxic substances present were being detected. Where 

chemical information during the preliminary assessment indicates the presence of a 

complex mixture of contaminants, two or more different toxicity tests should be run on each 

sample to increase the likelihood that toxicity from the different toxic materials would be 

July 2003 2-2 
HAZMAT 93-1 Role of Bioassessment

identified. If the compounds such as PCBs or dioxin are present that are known to 

bioaccumulate and are not acutely toxic to most organisms, bioaccumulation studies may be 

the best approach. 

Bioassessment procedures such as toxicity tests or benthic community studies provide a 

direct evaluation of the spatial distribution of toxic areas and an indication of the degree to 

which toxicity is associated with the distribution of one or more toxic substances. In the 

simplest case, the bioassessment acts as an adjunct to the chemical analyses. The chemical 

measurements provide the link between the spatial areas and the source(s) of 

contamination, while the bioassessment measurements determine the zones where the 

chemical contamination is sufficient to be toxic. In other situations, toxicity detected in the 

bioassessment may not vary spatially in the same manner as the majority of the chemicals 

measured; possibly because the biological test is responding to substances that are not 

detected in the standard chemical tests. In both cases, synoptic surveys of bioassessment 

and chemical measurements should be made. (Note that the spatial heterogeneity at 

hazardous waste sites is often very high, both horizontally and vertically. As a result, the 

bioassessment and chemical samples must, if possible, be taken as aliquots from the same 

homogenized sample.) 

Toxicity tests have been used effectively to determine the extent of contamination at the 

Rocky Mountain Arsenal waste site in Colorado (Thomas et al., 1986). Using the results 

from a lettuce seed soil toxicity test and a statistical mapping technique called kriging, the 

extent of contamination at the site was determined solely by mapping the levels of toxicity. 

In summary, the selection of the numbers of samples and the types of tests to perform must 

be considered on a case-by-case basis, weighing the trade off between collecting greater 

numbers of samples to improve spatial coverage and conducting more tests at each station 

to broaden the types of toxicity that will be detected. In general, a minimum of two toxicity 

tests should be performed. Additional toxicity tests should be included if the available data 

indicate the possible presence of organism-specific toxins or complex suites of substances. 

As resources allow, in situ bioassessments (e.g., benthic community assessments, incidence 

of disease, bioaccumulation) may be included in the site characterization. Although these 

types of tests tend to be less precise in their ability to define the spatial extent of problem 

areas, they are important in demonstrating impact to natural resources and supporting the 

results of the toxicity tests. 

The data needed for the characterization of ecological effects from a site differ from those 

needed for the earlier phases of the remedial process. Impact evaluation should be based on 

2-3 July 2003 
HAZMAT 93-1 Role of Bioassessment

in situ measures of response, i.e., community effects and/or disease incidence. Toxicity tests, 

even in situ, are measures of the presence of toxicity and hence indicate possible effects, but 

only direct assessment of resident organisms can establish and quantify those effects. (Note, 

however, that the toxicity test and chemical data that is obtained for other purposes may be 

invaluable in demonstrating that any measured in situ effects are related to the 

contamination, and are not a result of natural fluctuations.) The characteristics of the 

habitats and populations that may be affected are major determinants in selecting 

bioassessment procedures for impact evaluation. The procedure selected must consider 

whether sufficient individuals are present at the sampling locations to obtain the statistical 

precision capable of allowing toxic effects to be resolved from natural variability. For 

example, small streams may have too limited a fish population to make creditable 

population or disease measurements possible. 

The scope of work for ecological assessments is dependent on the balance that must be 

achieved between the resources available and the quantitative precision of the assessment. 

Both the selection of multiple assessment procedures and the number of samples collected 

directly affect the costs of the overall assessment. The selection task at most sites is 

generally simpler than implied above, however, because in most situations: 

a) The populations suitable for assessment are in fact limited. 

b) The area over which the exposure is expected is also usually limited, particularly 

with respect to the ranges of many organisms that would be tested. 

c) At least the minimum numbers of samples required to obtain statistically 

meaningful data can usually be readily determined. 

FEASIBILITY STUDY: BIOASSESSMENT AS A TOOL TO 

ESTABLISH SITE CLEANUP TARGET LEVELS.

The ultimate goal of site remedial activities is to eliminate exposing biological organisms to 

any toxic materials. Bioassessment procedures have obvious uses in meeting that goal by 

providing site-specific information on the distribution of toxic areas and the levels of siterelated contaminants that are toxic. Bioassessments can be used in two ways to help 

determine target levels for cleanup. 

The most frequently approach for using bioassessment procedures to establish cleanup 

levels is to determine the toxicity of a range of concentrations of the substances of concern at 

a site. The range of concentrations to be tested can be prepared by diluting a single sample 

July 2003 2-4 
HAZMAT 93-1 Role of Bioassessment

of contaminated media from the site in the laboratory with clean media, or by collecting a 

number of samples of contaminated media from different spatial areas that are known to 

have different chemical concentrations. The samples representing the range of chemical 

concentrations obtained by either approach are tested using one or more bioassessment 

techniques to determine which concentrations are toxic and which are not. The lowest 

contaminant concentration found to be toxic (induce the designated biological endpoint) is 

determined to be the target level for cleanup. 

One example of this approach is the apparent effects threshold (AET), which uses field 

chemistry data (concentrations of toxic substances in sediments) and at least one biological 

indicator of injury (sediment toxicity tests, altered benthic infaunal abundance, 

bioaccumulation, histopathology, etc.) to determine the concentration of a given 

contaminant above which statistically significant biological effects would always be 

expected (Tetra Tech, 1986). The AET approach was developed in Puget Sound, 

Washington to establish chemical criteria for disposal of dredged material, and is being 

considered by U.S. Environmental Protection Agency (EPA) Region 10 for use in 

establishing cleanup target levels in that area. As currently developed, the AET approach 

should be used on a site-specific basis and can require extensive data collection. However, 

by carefully selecting one or a few appropriate bioassessment procedures, the approach can 

be cost effective at many sites. Further, if similar data are collected at different sites within a 

region that have similar habitat characteristics, the data may be suitable to combine into a 

regional database that might be used to develop standards with broad application. 

A second approach that is particularly useful where the suite of chemicals is complex (e.g., 

landfills and hazardous waste recycling sites) is to use the biological responses measured by 

one or more of the bioassessment techniques directly to determine which areas of a waste 

site need to be cleaned up. For example, it might be agreed that all sediments in a stream 

that were significantly toxic in the applicable toxicity test (irrespective of any chemical 

measurement) would be treated to eliminate the toxicity. No remedy would be applied to 

sediments that were not significantly toxic. This approach was suggested by investigators 

at the Rocky Mountain Arsenal waste site where toxicity tests results showing toxicity above 

a pre-selected level were used with a statistical mapping technique (kriging) to define 

contaminated areas for potential cleanup. (Thomas et al., 1986). 

In both approaches discussed above, it is important to note that there are currently no 

guidelines for selecting appropriate tests to determine target levels or to select the endpoint 

of those tests that have more than one possible endpoint. To use bioassessment procedures 

2-5 July 2003 
HAZMAT 93-1 Role of Bioassessment

most effectively for this purpose, at least general agreement among the parties involved 

must first be obtained regarding the acceptability of the approach and the bioassessment 

measurements that will be the basis for decisions. Ideally, this agreement should be reached 

early in the investigations when the scope of work is planned. 

In many cases, the data obtained from the surveys to determine the extent of contamination 

can also be used to determine target levels, if the data originally collected included sufficient 

spatial coverage and/or sampling across gradients of chemical concentrations present. If 

these data needs are not met, additional sampling will be required. It is also possible that a 

biological endpoint or bioassessment procedure completely different from that used in site 

characterization may be selected to define the cleanup procedure. 

REMEDIAL DESIGN/REMEDIAL ACTION: BIOASSESSMENT AS A 

TOOL TO EVALUATE THE SUCCESS OF REMEDIAL ACTIONS 

In the same way that bioassessment procedures can be used to establish and map the 

toxicity of media associated with a site, they can also be used after remediation to confirm 

that toxicity has been eliminated. Bioassessment may also be used as a tool for monitoring 

any ongoing activities that are part of the remedy, such as discharges of treated 

groundwater. Toxicity tests are commonly used by EPA and other regulatory agencies to 

monitor effluents for National Pollutant Discharge Elimination System permits and for 

testing dredged sediments for open-water disposal. 

Where the remedy has involved treating or isolating contaminated soils/sediments, toxicity 

tests may be the only useful immediate measure of the success of such remedial activities in 

those areas where the remedy has resulted in substantial disruption (i.e., from construction 

or dredging) of the natural system. In any case, the most logical bioassessment procedures 

to use to determine the "cleanliness" of the site are the ones employed to determine the 

toxicity of the site prior to cleanup. This should be especially clear in the situations where a 

particular test was used in the selection of the target cleanup level. 

If post-remedy assessments are to be performed on sites where no previous bioassessment 

has been done, the selection of appropriate procedures can be developed following the 

guidelines for determining the extent of contamination (Preliminary Assessment and 

Remedial Investigation sections of this chapter). The level of effort will depend on the 

resources available and the level of confidence in the result that is needed. 

July 2003 2-6 
HAZMAT 93-1 Role of Bioassessment

REFERENCES 

Thomas, J.M., J.R. Skalski, J.F. Cline, M.C. McShane, and J.C. Simpson. 1986. 

Characterization of chemical waste site contamination and determination of its extent using 

bioassays. Environmental Toxicology and Chemistry 5: 487-501. 

Tetra Tech. 1986. Recommended protocols for measuring selected environmental variables 

in Puget Sound. U.S. Environmental Protection Agency, Puget Sound Estuary Program, 

Seattle, WA. 

2-7 July 2003 
HAZMAT 93-1 Role of Bioassessment 

July 2003 2-8 
HAZMAT 93-1–Toxicity Tests 

CHAPTER 3 

TOXICITY TESTS 

INTRODUCTION 

A toxicity test is a procedure that involves the exposure of organisms to complex 

environmental samples (water, sediment, or sediment extract) under controlled conditions 

to determine if adverse effects occur. Test samples usually contain unknown amounts of 

mixtures of contaminants. This procedure is sometimes referred to as a bioassay, but 

'toxicity test' is the more appropriate term because a bioassay is a test to determine the 

toxicity threshold of a specific substance, while this test is used to determine the toxicity of a 

whole sample, not its chemical components. Toxicity tests may be performed in the 

laboratory or with caged organisms in the field; however, protocols for in-situ toxicity tests 

are still in the developmental stage. Many organisms, from bacteria to mammals, have been 

used in such testing, and recently, toxicity tests using cell cultures and biomarker type 

indicators have been developed (see Chapter 2). These tests measure a variety of organism 

responses (endpoints) to determine the toxic effects of substances on biological organisms. 

While the most commonly measured endpoint is death of the organisms, other endpoints 

frequently measured include developmental abnormalities, behavioral changes, changes in 

reproductive success, and alteration of growth. Although some programs also measure the 

bioaccumulation of contaminants at the end of toxicity testing, the use of bioaccumulation 

as an endpoint is not discussed in this chapter. Toxicity tests are becoming increasingly 

important in bioassessment, in part, because they are relatively inexpensive and numerous 

tests can be conducted quickly. Although the following discussion applies to toxicity testing 

in general, most of the emphasis is on sediment toxicity tests since these are often 

recommended at waste sites. 

Objectives of Toxicity Testing 

Toxicity tests put environmental chemical data into perspective by acting as a measure of 

bioavailability. They can be used to document the extent of bioavailable contamination and 

to illustrate the potential for adverse effects at a waste site. Toxicity test results can be used 

early in the investigation of a site as a screening tool to indicate whether further 

bioassessment (for example, benthic community analyses) should be conducted. Results of 

toxicity tests can provide some information to indicate whether (and where) remediation 

should occur. Toxicity testing may help determine target levels for remediation. Results of 

toxicity tests alone will not be adequate for making remediation decisions; however, when 

 3-1 August 1997 
HAZMAT 93-1–Toxicity Tests 

combined with chemical analyses and other bioassessment results, they are a useful tool. 

Toxicity testing can also be useful in monitoring the success of remediation. 

Advantages of Toxicity Testing 

The principal advantage and reason for conducting toxicity tests at a site is to provide a 

direct quantifiable measure of the potential for the occurrence of bioeffects at the site (Table 

3-1). Because toxicity tests measure the relative toxicity of a mixture of chemicals, any 

synergistic or antagonistic effect between chemicals is automatically taken into account. 

Used in conjunction with chemical analysis, toxicity tests can be used to correlate toxic 

effects with concentrations of specific contaminants and thus serve as an indirect measure of 

bioavailability. Unlike chemical analyses, toxicity tests are not limited by a predetermined 

list of chemicals to be tested for. Therefore, if toxic effects are found and there is no 

correlation between the effects and the contamination levels, this could indicate that a 

chemical substance not analyzed for was causing the effect (i.e., you can't find something 

you don't look for). In general, toxicity test organisms are intended as generic 

representatives of sensitive organisms that could be present at a site, and thus can provide 

indications of whether conditions are toxic enough to kill or otherwise impact sensitive 

species. In other cases, a species of particular concern at a site can be used as the test 

organism to provide a more direct indication of potential effects to this species. 

Since toxicity tests are based on controlled procedures that minimize natural variability of 

conditions such as temperature, it is easier to detect differences between sites than with 

other more variable bioassessment methods, such as benthic community studies. Unlike 

measures of benthic community structure, toxicity tests are not dependent on the presence 

of any particular in-situ population. Because the test samples can be collected from small, 

well-defined areas, the spatial resolution of toxicity test results is better than for most other 

assessment approaches. Many toxicity tests have well-developed procedures for which 

widely accepted protocols have been developed. Finally, they are quick, relatively 

inexpensive and are available from a number of agency and contract laboratories. As a 

result of these factors, toxicity tests are often the first choice for bioassessment at waste sites. 

August 1997 3-2 
HAZMAT 93-1–Toxicity Tests 

Table 3-1. Advantages and disadvantages of toxicity tests. 

Advantages Disadvantages

Provides quantifiable information 

about the potential for bioeffects at a 

site. 

Indirect indicator of bioavailability of 

contaminants. 

Response not restricted by 

predetermined list of contaminants. 

Indicates potential effects to sensitive 

species or to species of particular 

concern. 

Performed under controlled test 

conditions (i.e., minimizes natural 

variability). 

Not dependent on the presence of 

any particular in-situ population. 

Spatial resolution of toxicity test 

results is better than for most other 

assessment approaches. 

Many have well-developed and 

widely accepted protocols. 

They are quick and relatively 

inexpensive. 

Not designed to mimic natural 

exposure, so may be difficult to relate 

directly to actual responses at a site. 

Response not necessarily directly 

related to specific contaminant(s). 

If test organisms do not naturally 

occur at the site it may be difficult to 

relate effects on test organisms to 

organisms occurring naturally at the 

site being tested. 

Tests are difficult to perform correctly 

by inexperienced laboratories. 

Not surrogates for determining 

population changes. 

Not appropriate for contaminants that 

cause subtle effects over long periods, 

or for those where the major concern 

lies in their potential to 

bioaccumulate or biomagnify. 

May observe toxicity in unexpected 

places (i.e., 'clean' sites) due to 

unknown or unquantified factors. 

Disadvantages of Toxicity Testing 

The principal disadvantage of toxicity tests is that while they may be a good measure of the 

potential for adverse environmental effects, they are rarely designed to precisely mimic 

natural exposure. As a result it may be difficult to relate the results directly to actual 

 3-3 August 1997 
HAZMAT 93-1–Toxicity Tests 

responses at a site (Table 3-1). Toxicity tests primarily determine whether the samples 

tested can interfere with the biota, not whether they have actually caused effects at a site. 

They are not true in-situ measures of toxicity. Toxicity tests by themselves give no 

information on what contaminants are present in a sample. Care must be taken when 

running a toxicity test to assure that any measured toxicity is the result of the presence of 

contaminants and is not due to naturally occurring substances such as ammonia and 

sulfides. Although toxicity tests may be a quick and inexpensive bioassessment technique, 

many of the tests are difficult to perform correctly by inexperienced laboratories. Toxicity 

tests are not surrogates for determining changes in benthic or other populations. They are 

not appropriate for contaminants that cause subtle effects over long periods, or for those 

where the major concern lies in their potential to bioaccumulate or biomagnify. 

TEST SELECTION 

Before selecting a toxicity test for application at a waste site, the questions to be answered 

by the test must be clearly defined. If a specific hypothesis can be formulated, test selection 

and interpretation become much easier. Identifying specific concerns at the site will help 

define the desirable test parameters. These parameters will include: sample matrix (soil, 

sediment, water); test species and, if appropriate, life stage; duration of the test; and 

measurement endpoints (e.g., death, growth, reproduction). The character of the 

environment (e.g., wetland, freshwater, estuarine), the receptors of concern, contaminants 

thought to be present at the site, and known exposure pathways will all contribute to the 

selection of an appropriate test or tests. Table 3-2 provides a general outline for selecting 

appropriate toxicity tests. While it is usually preferable to select a test that uses generally 

accepted protocols (e.g., ASTM), new or modified tests that are more suitable to the specific 

site conditions should not be dismissed Selecting a battery of tests will greatly improve the 

chances of correctly interpreting the potential for toxic effects at a waste site. 

August 1997 3-4 
HAZMAT 93-1–Toxicity Tests 

Testing Water, Soil, or Sediment 

Toxicity tests have been developed that 

expose test organisms to water, soil, or 

sediment. In water toxicity tests, the 

test organisms are placed directly in a 

sample of the water of concern. 

However, there are four different 

exposure scenarios for soil or sediment 

toxicity tests. First, organisms can be 

exposed to whole, intact soil or 

sediments ("bulk sediment" or the "solid 

phase") with, in the case of sediments, 

overlying clean water. Second, they can 

be exposed to soil or "sediment 

elutriate" where clean water is mixed 

with the test material. The mixture is 

then either allowed to settle or is 

centrifuged and the water phase is 

poured off to become the test sample. 

This elutriate sample contains the 

"suspended phase" if it still contains 

particulates, or is the "liquid phase" if 

the particulates have been removed by 

centrifugation or filtration. 

Table 3-2. Steps in toxicity test selection. 

The third scenario is exposing 

organisms to soil or sediment extracts, 

using a chemical extraction procedure similar to that used for sediment chemistry analyses. 

This extraction process isolates specific classes of contaminants (neutral, non-ionic organic 

compounds) while failing to extract others (metals and highly acidic and basic organic 

compounds). The fourth, relatively new technique involves exposing organisms to the 

interstitial or pore water present in a sediment sample. Pore water can be collected from 

wet bulk sediment through the use of centrifuges, squeezers and filters, or dialysis 

chambers. After the pore water is collected, water toxicity tests can be used. Although 

DEFINE TEST OBJECTIVES 

DETERMINE ECOSYSTEM TYPE 

Terrestrial 

Freshwater 

Brackish (Estuarine) 

Marine 

DETERMINE MATRIX TO BE TESTED 

Soil 

Water 

Sediment 

 Bulk 

 Elutriate 

 Extract 

 Pore Water 

DETERMINE TYPE OF TEST DESIRED 

Acute 

Chronic 

SELECT TEST ORGANISM 

DEFINE ENDPOINTS TO BE OBSERVED

Death 

Growth 

Reproduction 

Etc. 

 3-5 August 1997 
HAZMAT 93-1–Toxicity Tests 

techniques have been developed to use caged animals to measure changes in growth rates 

in the field, methods to expose organisms to sediment in-situ are still under development. 

The choice of whether to perform either soil, sediment, or water toxicity tests is dependent 

on conditions at the site under investigation. Surface water in wetland areas, pools, streams, 

and rivers is often present near waste sites. Water toxicity tests are well developed, have 

standard protocols, use a variety of species and endpoints, and are relatively easy to 

perform compared to sediment toxicity tests. However, contaminants in surface water may 

not occur at acutely toxic levels because many contaminants are hydrophobic and are 

rapidly removed from the water column by adsorption to suspended particulate matter and 

sediments. Also, levels of contaminants in surface water are often highly variable over time; 

this is especially true if inputs are periodic rather than continuous. Bulk soil toxicity tests, 

like those for water, are relatively well developed, fast, and inexpensive and have standard 

protocols. 

Sediments often act as reservoirs of contamination near waste sites. They generally have 

higher concentrations of contaminants than the overlying water and contaminant levels are 

less variable over time. Bulk sediment tests expose the test organisms to contaminated 

sediments covered with clean water. The test organisms can be either free swimming, 

recieving their exposure from contaminants that diffuse from the sediment into the 

overlying water, or they can be burrowing benthic organisms exposed through direct 

contact and sometimes ingestion of the contaminated sediments. However, there are a 

limited number of standard protocols currently available for sediment toxicity tests. 

Sediment elutriate and extraction tests involve the transfer, by either simple mixing or 

chemical extraction, of contaminants from the sediments to clean water or solvent that is 

then used as the test medium. Equilibrium partitioning theory1 suggests that the 

bioavailable toxic phase of sediments is the pore water phase, therefore it follows that a 

direct measure of sediment toxicity is one that is performed on the pore water phase (Long, 

personal communication). Since the resulting test matrix for elutriate and pore water tests is 

water, water toxicty test protocols have been adapted for use with these tests. 

Toxicities determined for the different routes of exposure to sediment samples (bulk 

sediment, elutriate, extracted or pore water) may not necessarily agree with each other. 

 

1 Equilibrium partitioning theory states that the chemicals in sediments are distributed between the particulate 

(solid) phase and the aqueous (pore water) phase; this can be expressed mathematically as a partitioning

coefficient. 

August 1997 3-6 
HAZMAT 93-1–Toxicity Tests 

Elutriate tests were originally developed for testing dredged material to simulate conditions 

occurring during open-water disposal and are not considered appropriate for testing the 

toxicity of in-situ sediments. The chemical extraction techniques, used for extraction testing, 

remove only certain contaminants so the test organisms are not exposed to the full suite of 

contaminants that are actually present in the contaminated sediments. Pore water 

techniques require specialized laboratory equipment, and contaminant concentrations may 

vary depending on the extraction technique. There are currently no generally accepted 

protocols for pore water extraction. 

Studies reviewed by Ankley et al. (1991) indicated that pore water exposures provide more 

information on sediment toxicity than elutriate exposures. They found that pore water 

samples were consistently more toxic than sediment elutriate samples. However, they also 

found that pore water samples were sometimes more toxic than bulk sediment samples, 

possibly due to pH differences or to the dilution of toxicants by the addition of clean water 

to bulk sediment samples (Ankley et al., 1991). Chapman and Fink (1984) also noted 

differences between toxicity of bulk sediment and sediment elutriate. Toxicity of bulk 

sediment to larval polychaetes was generally greater than that of sediment elutriate. 

However, elutriate samples from some stations were toxic, while bulk sediment from the 

same stations were not (Chapman and Fink, 1984). Contaminants that have low solubility 

in water generally have lower toxicities determined by elutriate tests. Bulk sediment testing 

is currently the preferred method for testing sediments at hazardous waste sites to 

determine the potential for biological effects at the site. The sediment toxicity tests in the list 

of recommended toxicity test protocols (Table 8-1) are based primarily on exposures to bulk 

sediments. 

Selecting a Test Organism 

A wide variety of organisms have been used in toxicity tests. The most commonly used soil 

toxicity tests are the seed germination test (typically using common crop species), the root 

elongation test (most often performed with lettuce), and the earthworm test. Freshwater 

organisms used for water and sediment toxicity tests include algae (Selenastrum), daphnids, 

chironomids, amphipods, and fish (especially fathead minnow and rainbow trout). The 

most commonly tested marine and estuarine organisms are amphipods, mysids, and bivalve 

or echinoderm larvae. Luminescent bacteria have also been used in tests of water, sediment, 

and soil. 

 3-7 August 1997 
HAZMAT 93-1–Toxicity Tests 

The choice of a test species need not consider whether the species is native to the area where 

the sample was taken. However, if an organism is available that is also native to the test 

site, its use can increase the ecological relevance of the results. If a test has not been 

developed using a species native to the test area, the use of a surrogate species still can 

provide valuable information. For example, a test using an amphipod native to the study 

area may be available, but the test may not evaluate reproductive effects. Combining the 

results of the amphipod test with results of a reproductive test using a surrogate species can 

provide a more useful suite of information. 

The relative sensitivity to specific contaminants varies greatly among different organisms 

(see Table 8-2). The physiology and behavior of the species probably influences its response 

to contaminants. This selective sensitivity to toxic materials should be considered when 

selecting an appropriate test organism. If the presence of a particular group of 

contaminants is known, a species thought to be sensitive to those contaminants can be used 

in a toxicity test. When a complex mixture or unknown mixture of contaminants is 

suspected, it is generally advantageous to test two or more different test organisms to 

improve the chances of correctly identifying the presence of toxic materials. Also, in the 

advent of an unexpected failure of one test, there will still be useful information available to 

assess the potential for toxicity. 

Some examples of test organisms are: Photobacterium phosphoreum., Selenastrum 

capricornutum, Daphnia magna, chironomid larvae, Pimephales promelas, Neanthes sp., and 

Arbacia punctulata. Each of these organisms have their advantages and disadvantages. The 

use of the Microtox® bacterial assay (P. phosphoreum) utilizing an organic solvent to extract 

contaminants appears to be consistently sensitive to some organic compounds (but not 

insecticides and herbicides). However, it is not very sensitive to metals (Munkittrick et al., 

1991) and was found to be relatively insensitive to Prudhoe Bay crude oil (Buchman, 

personal communication). Also, it may be hard to show the environmental relevance of 

Microtox® test results. Algae such as Selenastrum capricornutum appear to be sensitive to 

metals and some organic contaminants, especially herbicides (Giesy and Hoke, 1990). 

Daphnids, especially Daphnia magna, are very sensitive to metals (Munkittrick et al., 1991; 

Giesy and Hoke, 1990). Chironomid larvae are also thought to be very sensitive to metals, 

especially when growth is measured as the response (Giesy and Hoke, 1990). Fathead 

minnows (Pimephales promelas) may be sensitive to PAHs and creosote, cyanide, and some 

metals. Amphipods and bivalve larvae are thought to be similar to each other, but less 

sensitive than the Microtox® test, in their overall sensitivity to contaminants (Williams et al., 

August 1997 3-8 
HAZMAT 93-1–Toxicity Tests 

1986; Long et al., 1990; Pastorak and Becker, 1989). However, this relative sensitivity now 

appears to be site or species specific (Long, personal communication). Amphipods may be 

more sensitive to creosote and PAHs than the Microtox® test (Pastorak and Becker, 1989). 

The polychaete Neanthes appears to be sensitive to metals and PAHs (Johns, 1988). The 

embryos and sperm cells of the sea urchin Arbacia punctulata appear to be similar to oyster 

larvae in their sensitivity to metals (Nacci et al., 1986). 

In general, only healthy organisms of similar size and life history stage should be used in 

toxicity tests. The organism should be appropriate for the material to be tested. For 

example, a planktonic or pelagic organism would be appropriate for a water toxicity test 

and a benthic organism would be appropriate for a sediment toxicity test. Taxonomic 

identifications of organisms must be confirmed by a qualified taxonomist. 

Acute or Chronic Testing 

Toxicity tests can be classified as "acute" or "chronic" tests. These terms refer to the duration 

of the test with regard to the life cycle of the organism being tested, not to the endpoint of 

the test. However, there is no universal agreement on the precise definition of these terms. 

For short-lived organisms, for example, daphnids, choronimids, and amphipods, the 

definitions are fairly straight forward and universally accepted: an acute test is one that is 

completed within a small portion of the organism's life cycle, while a chronic test exceeds 

at least one life cycle (Chapman, 1989). The problem with the definitions arises when longlived organisms, such as fish, are tested. This is because it is impractical to run life-cyclelength toxicity tests when the life cycle is a year or more. Even if the life cycle is only a few 

months, life-cycle-length tests would be impractical on a routine basis. Therefore the 

question is: how long must the duration of a test be for it to be considered a chronic test 

when it is impractical to run it for one or more life cycles? There is currently no generally 

accepted answer to the question. 

Endpoints 

The endpoint of a toxicty test is the response of the organism that is used as a measure of 

toxicity. Endpoints can be classified as "lethal" or "sublethal." The only lethal endpoint is 

the death of the test organism and is reported as either percent survival or percent 

mortality. Sublethal endpoints do not involve the death of the test organism but are 

responses that could affect the survival of in situ populations. Sublethal endpoints include: 

developmental abnormalities, behavioral changes, changes in reproductive success, and 

physiological responses reflecting changes in enzyme activity and growth rates. While 

 3-9 August 1997 
HAZMAT 93-1–Toxicity Tests 

acute toxicity tests often test for lethality, they may also use sublethal endpoints. 

Conversely, chronic tests often are designed to measure sublethal endpoints, but lethality 

could be one of the observed results. Therefore it needs to be kept in mind that the terms 

acute and chronic are not interchangeable with the terms lethal and sublethal. 

Organisms can respond in many different ways to toxic materials. In selecting the response 

to be used as the endpoint of a toxicity test, consideration must be given to the ease with 

which it is quantifiable (counted, measured) and whether the response actually reflects an 

adverse impact on the organism. The use of a continuous response endpoint, such as 

growth, can increase the discriminatory power of a test (Giesy and Hoke, 1990). A range of 

responses then allows samples to be ranked by their relative toxicity. When death is the 

measured endpoint, samples can only be classified as toxic or nontoxic compared to control 

samples. In addition, growth and reproductive effects are usually more sensitive endpoints 

than lethality and may reflect the potential for community alterations. 

Sediment Test Conditions: Static or Flow-Through 

Sediment toxicity tests can be performed under "static" conditions where the entire test 

takes place in a closed container with test sediment and overlying water added only at the 

beginning of the test. These tests are simple measures of effects of acute (or sometimes 

chronic) exposures. However, waste product buildup could produce a toxic effect and cause 

erroneous results. Chronic tests can be done under static conditions, "flow-through" 

conditions, or with "static-renewal" conditions. 

Flow-through: Flow-through sediment toxicity tests have a constant flow of clean water 

through the container of test material. While this may better simulate actual test site 

conditions than static tests, the actual toxicity of bedded sediments may be underestimated 

by these tests due to the dilution of contaminant concentrations. 

Static renewal: Tests performed under static-renewal conditions involve the periodic 

replacement of water and/or sediment with clean water and/or fresh sediment from the 

test site during the course of the tests. Static-renewal test protocols have not yet been 

developed. 

Cost Effectiveness 

In a review of toxicity tests using seven different species Pastorak and Becker (1989) 

evaluated cost effectiveness as a combination of low cost and high overall sensitivity to 

August 1997 3-10 
HAZMAT 93-1–Toxicity Tests 

contaminants. The Microtox® test was determined to be the most cost effective. The use of 

two amphipod species (Rhepoxynius and Eohaustorius) and the measurement of 

developmental abnormalities in echinoderms (Dendraster) were determined to be 

moderately cost effective. Tests using polychaetes (Neanthes) and geoduck clams (Panope) 

were determined to be the least cost effective. 

Although cost can be an important factor in the selection of toxicity tests, it should not be 

the primary criterion. Ideal tests should have high discriminatory power, low withinsample variability, and strong positive correlation with measured concentrations of 

contaminants (Long et al., 1990). Although the Microtox® test is relatively inexpensive and 

may be very sensitive to some contaminants, this does not necessarily make it the test of 

choice for all situations. 

SAMPLING DESIGN CONSIDERATIONS 

Designing a sampling plan for toxicity testing should be done in conjunction with a 

chemical analysis sampling plan. Ideally the samples to be used for chemical analysis 

should be split (subsampled) with one portion being chemically analyzed and the other 

portion being used for toxicity testing. At the very least samples for chemical analysis and 

toxicity testing must be taken at the same time and location. Without this conjunctive 

sampling it would be impossible to correlate toxicity to contaminant concentrations.

Another consideration when designing a sampling plan is the holding time for samples; for 

example, the time between sample collection and the beginning of the toxicity test. Many 

chemical and toxicity test protocols specify maximum holding times for environmental test 

samples in order to insure that the properties of the sample do not significantly change 

between sampling and analysis. For example, the prolonged storage or exposure to air of 

sediment samples will volatilize acid volatile sulfides (AVS) thus increasing the availability 

and toxicity of metals which are normally bound to AVS. However, the exact implications 

of exceedances of holding times are unknown for most chemicals. Resident infauna in 

sediments will eventually die, decay, and may produce lethal levels of ammonia. Bacteria 

present in the sample may continue to alter contaminants such as PAHs. Mercury for 

example, is thought to change more quickly than other metals. Since the different forms of 

many metals vary in toxicity, the response to test samples might change if holding times are 

exceeded. A basic rule of thumb is not to exceed holding times of two weeks for sediment 

toxicity tests when the samples are maintained at 4 degrees Celsius. While sediments for 

 3-11 August 1997 
HAZMAT 93-1–Toxicity Tests 

chemical analysis can be kept frozen for several years before being analyzed, toxicity test 

sediments should not be frozen since this is thought to alter toxicity. 

INTERPRETATION OF RESULTS 

The interpretation of toxicity test results can be difficult. An observed toxic response may 

not correlate with measured chemical concentrations. If toxicity does not correlate with 

measured contamination it does not necessarily mean that the toxicity results are incorrect. 

Contaminants present in the sample may not be bioavailable. Also, a response may be 

caused by contaminants that were not measured in the chemical analyses. Natural factors 

such as grain size, ammonia, or sulfides also can produce a toxic response in some 

organisms. When two or more different toxicity tests are conducted, the results may not 

correlate with each other. This may be due to the differences in sensitivity between species 

to the mixture of chemicals in the sample. However, the most toxic samples will be those in 

which all the tests and endpoints showed significant effects. 

Comparison with Control Samples 

The interpretation of the results of toxicity tests is centered around detecting statistical 

differences between responses to test materials and to "negative controls.” 

Negative control: A negative control is a sample known to be nontoxic to the test 

organisms and in which they can function normally. Negative controls are a critically 

important factor in toxicity test studies. Negative controls should not be confused with 

reference samples. Reference samples are samples generally taken from the same system (i. 

e. stream, lake, estuary) as the test samples, but from an area not impacted by the hazardous 

waste site. Negative controls are used to evaluate the health and viability of the test 

organisms, the effects of handling the organisms in the laboratory environment, and the 

proper running of the toxicity tests. Therefore, they must not cause a significant response in 

test organisms (for example, death should occur in less than 10 percent of control 

organisms). Water toxicity tests can use distilled or clean seawater as negative control 

samples. The choice of an uncontaminated site to provide acceptable control sediment or 

soil is critical and often difficult. If the test organisms are collected from the wild, as 

opposed to cultured in a laboratory, then the sediments from their collection site can be 

used as a negative control. 

Positive controls: Also, test organisms are usually exposed to "positive controls.” Positive 

controls consist of a dilution series of water spiked with a toxic compound that produces a 

August 1997 3-12 
HAZMAT 93-1–Toxicity Tests 

response (the endpoint) in the species used for the test. Positive control samples help 

establish a dose-response relationship for the test species. These tests demonstrate the 

responsiveness of the test organisms for the endpoint of the test. 

EC50 and LC50

Toxicologists often report the toxicity of a substance as either an EC50 or an LC50 value. An 

EC50 is the concentration of a particular chemical associated with a sublethal response in 50 

percent of the test organisms; EC stands for effective concentration. An LC50 is the 

concentration associated with death of 50 percent of test organisms; LC stands for lethal 

concentration. The results of toxicity tests on environmental samples are rarely reported as 

either EC50 or LC50 values because the samples consist of a mixture of chemicals, often 

unknown, and the toxicity cannot be associated with one specific chemical. However, if 

tests are run on a series of dilutions of the test sample, then the results can be reported as 

either an EC50 or an LC50 with regard to the sample dilution, not the chemical 

concentrations in the sample. For example, if toxicity tests were performed on a dilution 

series of a contaminated water sample and 50 percent of the test organisms were killed by 

the solution containing 40 percent test sample, the LC50 would be 40 percent. 

Other Factors Influencing Toxicity Tests 

In all experimental measurements, and especially those involving living organisms, it is 

important to identify outside factors that may interfere with a correct interpretation of the 

test results. The effects of unknown co-contaminants, impurities, and degradation products 

(such as ammonia or sulfides) in the test material can further complicate interpretation of 

the toxicity of the test material. Another factor that can confuse interpretation of toxicity 

test results is seasonal variation in the test organism's sensitivity to the substances being 

tested. The physical characteristics of the sample matrix can influence toxicity test results 

both by controlling the bioavailability of the contaminants and by directly affecting the test 

organisms. The latter is of special concern when the test organisms do not occur naturally 

in the area from which the test samples were taken. In the case of water these physical 

characteristics include pH, salinity, and temperature, while in sediments grain size, total 

organic carbon (TOC), and water content must also be considered. Very high concentrations 

of fines in apparently uncontaminated sediments have been found to be toxic to the the 

amphipod, Rhepoxynius abronius, who prefers fine sandy sediments (DeWitt et al., 1988). 

The toxicity was believed to be due to either the fine grain size, the high sediment water 

 3-13 August 1997 
HAZMAT 93-1–Toxicity Tests 

content or high TOC, but because of the interrelatedness of these characteristics, the exact 

cause could not be identified. 

There has been some research conducted to determine factors that control bioavailability 

(and presumably toxicity) of contaminants in marine sediment samples. AVS 

concentrations appear to reduce and preclude the bioavailability and, therefore, the toxicity 

of cadmium, and possibly other divalent metals, to two amphipod species (DiToro et al., 

1990). Sediments containing a high percentage of fine grain material (silts and clay) and/or 

a high percent of TOC have the potential for containing higher contaminant concentrations 

than do coarser or lower TOC sediments. However, the contaminants may be less 

bioavailable due to binding by the fine grains and TOC material. These factors and the 

oxidation-reduction potential (Eh) in sediment can influence the distribution of chemicals 

between solid and aqueous phases, and can therefore influence availability to organisms. 

Disturbing a sample can also change the distribution of contaminants between solid and 

liquid phases. Other factors such as lighting, temperature, and pH influence the behavior of 

the test organism and can increase or decrease apparent toxicity. A well-developed protocol 

will include the control and measurement of as many of these factors as appropriate 

(especially grain size, TOC, AVS, ammonia, pH, dissolved oxygen, and temperature) to 

correct for influences that might lead to misinterpretation of the results. Accepted protocols 

should specify the use of both positive and negative control samples to show that response 

is caused by some toxic agent present in the sample, and not due to the laboratory 

environment or defects in the test organisms. 

Determining the Cause of Toxicity 

When attempting to determine which contaminants are causing toxicity at a site, it is 

imperative that chemical analyses are conducted on portions of the samples tested for 

toxicity. Correlation between toxicity and chemical concentrations may provide some firstorder clues as to which chemicals are most highly associated with the observed toxicity. 

However, cause and effect relationships are not determined by correlation analysis. It may 

be impossible to identify the toxic component of a sample that contains high levels of a 

variety of contaminants. In such cases, toxicity identification evaluation (TIE) techniques 

can be used to identify the class of contaminant most responsible for observed toxicity 

(Figure 3-1). TIE procedures use chemical and physical fractionation techniques and toxicity 

testing to isolate the chemical fraction most responsible for observed toxicity. The fraction 

with the greatest observed toxicity can be chemically analyzed to determine compounds 

that are present at high levels. Although these techniques have been most widely used with 

August 1997 3-14 
HAZMAT 93-1–Toxicity Tests 

complex wastewater effluents, similar approaches have been taken with sediment pore 

water or sediment elutriate. At the present time, there are no TIE procedures available to 

directly test bulk sediment (Ankley et al., 1992). When the cause of toxicity in bulk sediment 

is to be determined, TIE procedures can be conducted on sediment elutriates or pore water 

samples. However, toxicity of elutriate or pore water must first be confirmed. TIEs are 

currently only in the research and development stage. 

Figure 3-1. TIE strategy to evaluate contributions of contaminant groups (Ankley et 

al., 1991). 

For sediment elutriates and pore water, a phased approach to eliminate possible groups of 

chemicals causing toxicity can be useful (Ankley et al., 1992; Giesy and Hoke, 1990). 

Ammonia can be eliminated as the cause of toxicity if toxicity does not occur in samples 

after pH has been increased, or if measured concentrations of ammonia are known to be 

below toxic levels. Similarly, hydrogen sulfide is more toxic at low pH values and toxic 

levels of hydrogen sulfide have been identified for many species. Cationic metals can be 

implicated as a cause of toxicity by testing the toxicity of a chelated sample. If toxicity 

decreases after chelation of the sample, cationic metals are implicated and further chemical 

analyses of the sample may indicate which metals are responsible. Nonpolar organic 

compounds such as PAHs, pesticides, and PCBs are implicated as a cause if toxicity is 

 3-15 August 1997 
HAZMAT 93-1–Toxicity Tests 

reduced after the sample is passed through an extraction column to remove organic 

compounds (Ankley et al., 1992). 

SUMMARY 

Toxicity tests are a relatively quick and inexpensive means of determining if the 

environmental media (soil, water or sediment) in the vicinity of a hazardous waste site has 

the potential for adversely affecting biological organisms. The sensitivity of the various 

available tests is dependent on site conditions, species being tested, and duration of the test. 

The selection of an appropriate test and test organism at a particular waste site is dependent 

on the contaminants of concern, habitat types (terrestrial, freshwater, estuarine, and marine) 

present, and the media being tested. Once appropriate tests have been selected, both 

negative and positive controls as well as reference site samples need to be incorporated as 

part of the testing program. Finally, because test sensitivity is highly variable, depending 

on the test and test organism, a minimum of two different tests should be performed at each 

site, and when there is a wide range of contaminant types (metals, organics, etc.) more tests 

should be used.

REFERENCES 

Ankley, G.T., M.K. Schubauer-Berigan, and J.R. Dierkes. 1991. Predicting the toxicity of 

bulk sediments to aquatic organisms with aqueous test fractions: pore water vs. elutriate. 

Environmental Toxicology and Chemistry 10(10) 1359-1366. 

Ankley, G.T., M.K. Schubauer-Berigan, and R.A. Hoke. 1992. Use of toxicity identification 

evaluation techniques to identify dredged material disposal options: A proposed approach. 

Environmental Management 16(1). 1-6. 

Buchman, M.F. 1992. personal communication. Hazardous Materials Response and 

Assessment Division, Office of Ocean Resources Conservation and Assessment, National 

Oceanic and Atmospheric Administration. Seattle, WA. 

Chapman, P.M. and R. Fink. 1984. Effects of Puget Sound sediments and their elutriates on 

the life cycle of Capitella capitata. Bulletin of Environmental Contamination and Toxicology. 

33(4). 451-459. 

DeWitt, T.H., G.R. Ditsworth, and R.C. Swartz. 1988. Effects of natural sediment features on 

survival of the phoxocephalid amphipod, Rhepoxynius abronius. Marine Enviromental 

Research 25. 99-124. 

DiToro, D.M., J.D. Mahony, D.J. Hansen, K.J. Scott, M.B. Hicks, S.M. Mayr, and M.S. 

Redmond. 1990. Toxicity of cadmium in sediments: the role of acid volatile sulfide. 

Environmental Toxicity and Chemistry 9(12). 1487-1502 

August 1997 3-16 
HAZMAT 93-1–Toxicity Tests 

Giesy J.P. and R.A. Hoke. 1990. Freshwater sediment quality criteria: toxicity bioassessment. 

In: R. Baudo, J.P. Giesy and H. Muntau, eds. Sediments: Chemistry and Toxicity of In-Place 

Pollutants. Chelsea, Michigan, Lewis Publishers, Inc. pp 265-348. 

Johns, D.M. 1988. Puget Sound dredged disposal analysis sublethal test demonstration. 

Report for U.S. Army Corps of Engineers, Seattle, by PTI Environmental Services. Contract 

Number DACW67-86-D-0047. 94 pp. 

Long, E.R. 1992. personal communication. Coastal Monitoring and Bioeffects Division, 

Office of Ocean Resources Conservation and Assessment, National Oceanic and 

Atmospheric Administration. Seattle, WA. 

Long, E.R., M.F. Buchman, S.M.Bay, R.J. Breteler, R.S. Carr, P.M. Chapman, J.E. Hose, A.L. 

Lissner, J. Scott, and D.A. Wolfe. 1990. Comparative evaluation of five toxicity tests with 

sediment from San Francisco Bay and Tomales Bay, CA. Environmental Toxicology and 

Chemistry 9(9). 1193-1214. 

Munkittrick, K.R., E.A. Power, and G.A. Sergy. 1991. The relative sensitivity of Microtox®, 

Daphnid, rainbow trout, and flathead minnow acute lethality tests. Environmental 

Toxicology and Water Quality, 6. 35-62. 

Nacci, D., E. Jackim, and R. Walsh. 1986. Comparative evaluation of three rapid marine 

toxicity tests: Sea urchin early embryo growth test, sea urchin sperm cell toxicity test, and 

Microtox. Environmental Toxicology and Chemistry. 5(6). 521-525. 

Pastorak, R.A. and D.S. Becker. 1989. Comparisons of bioassays for assessing sediment 

toxicity in Puget Sound. Final Report to EPA Region 10. EPA 910/9-89-004. 85 pages. 

Williams, L.G., P.M. Chapman, and T.C. Ginn. 1986. A comparative evaluation of marine 

sediment toxicity using bacterial luminescence, oyster embryo, and amphipod sediment 

bioassays. Marine Environmental Research 19(3). 225-249. 

 3-17 August 1997 
HAZMAT 93-1–Toxicity Tests 

August 1997 3-18 
HAZMAT 93-1–Bioaccumulation

CHAPTER 4 

BIOACCUMULATION 

INTRODUCTION 

Bioaccumulation is the net result when the uptake of a chemical by a biological organism 

exceeds the depuration of the chemical from the organism. Uptake may occur directly from 

the air, water, soil, or sediment via absorption or indirectly through the ingestion of food 

containing the chemical. Bioconcentration is the process by which a chemical is directly 

taken up (by absorption only) from water and is accumulated to levels greater than those 

found in the surrounding water. Biomagnification is the increase in tissue concentrations 

of a bioaccumulated chemical as the chemical passes up through two or more trophic levels. 

A chemical is usually considered capable of being biomagnified if concentrations of the 

chemical increase by more than an order of magnitude at each step up the food chain. 

DDTs, polychlorinated biphenyls (PCBs) and mercury are among the few chemicals for 

which there is evidence of biomagnification. 

Bioaccumulation studies take many forms. They may simply involve the measurement 

of tissue residues in indigenous organisms, or they may involve the measurement of tissue 

residues in test organisms exposed to contaminated environmental media (water or 

sediment) for a specific length of time. This exposure may be accomplished either by 

transplanting the test organisms to a contaminated area or by exposing them in a laboratory. 

The results of these types of studies are generally reported as the concentration of the 

chemical per unit weight of the organism (body burden) or some component of the 

organism. 

Another type of bioaccumulation study uses artificial organisms (e.g., lipid bags). These 

artificial organisms are exposed to contaminated water or sediments for a specific length of 

time and then the concentration of the chemical(s) of concern in the lipid are measured. 

Since artificial organisms only take up chemicals through absorption, they only measure the 

potential for bioconcentration of the chemical(s). In studies with artificial or real organisms 

when the only route of uptake is through absorption (not through ingestion) the results are 

often reported as a unitless bioconcentration factor (BCF). The BCF relates the 

concentration of a chemical in an organism (real or artificial) or component of an organism 

to the average concentration found in the surrounding water. 

 4-1 November 1992 
HAZMAT 93-1–Bioaccumulation

Advantages of Bioaccumulation Studies 

The principal advantage and reason for conducting a bioaccumulation study at a site is to 

provide a direct measure of the bioavailability of contaminants there (Table 4-1). The 

chemical analysis of sediments may indicate that a site is highly contaminated, but it does 

not indicate that the contaminants are available to the biota. In the case of metals, they may 

be bound up in the crystal lattice of minerals making them totally unavailable for biological 

uptake. Also, like sediments, bioaccumulation analyses are an integrated measure of 

contamination levels over time, while water analyses give concentrations for instants in 

time. In addition to being extremely variable over time, contaminant concentrations in 

water are also relatively low. The measurement of these low concentrations requires 

laborious multistep techniques that are not only expensive, but increase the risk of 

laboratory contamination and dilution. Biota that concentrate contaminants with respect to 

the surrounding water, permit less complex and therefore less expensive analyses. 

Bioaccumulation studies can also indicate the potential for human health risks by analyzing 

organisms consumed by humans. 

Disadvantages of Bioaccumulation Studies 

Among the disadvantages of bioaccumulation studies (Table 4-1) is the lack of direct 

correlation between body burdens and bioeffects. Bioeffect, as used here, is a change in the 

condition or functioning of an organism resulting from exposure to a toxic chemical(s) that 

reduces its potential viability. While bioaccumulation does not necessarily indicate 

bioeffects, at least in the case of metals, bioeffects cannot occur without bioaccumulation 

(Phillips, 1977). This simply means that unless metals are retained in organisms at 

concentrations higher than normally found in a healthy organism (many metals are 

micronutrients), the organism will not be negatively affected by the metal. The same is not 

true for organic contaminants, some of which are metabolized with the metabolites being 

more toxic than the original chemical, e.g., polycyclic aromatic hydrocarbons (PAHs) in fish. 

The high natural variability between individuals and species can sometimes make the 

interpretation of bioaccumulation data difficult. Some contaminants are readily 

metabolized by some organisms (PAHs in fish), while others are actively regulated so the 

uptake by an organism is not related to the environmental concentration of the contaminant 

(copper [Cu] in mussels, Phillips, 1977). Also, different contaminants compete with each 

other for uptake by organisms. Therefore, the presence of a particular contaminant or group 

of contaminants may inhibit the bioaccumulation of another contaminant. For example, the 

November 1992 4-2 
HAZMAT 93-1–Bioaccumulation

uptake of Cu in mussels is influenced by the concentrations of zinc (Zn), cadmium (Cd), and 

lead (Pb) present (Phillips, 1977). Contaminants can inhibit the rate of biological processes 

that can then reduce the uptake of contaminants. For example, generally the faster an 

organism grows, the more rapidly it accumulates contaminants; so, if the growth rate is 

slowed by the presence of contamination, the rate of bioaccumulation will also be decreased. 

While this seems a formidable list of disadvantages, many can be minimized by the proper 

selection of test organisms and methodologies. 

Table 4-1. Advantages and disadvantages of bioaccumulation studies. 

Advantages Disadvantages

Direct measure of bioavailability. Relationship between body burdens 

and bioeffects uncertain. 

Integrates contamination levels over 

time. 

High natural variability between 

individuals and between species. 

Concentrates chemicals from water 

allowing easier and less expensive 

analyses. 

No direct relationship between body 

burdens and environmental levels for 

some contaminants due to 

bioregulation or metabolism. 

Potential for determining human health 

risks. 

Difficult to associate contamination in 

mobile species to area of 

environmental contamination. 

Uptake of one contaminant may be 

inhibited by the presence of other 

contaminants. 

Rates of biological processes maybe 

reduced by contamination thus 

reducing rates of bioaccumulation. 

 4-3 November 1992 
HAZMAT 93-1–Bioaccumulation

TEST ORGANISMS 

Bioaccumulation field studies can be conducted by analyzing indigenous organisms, 

transplanted caged organisms, or artificial organisms. Each of these individual 

methodologies has advantages and disadvantages (Table 4-2), and the choice of which one 

to use at a particular site will be dependent on site conditions, contaminants of concern, and 

the specific objectives or purpose of the study. In addition, standard protocols currently 

exist for bioaccumulation studies with resident and transplanted organisms (e.g., NOAA's 

National Status and Trends (NS&T) Mussel Watch and California's Mussel Watch, 

respectively), while the use of artificial organisms is still in the experimental stages. 

Type of Organism 

The simplest and most straightforward bioaccumulation study is one involving the use of 

organisms that are indigenous to both the site of concern and the proposed reference site. 

The use of indigenous organisms permits the correlation of site contamination and the 

bioavailability of contaminants to the resident biota. While this method may appear to be 

ideal, the use of indigenous organisms does have certain drawbacks. The first and probably 

the most serious drawback is that the use of indigenous organisms requires that an 

appropriate organism (see below) is present and sufficiently plentiful at the contaminated 

site as well as at an appropriate reference site. There is also a high degree of variability 

between individuals of indigenous populations, and the locations of test stations are 

restricted by the presence or absence of the chosen test organism. 

The use of transplanted organisms in bioaccumulation studies can overcome some of the 

drawbacks of indigenous organisms. A transplanted organism can be selected based on its 

ability to bioaccumulate the chemical or chemicals of concern at the study site and is not 

dependent on its actual presence at the site. Sufficient biomass for analysis is assured from 

the start by transplanting excess organisms to allow for any mortality. With transplanted 

organisms, investigators have more freedom in the selection of sampling locations. For 

example, organisms can be randomly or evenly spaced along a transect moving away from 

the site to determine the existence of a gradient. 

November 1992 4-4 
HAZMAT 93-1–Bioaccumulation

Table 4-2. Advantages and disadvantages of the various types of 

organisms used in bioaccumulation studies.

Type of Test 

Organism 

Advantages Disadvantages

Indigenous Indicator of what in situ

organisms are actually 

accumulating 

Integrates accumulation over 

an extended time period 

Locating an appropriate 

organism on-site and at 

reference site 

Collecting sufficient biomass 

for analysis 

Possibility of higher individual 

variability 

Test stations restricted by 

presence of organism 

Transplanted Direct indication of 

bioavailability 

Reduced variability between 

individuals 

Organism can be selected 

based on environment and 

contaminant(s) of concern 

Broader coverage of area of 

concern, gradients more 

readily determined 

Relatively short exposure time 

More involved (expensive) 

methodology 

More difficult to relate results 

to in situ organisms 

 

Artificial Minimal variability between 

individuals 

Broader coverage of area of 

concern, gradients more 

readily determined 

Does not actively select for 

contaminants

 

Only measures 

bioconcentration component 

Only a simplified model of an 

organism 

May be difficult to relate data 

to real world 

 4-5 November 1992 
HAZMAT 93-1–Bioaccumulation

The principal drawbacks of transplanted organisms can be the relatively short exposure 

time and more involved and expensive methodology (increase in labor costs). While 

indigenous organisms may be accumulating site-related contaminants during their entire 

life cycle, transplanted organisms are generally only exposed during a small portion of their 

life cycle. The results of transplant bioaccumulation studies can indicate whether the 

transplanted organism is accumulating more contaminants at the site than at a reference site 

and thus infer that there are more bioavailable contaminants at the test site than at the 

reference site. However, the data may not be representative of the degree of 

bioaccumulation by indigenous organisms. When transplanted organisms are used for 

bioaccumulation studies, it is mandatory that a sample of the test organisms taken directly 

from the source of the organisms be analyzed for levels of the contaminants of concern. 

While not mandatory, it is highly desirable to have chemical data for the water and/or 

sediments from the source of the transplanted organisms. 

Whether using indigenous or transplanted organisms, other parameters, in addition to 

chemical concentrations, need to be recorded. These parameters include: species, organism 

size, age, sex, spawning condition, lipid content, moisture content, and specific tissue type 

analyzed. All of these parameters will affect contaminant body burdens. For example, if the 

contaminant of concern is lipophilic, an organism with a higher lipid content will have 

higher contaminant concentrations than one with a lower lipid content (if they were both 

exposed to the same level of contamination). A large proportion of the lipids in a ripe 

female are located in the egg mass; when the female spawns the lipids are lost resulting in a 

reduced contaminant body burden for the female. Therefore, if recently spawned females 

were sampled without recording their reproductive condition the results would suggest that 

contaminant levels were lower than they actually were. 

Artificial organisms are usually semipermeable membrane bags containing a lipid. These 

artificial organisms are passive accumulators while biota are active accumulators, and what 

the artificial organisms are actually measuring is the partitioning of contaminants between a 

water and lipid phase. Therefore, the results of artificial organism studies indicate the 

potential for the bioconcentration of lipophilic contaminants, i.e., the quantity of lipophilic 

contaminants that are available for direct uptake by absorption. The principal advantage of 

artificial organisms is the lack of variability between individual organisms in a study. As a 

result, all differences in accumulation of contaminants between test sites are due to site 

differences and not test organism differences. The major drawback to artificial organisms is 

that they only measure the level of contaminants available through passive absorption and 

November 1992 4-6 
HAZMAT 93-1–Bioaccumulation

do not measure the level of contaminants that are available through active uptake 

mechanisms and through the ingestion of particulates, sediments, and food. Particulates 

and sediments may be significant sources of contaminants for filter and deposit feeders, 

respectively. Also, food is a particularly important source for those contaminants that 

biomagnify. Therefore, it must be kept in mind that artificial organisms are just simplified 

models of real organisms and, as such, are not able to indicate exactly what real organisms 

accumulate. 

Test Organism Selection 

The selection of an appropriate organism, whether indigenous or transplanted, for a 

bioaccumulation study is extremely important. Table 4-3 gives a list of criteria that should 

be considered when selecting an organism for a bioaccumulation study. The ideal organism 

should meet all of these criteria, but since bioaccumulation studies are conducted in the real 

world, that organism doesn't exist. Possibly the two most important criteria are numbers 1 

and 8. The test organism should be able to tolerate the expected levels of contamination 

without any significant changes in its viability, including changes in its metabolic rate. Any 

such changes may reduce the bioaccumulation rate. The organism should also concentrate 

the contaminant or contaminants of concern. For example, while leeches meet most of the 

selection criteria, they would be a poor choice of organism for PCB studies because they do 

not accumulate organochlorides (Environment Ontario, 1988). 

Sedentary organisms are preferred (criterion 2) because they can be associated with a 

specific location and the bioaccumulation data can be related to sediment chemistry. If 

biomagnification or routes of human exposure are of concern, then non-sedentary 

organisms from higher trophic levels might be preferable. However, it is difficult to 

associate non-sedentary organisms (e.g., fish) with specific locations and thus specific levels 

or sources of contamination. If they are used, then this lack of precision should be noted. 

The organism needs to be hardy enough (criterion 7) to transport to and survive in a 

laboratory in case depuration before analysis is necessary; this is particularly true of deposit 

feeders whose gut content could be a significant proportion of total body concentration of a 

contaminant (Chapman, 1985). In the case of transplant organisms, they need to be hardy 

enough to survive collecting, handling, and caging. Some organisms actively regulate levels 

of certain contaminants, especially those that are also essential micronutrients. This 

regulation can confound the correlation between organismal and water concentrations 

(criterion 9). For example, Cu is regulated by mussels, and, therefore mussels are not a good 

indicator of its bioavailability (Phillips, 1977). 

 4-7 November 1992 
HAZMAT 93-1–Bioaccumulation

Table 4-3. Organism selection criteria for bioaccumulation studies, adapted 

from Phillips, 1977. 

1 The organism should accumulate the pollutant without being killed by the 

levels encountered. 

2 The organism should be sedentary in order to be representative of the area 

of collection. 

3 The organism should be abundant in the study area. 

4 The organism should be long lived in order to allow sampling of more than 

one year class. 

5 The organism should be of reasonable size, giving adequate tissue for 

analysis. 

6 The organism must still be growing, i.e., producing new tissue. 

7 The organism should be easy to sample and hardy enough to survive in the 

laboratory allowing depuration before analysis ( if desired). 

8 The organism should exhibit a high concentration factor for contaminants of 

concern, allowing direct analysis without preconcentration. 

9 A simple correlation should exist between contaminant content of the 

organism and the average contaminant concentration in the surrounding 

waters. 

10 All organisms in a survey should exhibit the same correlation between their 

contaminant contents and those in the surrounding water at all locations 

studied. 

If a specific tissue of an organism is going to be analyzed for bioaccumulation then criteria 5, 

8, and 9 should be applied in the selection of the tissue. Many organic contaminants are 

lipophilic and tend to accumulate more in liver than in muscle tissue because of the higher 

lipid content in liver. Because of this lipophilicity of some contaminants, lipid content of 

test organisms should be determined, especially if organic contaminants are of concern, and 

the chemical concentrations normalized for lipids. Since different metals tend to accumulate 

in different tissues depending on the organism (Hawker, 1990), it may be desirable to 

analyze more than one tissue type if a suite of metals is of concern. 

November 1992 4-8 
HAZMAT 93-1–Bioaccumulation

While bivalves are possibly the closest thing to a perfect class of bioaccumulation organism 

and are frequently used in biomonitoring studies throughout the world, they are not 

appropriate for all hazardous waste sites. There is no one organism that is appropriate for 

all waste sites; the selection of an appropriate organism needs to be done on a site-specific 

basis. 

SUMMARY 

Bioaccumulation studies are a relatively straight forward method of measuring the 

bioavailability of specific contaminants. While there are three basic approaches to these 

studies, using indigenous organisms, transplanted organisms, or artificial organisms, the 

preferred approach at hazardous waste sites is the use of indigenous organisms. However, 

decisions on the approach as well as the specific organism or organisms to use must be 

based on site specific conditions. 

Finally, bioaccumulation studies should not be performed in a vacuum; contaminant 

concentrations in sediments and/or water should be determined at the same sites where 

organisms are collected. A full-scale bioassessment study should include: 

• Chemical analysis of pertinent media to determine levels of contamination present. 

• Bioaccumulation studies to determine the availability of contaminants; toxicity 

testing to determine the toxic effects of contaminants. 

• Benthic community studies to determine if the indigenous community has been 

affected. 

REFERENCES 

Chapman, P.M. 1985. Effects of gut sediment contents on measurements of metal levels in 

benthic invertebrates–a cautionary note. Bulletin of Environmental Contamination and 

Toxicology 35. 345-347. 

Environment Ontario. 1988. The investigation, evaluation, and recommendations of 

biomonitoring organisms for procedures development of environmental monitoring. 

Ontario, Canada, Environment Ontario. 95 pp plus appendices. 

Hawker, D. W. 1990. Bioaccumulation of metallic substances and organometallic 

compounds. D.W. Connell, (ed) Bioaccumulation of Xenobiotic Compounds. Boca Raton, 

LA CRC Press, Inc. pp 187-205. 

Phillips, D. J. H. 1977. The use of biological indicator organisms to monitor trace metal 

pollution in the marine and estuarine environments–A review. Environmental Pollution 13. 

281-317. 

 4-9 November 1992 
HAZMAT 93-1–Bioaccumulation

November 1992 4-10 
HAZMAT 93-1–Biomarkers

CHAPTER 5 

BIOMARKERS 

INTRODUCTION 

A biomarker is a direct biological measure of the response of an organism to exposure to a 

contaminant. More specifically, biomarkers are "...biochemical, physiological, or histological 

indicators of either exposure to, or effects of, xenobiotic chemicals at the suborganismal or 

organismal level" (Huggett et al., 1992). 

Exposure to a contaminant means that an organism has contact with and has taken up a 

contaminant. Under this definition four conditions must be fulfilled for an organism to be 

exposed to a contaminant: 

1. The contaminant must be present in the same environment as the organism 

2. The contaminant must be in a bioavailable form. 

3. The organism must be able to come into physical contact with the contaminant. 

4. Uptake of the contaminant by the organism must have occurred. 

Uptake of a contaminant is defined as the passing of the contaminant from the external 

environment surrounding the organism, across a cell boundary layer, and into the internal 

environment of the organism. There are three potential routes of uptake: through the skin, 

through the lining of the respiratory organs, and through the lining of the gastrointestinal tract 

of the organism. Ingestion, the intake of substances into the gastrointestinal tract is not 

considered uptake. 

Once contaminant uptake has occurred the organism can respond in various ways at various 

levels. The first level of response involves the accumulation1 or excretion of the relatively 

unchanged contaminant; or, it involves the metabolism of the contaminant, with the metabolites 

being either accumulated or excreted. Accumulation of the contaminant or its metabolites can 

result in higher level responses by the organism. These responses can range from the molecular 

(e.g., genetic abnormalities) to the histopathological (e.g., lesions). It is these responses to 

contaminant uptake that are measured by the use of biomarkers. Figure 5-1 illustrates the 

general exposure-effects pathway and examples of measures of exposure, response, and effects 

that can be performed on indigenous aquatic organisms after contaminant exposure. 

 

1 Some environmental scientists consider bioacummulation of contaminants, as well as population, 

commmunity, and ecosystem measures of response or effects as biomarkers, but the most common usage 

restricts the term to suborganismal and organismal measures (Huggett et al., 1992; McCarthy and Shugart, 

1990), and that is how the term will be used in this manual. 

 5-1 November 1992

 
HAZMAT 93-1–Biomarkers 

November 1992 5-2 
HAZMAT 93-1–Biomarkers

It is important to remember that a biomarker is a measure of a response of an organism to 

exposure to a contaminant or class of contaminants; it may or may not be a measure of an 

adverse effect caused by the contaminant. However, the response measured may be a 

potential precursor of adverse effects, such as reduced fecundity or death, should exposure 

continue or increase. Exposure to contaminants may trigger the release of enzymes and 

other proteins that can catalyze further reactions. Measurement of these reaction products 

may indicate that an animal's natural detoxification mechanisms are functioning normally 

or, conversely, that they are overloaded. Furthermore, these reactions can indicate that the 

animal is stressed, that genetic material has been altered, or that reproduction might be 

affected. It should be noted that detoxification and metabolism are not synonymous since 

some metabolites are more reactive and more toxic than the parent compound. This is the 

case with metabolites of PAH, many of which are potential mutagens (Melancon et al., 

1992). Histopathological changes within organs and other, higher-level physiological 

disorders such as skin tumors, fin erosion, skeletal defects, reductions in growth rate, and 

measures of reproductive health are effects biomarkers that have also been correlated with 

exposure to contaminants. These biomarkers have been measured in feral or caged 

organisms from contaminated areas to document exposure to contamination or actual 

adverse effects. 

There are wide ranges of pathways by which contaminants can interact with biological 

organisms. Some contaminants (metal ions, for example) are free to bind with sensitive 

cellular components directly after uptake. Many organisms actively regulate uptake of 

essential metals. Other contaminants are transformed into more reactive components (the 

first phase of biotransformation), some of which are more toxic than their parent 

compounds. These toxic metabolites may then be free to bind with DNA or other proteins. 

Contaminants may be excreted through bile or urine, or may accumulate in fat stores or as 

granules (Luoma et al., 1991). A second phase in the biotransformation of xenobiotic 

compounds links metabolites to water-soluble conjugating compounds naturally present in 

cells. The binding of reactive contaminants with DNA and other proteins probably provides 

the subcellular mechanism for many adverse effects. Although the progression of the 

development of physiological disorders from subcellular reactions through gross 

pathologies has not been conclusively demonstrated, some evidence exists to link such 

conditions to exposure to toxicants. 

Because of their recent development, most work with biomarkers has been done at the 

research level and not at the practical application level, although NOAA has a history of 

 5-3 November 1992

 
HAZMAT 93-1–Biomarkers 

involvement with their use in Puget Sound and elsewhere (Long, personal communication). 

This lack of a history of practical application coupled with a lack of specific protocols for 

their use and the controversy over the exact relationships between contaminants, 

biomarkers, and significant ecological effects, has resulted in the infrequent use of 

biomarkers at waste sites. However several biomarkers show great promise as 

bioassessment tools for evaluating effects of specific groups of contaminants found at many 

waste sites. This chapter attempts to identify those biomarkers with the greatest promise 

for issues that apply to waste sites. 

Objectives and Purpose 

There are two purposes for the measurement of biomarkers at waste sites. The use of 

biomarkers can indicate that organisms have been exposed to bioavailable contaminants 

and may indicate that adverse effects are occurring. Biomarkers can also illustrate the 

extent of contamination through the measurement of biological responses. Organisms must 

be exposed to bioavailable contaminants in order to produce a response. Some biomarkers 

(for example, liver tumors in fish) can be considered a direct measurement of an adverse 

effect. A demonstrated reduction in growth rates of organisms near a site when compared 

with a reference site also would be considered an adverse effect. When used in combination 

with other assessment tools, biomarkers can help evaluate the need for remediation. If 

indicators of exposure and adverse effects are seen prior to remediation, these measures can 

be repeated during and/or after remediation to monitor its success. 

Advantages of Biomarkers 

Biomarkers have the advantage of measuring actual biological responses to environmental 

conditions (Table 5-1). They can provide indications of biological effects occurring near a 

site. These measures can integrate the patchy temporal nature of exposure, and can provide 

information that is ecologically relevant. Some biomarkers (e.g., aryl hydrocarbon 

hydroxylase (AHH) induction) are very dose dependent and diminish rapidly upon 

removal of the toxicant(s) (Long, personal communication), thus providing temporal 

information on contaminant distribution. They can account for actual environmental 

conditions that cannot be reproduced in laboratory toxicity tests. Biomarkers can indicate 

sensitive subcellular effects that may not be measured after short-term laboratory exposures 

(McCarthy and Shugart, 1990). For contaminants that are rapidly metabolized or not 

accumulated, biomarkers may offer the only direct measures of their uptake. The greatest 

advantage of biomarker measurements is that they can demonstrate that organisms actually 

November 1992 5-4 
HAZMAT 93-1–Biomarkers

present near a waste site have been adversely affected by contamination, i.e., they can 

answer the 'So what?' question. 

A further advantage is that a variety of levels of severity of effects can be assessed. Some 

measures simply indicate that exposure has occurred. Other measures can indicate a broad 

spectrum of responses. Liver lesions, for example, can indicate mild or severe biological 

effects depending on the type of lesion that is most prevalent. Different measurements can 

also indicate that animals are stressed or diseased, or that reproduction or growth has been 

impaired. In addition, biomarkers can provide a more sensitive measure of response than 

community changes and can provide some indication of the cause of the effects (McCarthy 

and Shugart, 1990). Biomarkers can be chosen for their known sensitivity to particular 

groups of contaminants and for their applicability to some particular species of concern. For 

example, mixed function oxidase (MFO) enzymes are induced by hydrocarbons and 

metallothioneins are induced by metals. Biomarkers that are known to be sensitive only to 

certain classes of contaminants offer advantages over other measures (such as community or 

population measures) that are known to be affected by a broad spectrum of toxicants and 

natural factors. 

Disadvantages of Biomarkers 

Although biomarkers and disorders discussed here have been well researched and applied 

in the natural environment, most of these measures have not been widely used at waste 

sites, and there are no accepted protocols for their use (Table 5-1). The perception that these 

measures are experimental will probably continue to limit their use at most waste sites. A 

major limitation of these approaches is the difficulty in determining the actual extent of 

exposure to specific contaminants, particularly for fish and other mobile organisms. It 

should be noted that for most biomarker measurements, there is no absolute measure of 

unacceptable response. Because some responses are also produced by natural conditions 

such as reproductive state and season, interpreting the significance of results depends very 

heavily upon the expertise of the investigator and the availability of an uncontaminated 

reference area that is otherwise very similar to the test site. Responses may vary by species, 

even between closely related species. Some disorders can take years to develop within the 

organism, and possibly years to disappear. Biomarker tests can be expensive and require 

specialists to conduct, although some tests are less expensive to conduct than evaluations of 

community effects (McCarthy and Shugart, 1990). Also specific tests are not available for all 

groups of contaminants and for all groups of organisms. 

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Table 5.1. Advantages and disadvantages of using biomarkers at waste sites. 

Advantages Disadvantages

Measure actual biological responses to 

contaminants. 

Little history of use at waste sites. 

May integrate pathcy temporal exposure. No existing EPA or other accepted 

protocols. 

Demonstrate effects on indigenous 

organisms. 

No absolute mesure of unacceptable 

response. 

Assess a variety of severity levels. Responses may be caused by natural 

factors. 

Measure more sensitive responses than 

other bioassessment methods. 

May require experienced expert 

investigators. 

Selective for particular contaminant or 

class of contaminant. 

Not always a known relationship between 

response and significant ecological effects. 

Selective for a particular species of concern. Responses may take years to develop or 

disappear (after remediation). 

May be cheaper than higher level 

ecological studies. 

Not yet feasible for all groups of 

organisms or contaminants. 

Few commercial laboratories can perform 

the tests. 

SELECTING A TEST 

The response of aquatic organisms to contaminants provides opportunities to measure 

actual environmental exposure before bioeffects have occurred as well as measuring actual 

biological effects. Selecting an appropriate test will depend entirely on the questions to be 

answered at the site. Certain biomarkers can demonstrate that contaminants are 

bioavailable, and can help determine the extent of contamination. Other measures are more 

useful for indicating specific biological effects (e.g., growth, reproductive, or disease). 

Knowledge of the contaminants present at the site will help define which tests might be 

useful. Site-specific concerns for particular types of effects will also aid in the choice of a 

test. Finally, the species to be tested must be present at the site in adequate numbers. The 

species chosen can also provide a link to human health or to other organisms of concern 

November 1992 5-6 
HAZMAT 93-1–Biomarkers

through food-web pathways. Selection of a test will thus be based on the contaminants of 

concern (Table 5-2) and the species to be tested (Table 5-3). 

TYPES OF BIOMARKERS 

Enzyme and Protein Systems 

Cells are known to respond to 

environmental stress with the 

production of enzymes and other 

proteins. Most of these proteins are 

normally found in cells that are not 

stressed, where they play a role in 

normal cellular function (Sanders, 

1990). These proteins include 

enzymes responsible for catalyzing 

transformation reactions, for 

metabolizing and excreting steroids 

produced during gametogenesis, 

and proteins that bind with 

contaminants, making them 

unavailable for further reactions 

with sensitive cellular components. 

These biochemical changes are 

usually the first detectable 

responses to changes in the 

environment (Stegeman et al., 

1992). These changes are usually 

highly sensitive and may lead to 

more severe effects within an 

organism. 

Table 5.2. Sensitivity of biomarkers to 

specific contaminant groups. 

Contaminant

Group 

Tests 

PAHs Kidney, intestinal, and 

 liver lesions. 

DNA adducts. 

MFO enzymes. 

Bile Metabolites. 

Reproductive hormone 

 measures. 

PCBs and 

DDTs 

MFO enzymes. 

Binding proteins. 

DNA adducts. 

Growth reduction. 

Reproductive hormones. 

Metals Binding proteins.

Growth reduction. 

Lesions, fin erosion. 

MFO enzymes (weak). 

Detoxification Enzymes 

Under normal conditions, animals have some capacity to process contaminants so that they 

are kept from sensitive cellular sites of toxic action. The first phase in the metabolism of 

absorbed lipid-soluble contaminants is the production of enzymes to catalyze 

transformation reactions. The most important enzymes that catalyze detoxification 

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HAZMAT 93-1–Biomarkers 

processes are those using the cytochrome P-450 electron transfer system. These enzymes are 

known as MFO enzymes. MFO enzymes synthesize and degrade natural compounds such 

as steroids and fatty acids and also transform the structure of foreign compounds (Stegeman 

et al., 1992). This family of enzymes includes AHH and ethoxyresorufin-O-deethylase 

(EROD). MFO enzymes are present in the endoplasmic reticulum of cells of many organs 

(gills, liver, intestines, and kidneys for example). They are usually most active in the liver 

on a whole organ basis and per gram of tissue. 

Table 5-3. Biomarker tests currently applied to specific groups of 

organisms. 

Mollusks Fish Other Species

MFO enzymes (weak) MFO enzymes MFO enzymes 

Binding proteins Binding proteins Binding proteins 

Neoplasms

Lesions and other 

histopathological 

disorders 

Lesions and other 

histopathological 

disorders 

Growth reduction 

DNA adducts

Skeletal defects

PAH metabolites in bile 

Reproductive measures

The activity of some specific MFO enzymes (such as AHH and EROD) has been shown to 

increase in response to exposure to many lipid soluble organic contaminants such as PAHs, 

PCBs and dioxin (Stegeman et al., 1992). Fish liver and kidney tissue have shown increases 

in MFO enzyme levels in response to 3-methylcholanthrene and similar compounds, 

including PAHs, DDTs, dioxins, and some PCBs (Leech et al., 1982; Stegeman, 1981; Gruger 

et al., 1977; Kurelec et al., 1977; Stegeman et al., 1992). However, Cd apparently decreases 

November 1992 5-8 
HAZMAT 93-1–Biomarkers

the activity of MFO enzymes by interfering with protein groups on the enzymes (Sorenson, 

1991). DDT does not appear to be an active inducer of MFO enzymes (Stegeman et al., 

1992), but was associated with elevated MFO induction in San Francisco Bay starry flounder 

(Spies et al., 1988). 

Other enzymes are known to be affected by exposure to metals. Cd, Zn, and silver (Ag) 

increase the activity of delta-aminolevulinic acid dehydratase (ALAD), a liver enzyme 

controlling formation of hemoglobin; while lead (Pb) decreases activity of ALAD 

(Johansson-Sjobeck and Larsson, 1979). Mayer et al. (1992) consider ALAD measurements 

in fish and birds to be an efficient and inexpensive measure of Pb exposure (but not 

necessarily a measure of Pb toxicity). However, measures of enzyme levels are not 

considered to be reliable indicators of exposure to other metals because of inconsistent 

responses (Sorenson, 1991). 

MFO enzymes are produced to metabolize foreign compounds following exposure. Their 

activity can show that organisms have been both exposed to and have responded to 

contaminants. They are not a good indicator of actual adverse effects, however, because the 

organism may be effectively metabolizing, binding, and/or excreting contaminants, thus 

preventing adverse effects. Another difficulty in the interpretation of enzyme activity levels 

is that some of these enzymes may also be produced in response to changes in temperature, 

reproductive state, or capture stress. In spite of these difficulties, under conditions where 

exposure to contaminants cannot be demonstrated with other measures, MFO enzyme 

activity is a useful measure of contaminant exposure that may be applied at waste sites. 

When compared to reference areas, elevated levels of these proteins can indicate that 

contaminants are bioavailable near the site. Some examples of these conditions include sites 

where contaminants do not bioaccumulate (PAHs in fish, for example), sites where 

concentrations in water are not measurable and fine grained sediments are not available for 

sampling, or as an indicator of contaminant bioavailability. Currently, MFO enzyme 

measurements are not recommended for invertebrates because rapid induction of 

appropriate enzymes has not yet been conclusively demonstrated (Stegeman et al., 1992), 

and more importantly, the physical mechanism of MFO induction in invertebrates is not 

understood (Stegeman personal communication to Long). 

Binding Proteins 

The production of MFO enzymes generally increases the organism's detoxification capacity 

for the type of chemicals to which it is exposed. Detectable increases in an organism's 

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HAZMAT 93-1–Biomarkers 

enzymatic activity may therefore be an indicator of its recent exposure to contaminants. The 

second phase in the transformation of contaminants (conjugation reactions) depends on the 

availability of conjugating substances produced within the organism, for example, the 

production of MFO enzymes, the concentration of binding or scavenging proteins. For 

example, in rats, death of liver cells occurred after GSH levels were depleted (Reid et al., 

1973). This was attributed to binding of oxygenated metabolites with genetic material and 

other proteins. However, levels of binding proteins do not indicate that actual adverse 

effects are occurring because the organism may be effectively binding, and/or excreting 

contaminants, thus preventing adverse effects. 

For organic contaminants, one of the more important conjugating compounds is the peptide, 

glutathione (GSH), which sequesters oxygenated metabolites away from sensitive cellular 

sites. The activity of this and other conjugating substances may be increased or induced by 

exposure to various classes of organic compounds (Meister, 1983; Thomas and Wofford, 

1984; Varanasi et al., in press). Enzymes that catalyze reactions with GSH have also been 

studied as biomarkers. These glutathione transferases (GST) also bind to contaminant 

metabolites and appear to be elevated in fish, crabs, and mussels from sites contaminated 

with PAHs (Stegeman et al., 1992). However, further research is needed to identify 

compounds that can induce GSH and GST levels before these measures should be applied at 

waste sites. 

For trace metals, the most important group of scavenging proteins is metallathioneins (MT), 

of which a variety of forms exist. MT is involved in a variety of processes concerned with 

metal metabolism including the regulation of the uptake of essential metals (especially Zn 

and Cu) and metal detoxification (Stegeman et al., 1992). MT appears to be a promising 

indicator of exposure to Cd, Cu, Zn, and mercury (Hg) (Stegeman et al., 1992). However, 

the concentration of MT in cells may be increased not only by exposure to contaminants, but 

also by sexual maturation, temperature, and nutritional status (Benson et all, 1990; 

Stegeman et al., 1992). MT synthesis and induction has been well studied in fish, bivalves, 

and other organisms. However, further research is probably required before MT 

concentrations will be useful as biomarkers (Melancon et al., 1992). 

The activity of conjugating substances such as MT and GSH can indicate that organisms 

have been exposed to contaminants. Some contaminants are known to be more effective at 

inducing the production of conjugating substances. The consequences of inadequate 

production of binding proteins on an organism’s health can be severe. When binding 

protein levels are depleted, contaminants are free to bind with more sensitive proteins. For 

November 1992 5-10 
HAZMAT 93-1–Biomarkers

example, in rats, death of liver cells occurred after GSH levels were depleted (Reid et all, 

1973). This was attributed to binding of oxygenated metabolites with genetic material and 

other proteins. However, levels of binding proteins do not indicate that actual adverse 

effects are occurring because the organism may be effectively binding, and/r excreting 

contaminants, thus preventing adverse effects. 

Contaminant Metabolites in Bile 

Some contaminants are rapidly metabolized in higher organisms and do not accumulate in 

tissues. PAHs for example, do not generally accumulate in fish tissue. However, PAH 

contamination of sediment has been correlated with some adverse effects in benthic fish. 

Although the development of these adverse effects may take years, the metabolites of PAH 

compounds can be detected in fish bile very quickly after exposure to PAHs (a matter of 

hours to days) and the metabolites remain detectable for weeks after an exposure (Melancon 

et al., 1992). Therefore, the measurement of these metabolites can serve as a good indicator 

of recent exposure to PAHs. It should be kept in mind however, that these methods are 

only semi-quantitative and appear to be most responsive under conditions with relatively 

high levels of contamination (Varanasi et al., in press). These measurements have been 

applied near waste sites contaminated with creosote (e.g., Eagle Harbor, in Puget Sound). 

Genetic Disorders 

Mutagenic, promutagenic, and carcinogenic compounds may not be detected in acute 

toxicity tests, but may be equally lethal as any acute toxin in the long run. There is a family 

of subcellular biomarkers that can be used to detect the effects of these compounds. The 

metabolism of many contaminants results in the creation of oxygenated metabolites that are 

known to react with genetic material. The outcome and implications of interactions with 

DNA are difficult to predict, especially since organisms have some limited ability to repair 

DNA. However, interactions with DNA are usually evidenced by abnormalities at the 

chromosome level and the effects of these types of disorders can be far reaching. Studies of 

liver pathologies in rats show that cellular death was associated with excess metabolites 

binding to DNA, RNA, or other proteins (Reid et al., 1973). 

It is possible to measure outcomes of reactions between DNA and contaminants. One 

outcome, known to be the result of chromosome breakage, is the formation of micronuclei 

(MN) in red blood cells of fish (Schmid, 1976). MN formation is also a very widely used 

biomarker in mammalian, industrial hygiene research (Long, personal communication). 

MN are small cytoplasmic chromatin masses that resemble small nuclei. These irregularities 

 5-11 November 1992

 
HAZMAT 93-1–Biomarkers 

are the result of lagging chromosomes during cell division or from unbalanced 

chromosomal fragments. MN formation has been induced in the laboratory by exposure to 

chemicals such as benzo(a)pyrene and ethylmethanesulfonate (Hooftman and de Raat, 1982; 

Hose et al., 1984). However, because of questions about other causes of MN formation, it 

has not been widely used and is not recommended for waste site investigations at this time. 

A more direct approach to assess contaminant effects on genetic material has been recently 

developed. The binding of contaminant metabolites with DNA molecules (the formation of 

DNA adducts) is measured through the labeling of DNA with 32P (Randerath et al., 1981) 

and autoradiography. DNA adducts have been measured in English sole from Puget Sound 

(Stein et al., 1990), and in winter flounder from Boston Harbor and Long Island Sound (Stein 

et al., 1989). DNA adducts have been demonstrated for metabolites of PCBs and PAH 

compounds. These direct measures of adverse effects have great potential for application at 

waste sites contaminated with PAHs or PCBs. 

Other measures of genetic effects include chromosome analysis (recording visible 

chromosomal aberrations) and sister chromatid exchange (SCE). SCE have been detected 

and quantified in polychaetes, mollusks, and fish in field surveys and laboratory exposures. 

It was also one of the methods used by the United States Environmental Protection Agency 

(EPA) at the Davisville, Rhode Island Superfund site (Munns et al., 1991) These are both 

labor intensive procedures that may have some applications to further document effects 

where other screening methods indicate the potential for effects (Shugart et al., 1992). 

Immune System Responses 

A variety of measures of immune system response are under investigation for use as 

indicators of contamination. These include blood cell counts, kidney macrophage function 

(known to be sensitive to PAHs in fish), and specific antibody counts (Benson and DiGiulio, 

1992). NOAA's NS&T Program is currently testing immune responses in oysters from 

Tampa Bay and mussels from San Diego Bay (Long, personal communication). Since these 

measures are still in the research stage of development, they are not recommended for 

analysis at waste sites. 

Histopathology 

Histopathology, the study of tissue responses to injury or disease, can indicate early 

warning conditions in organisms such as fish and shellfish long before more advanced 

abnormalities appear. The nature of disorders can also help identify contaminants 

November 1992 5-12 
HAZMAT 93-1–Biomarkers

responsible for them. For field bioassessment, histopathology is the most rapid method of 

detecting adverse acute and chronic effects of exposure (Hinton et al., 1992). 

Histopathological changes reflect prior alterations in biochemical function (Hinton et al., 

1992). Most histopathological studies have focused on the liver of higher organisms such as 

fish. Although there have been many attempts to discover reliable histopathological 

biomarkers in bivalve mollusks, further research is needed to support associations between 

lesions and contaminant exposure. 

After an exposure that causes cell death, the cell is acted upon by enzymes that cause 

nuclear and cytoplasmic changes that are easily detected. These alterations are referred to 

as degeneration and necrosis. Other types of injuries result in disturbances to the normal 

growth, repair, or replacement of cells. Growth anomalies may be non-cancerous or 

neoplastic (resulting in tumors). Neoplasms, or cancerous tumors, are abnormal masses of 

tissue that are obviously different from surrounding tissues and grow excessively even after 

removal of the causative factor. Tumors absorb nourishment at the expense of normal 

tissues, yet provide no beneficial service. Non-neoplastic growths are characterized by 

disturbances in tissue differentiation, diminished growth, or excessive, but not limitless, 

growth. This latter category is distinct from neoplasms and is referred to as "proliferative 

disorders." 

There are some histopathological conditions seen in fish that are highly suspected to be 

caused by chemical contamination. Those conditions are: 

1. Neoplasms (cancerous tumors), including both adenomas (benign tumors) and 

carcinomas (malignant tumors). 

2. Foci of cellular alteration (FCA), locations of altered, pre-neoplastic cells. 

3. Proliferative disorders (areas of excessive growth). 

4. Specific degenerative or necrotic (SDN) lesions (Myers et al., 1990). 

This latter category includes nuclear pleomorphism (where cell nuclei are misshapen), and 

megalocytic hepatosis (where cells and nuclei are abnormally enlarged and other 

degenerative effects are noted). Only liver and kidney conditions are highlighted here 

because these organs play the major role in eliminating waste products and subsequently 

have a greater susceptibility to lesions. Other conditions, such as, vacuolation (where cells 

contain empty space) and hypertrophy (where cells and surrounding tissues are enlarged) 

may also be related to contamination, but no direct correlation with sediment contamination 

 5-13 November 1992

 
HAZMAT 93-1–Biomarkers 

has been demonstrated. A small percentage of fish from uncontaminated areas also exhibit 

some signs of these lesions. 

Results from a growing number of studies indicate that there is a causal link between certain 

fish liver lesions, especially tumors and pre-tumorous conditions, and exposure to some 

contaminants, especially PAHs, and possibly PCBs. A sequential scheme of lesions that 

progress toward neoplasms, similar to that developed from laboratory rat or mice studies, is 

now being demonstrated for some species of benthic fish (Myers et al., 1990). Laboratory 

exposures of healthy bottom fish to chemical extracts of contaminated sediments have 

successfully induced liver lesions similar to those observed in feral fish (Varanasi et al., 

1987). 

Evidence exists that the frequency and type of liver lesions changes as the fish ages 

(Varanasi et al., in press). In fact, the prevalence of most lesions increases with the age of the 

fish population, so histopathological data analyses must take into account the age of the fish 

when comparing sites (Long, personal communication). Liver lesions have been 

demonstrated to adversely affect liver function (Casillas et al., 1985). Liver disorders have 

been measured at waste sites contaminated with PAHs (in Eagle Harbor, for example) to 

demonstrate that site related contamination has caused adverse biological effects. These 

measurements show great promise for other marine or estuarine sites contaminated with 

PAH compounds. However, the detection of histopathological disorders, such as liver 

lesions, requires dissection of the organ, thin tissue section preparation, and examination by 

an expert While several Federal laboratories have this capability, few commercial or 

academic laboratories are able to perform these analyses (Long, personal communication). 

Pathology 

When cellular injury proceeds unchecked and major portions of a tissue or an entire organ 

become affected, it is sometimes possible to observe the damage without dissecting an 

organism. This is certainly the case for conditions affecting the skin, lips, and eyes of fish. 

Since the skin is one route of exposure to contaminants for fish, it is not surprising that some 

pathological conditions may be observed in this tissue. 

Fin erosion is one of the most easily detected external abnormalities in fish, and therefore, is 

one of the most commonly documented. It is characterized by loss of epidermal, dermal, 

and fin ray tissues. This condition must be accurately distinguished from net damage or 

wounds from predators. Because its absolute identification must be confirmed with 

November 1992 5-14 
HAZMAT 93-1–Biomarkers

histological examination, the significance and accuracy of observations are difficult to 

determine. 

Though commonly reported, the cause of fin erosion is poorly understood. Laboratory tests 

have verified that exposure to contaminated sediments can induce fin erosion in healthy fish 

(Sinderman, 1979). Causative agents identified to date include PCBs, crude oil, Pb, Zn, and 

Cd (Sinderman, 1979; Tetra Tech, 1986). Fin erosion is most likely the result of a 

combination of factors including chemical contamination, secondary bacterial infection, 

mechanical injury, and poor water quality (low-dissolved oxygen) (Sinderman, 1979). Skin 

and lip lesions in bullheads have been induced by exposures to PAH-contaminated 

sediments in laboratory tests (Black, 1983). Because of the controversy over the causes of fin 

erosion, this measure of effects would be difficult to attribute to contaminants from a 

specific waste site. Until further research illuminates the causes of this disorder, it is not 

recommended for application at waste sites. 

The cause of epidermal tumors is also not completely understood. Some lesions are thought 

to be caused by contaminants, while others are more clearly related to viruses or parasites. 

Three particular skin lesions (papillomas, squamous carcinomas, and chromatophoromas) 

have been recommended for further study in contaminated areas (Hinton et al., 1992). As 

with fin erosion, this disorder would currently be difficult to attribute to contamination 

from a waste site. 

Skeletal anomalies are found in fish from areas of highly contaminated sediments. Most 

observed skeletal anomalies involve the spinal column and include fusion, flexures, and 

vertebral compressions. Skeletal anomalies also include abnormalities of the head, fins, and 

gills. Skeletal anomalies have been induced in fishes after lab exposure to the chlorinated 

pesticide kepone and heavy metals (Sinderman et al., 1980). However, some of these 

deformities can also be caused by nutritional deficiencies (Hinton et al., 1992). Mayer et al. 

(1992) recommend that further research be conducted to assess the degree of abnormality 

compared to normal occurrences that could be attributed to the effects of contaminants. 

 5-15 November 1992

 
HAZMAT 93-1–Biomarkers 

Growth 

Metabolic energy is required for movement, active transport of substances across cellular 

membranes, biosynthesis of compounds, and reproduction. An organism expends a great 

deal of energy just maintaining its normal functioning. This metabolic energy is produced 

and stored in the form of chemical bonds within a high-energy molecule (adenosine 

triphosphate). Energy from nutrition in excess of the normal maintenance requirement is 

what is available for the organism to use in growth processes. However, if an organism is 

responding to some environmental stress (including exposure to contaminants) it must 

expend energy to compensate for the stress, thereby reducing the amount available for 

growth. 

Linear growth rates in caged animals have been used as a simple indication of physiological 

stress. Growth measurements of organisms caged near waste sites can be a useful indicator 

of effects due to contaminants. Caged juvenile mussels have been used in San Diego Bay to 

evaluate effects of marinas and naval facilities on biota (Salazar and Salazar, 1991). This 

technique has been applied at two sites in the Puget Sound area, Commencement Bay and 

Elliott Bay. 

A scope for growth (SFG) index, defined as the theoretical amount of energy available to an 

organism for growth and reproduction (Warren and Davis, 1967), may also be used as an 

indicator of physiological stress. The SFG index is expressed as the difference between the 

energy value of all the food consumed and the energy value of all processes other than 

growth (respiration and excretion). It is an extremely labor-intensive method. This 

technique has not previously been used at waste sites, but has been used a great deal in 

Europe and the United Kingdom, especially on or near oil platforms and, in this country, in 

Narragansett and San Francisco bays and in southern California (Long, personal 

communication). SFG may have some potential for application using transplanted bivalves 

during environmental assessment investigations. 

Reproduction 

Reproduction in female fish is governed by an intricate system of hormones, proteins, and 

external signals (Nagahama, 1987). During maturation, eggs pass through two phases: 

primary growth and vitellogenesis. Primary growth results in large increases in oocyte 

volume and development of numerous sub-cellular structures or organelles. Vitellogenesis 

is the sequestering of a protein, vitellogenin, into the yolk. Vitellogenin is produced in the 

November 1992 5-16 
HAZMAT 93-1–Biomarkers

liver and released into the blood in response to the reproductive hormone, estradiol. Once 

vitellogenesis ends and the oocytes have reached their fully developed size, a surge of 

hormones induce the follicles to release steroids. This in turn stimulates resumption of 

oocyte cell division, hydration of the oocytes, and eventually, ovulation (discharge of the 

oocytes). This entire process is initiated in response to external signals of light and 

temperature. 

Because of the complexity of the reproductive system, there are a number of disturbances 

that could be a result of contamination. Not all vitellogenic oocytes mature and ovulate. 

The ovarian follicle may lose its integrity and its oocyte may not be released (known as 

atresia). This condition is frequently observed in species that spawn more than once per 

year, during the end of the spawning season when unspawned eggs are resorbed 

(Braekevelt and McMillan, 1967). Stress, altered photoperiod, temperature regimes, and 

poor diet have all been correlated with atresia (Cross et al., 1984). However, any factor that 

lowers gonadotrophin2 levels (including exposure to contaminants) could induce atresia 

(Braekevelt and McMillan, 1967). Oocyte atresia has great potential to indicate adverse 

effects that are highly significant, but it has not been verified in contaminated areas. 

Contaminants that induce production of MFO enzymes can also affect enzymes that 

regulate the production of steroids controlling spawning (Spies et al., 1984). Exposure of 

English sole to extracts of contaminated sediment (containing PAHs and PCBs) reduced 

plasma levels of estradiol (the steroid controlling vitellogenesis) in sexually maturing 

females (Stein et al., 1991). Similar effects have been seen in Atlantic croaker, Micropogonias 

undulatus, (Thomas, 1988). Depressed plasma estradiol levels have been linked with 

impaired ovarian development and reduced ability to spawn (Johnson et al., 1988; Varanasi 

et al., in press). Other laboratory studies have also linked decreased fertility with exposures 

to contaminants, including PAHs, PCBs, and pesticides (Nagler et al., 1986; Cross et al., 

1984; Hose et al., 1981). Some investigators theorize that MFO enzymes produced after 

exposure to PAHs, PCBs, and dioxins result in the metabolism of steroids (such as estradiol 

and testosterone) that control reproduction; however, the exact cause of depressions in 

estradiol levels are not known (Johnson et al., 1988; Johnson et al., in press). 

Measures of reproductive success that may be affected by contamination include hormone 

levels in plasma (estradiol, for example) and oocyte condition (atresia, for example) in feral 

fish. Fish can also be taken from the environment and held in the laboratory to induce 

 

2 Gonadotrophins are a class of gonad-stimulating hormones required for the development and maintenance of 

the gonads in seasonal breeders. 

 5-17 November 1992

 
HAZMAT 93-1–Biomarkers 

spawning. Fertilization success, hatching success, egg condition, and estradiol and 

vitellogenin levels can then be measured and correlated to contamination at the site where 

the fish were taken, or they can be correlated with contaminants measured in fish tissue or 

bile. However, the measurement of fertilization success is very labor intensive, has a high 

chance of failure, and takes a long time, therefore it is not recommended for application to 

problems at waste sites. Measurements of reproductive hormone levels in fish near waste 

sites may be a useful technique for predicting reproductive effects (Johnson et al, 1988). 

They may be particularly useful at estuarine sites contaminated with PAHs or PCBs where 

white croaker, Atlantic croaker, English sole, flathead sole, or rock sole are present. 

Although these tests have been applied to winter flounder from Boston Harbor and Raritan 

Bay, there was little evidence that steroid metabolism was altered by contaminants in this 

species (Johnson et al., in press). 

DATA INTERPRETATION 

The interpretation of results of biomarker studies rests heavily on comparisons between test 

sites and appropriate reference sites. These indicators of exposure and response to 

contamination can also reflect stress generated by seasonal factors, reproductive state, and 

other natural factors. For these reasons, it is essential to collect organisms from reference 

sites at the same time and with exactly the same methods as from test sites. Natural 

variability can be more easily evaluated if age, sex, and reproductive state are noted when 

organisms are collected. The statistical significance of responses will vary by the parameters 

measured by individual tests. In general, however, results of tests will indicate that 

organisms at the site are (or are not) different from animals upstream, downstream, or at the 

reference site. 

SUMMARY 

Biomarkers are direct biological measures of the response of an organism to exposure to a 

contaminant. They consist of a wide range of measurable responses from the biochemical to 

the physiological. Biomarkers not only indicate that contaminants are present and 

bioavailable, but, they also indicate that the organisms are responding to the contaminants. 

However, while they can indicate that the contaminants are causing adverse biological 

effects, they do not necessarily do so. They may just indicate that the organisms are 

detoxifying and eliminating the contaminants. There are numerous biomarker tests being 

worked with and some even used at waste sites (e.g., SCE, growth), but the majority are still 

in the research and development stage. Therefore, while biomarkers hold great promise for 

November 1992 5-18 
HAZMAT 93-1–Biomarkers

use at future hazardous waste sites, only a few are sufficiently developed to be currently 

considered for use at hazardous waste sites.

REFERENCES 

Benson, W.H., K.N. Baer, and C.F. Watson. 1990. Metallothionein as a biomarker of 

environmental metal contamination. In: McCarthy, J.F. and L.R. Shugart. 1990. 

Biomarkers of Environmental Contamination. Boca Raton, FL, Lewis Publishers. pp. 255-

266. 

Benson, W.H. and R.T. DiGiulio. 1992. Biomarkers in hazard assessments of contaminated 

sediments. In: Burton, G. A. (ed). Sediment Toxicity Assessment. Boca Raton, FL, Lewis 

Publishers. pp. 241-266. 

Black, J. 1983. Field and laboratory studies of environmental carcinogenesis in Niagara 

River fish. Great Lakes Research 9: 326=334. 

Braekevelt, C. R. and D. B. McMillan. 1967. Cyclic changes in the ovary of the brook 

stickleback, Eucalia inconstans. Journal of Morphology 123: 373-396. 

Casillas, E., M.S. Myers, L.D. Rhodes, and B.B. McCain. 1985. Serum chemistry of diseased 

English sole, Parphrys vetulus Girard, from polluted areas of Puget Sound, Washington. 

Journal of Fish Diseases 8. pp 437-449. 

Cross, J.N., V.E. Raco, and D.W. Diehl. 1984. Fish reproduction around outfalls. In: 

Bascom, W (ed). Southern California Coastal Water Research Project Biennial Report, 1983-

84. Long Beach, CA, Southern California Water Research Project. pp. 211-228. 

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November 1992 5-24 
HAZMAT 93-1–Benthic Community Studies 

CHAPTER 6 

BENTHIC COMMUNITY STUDIES 

INTRODUCTION 

Most coastal CERCLA sites are located next to or near aquatic systems (e.g., streams, rivers, 

estuaries, bays). These aquatic systems play an important role in site investigations because 

they are ecologically important to NOAA trust resources or because they serve as 

contaminant pathways to habitats used by NOAA trust resources. Assessing aquatic 

biological communities permits the determination of the ecological condition or 'status' of 

an aquatic environment (Hunsaker and Carpenter, 1990). Aquatic biological community 

studies can expand and improve the assessment of the impacts of CERCLA sites, help 

determine sites of major concern, identify impaired beneficial uses, determine program 

priorities, and help monitor the overall success of a remediation program. 

While any combination of taxonomic group (algae, invertebrates, or fish) and level of 

biological organization (individual, population, community, or ecosystem) can be used to 

assess the biological health of an aquatic system, this chapter focuses on the use of benthic 

invertebrate communities. Benthic invertebrates are commonly used in environmental 

monitoring because they show cumulative effects of present and past conditions, they have 

low mobility and relatively long life cycles, and their ecological relationships are relatively 

well understood (Herricks and Cairns, 1982; Wilhm, 1975). In addition, sampling 

procedures are relatively well developed, the group is heterogeneous in that a single 

sampling technique collects a considerable number of species from a wide range of phyla, 

and macroinvertebrates are generally abundant (Mason, 1981). Macroinvertebrates are 

those invertebrates retained by a 0.5-millimeter (mm) mesh screen (McIntyre et al., 1984) 

and are preferred for environmental monitoring studies over microinvertebrates because 

their taxonomy is better known. However, there are many sites where invertebrates <0.5 

mm may need to be targeted, e.g., lakes and large rivers. 

Community level studies provide the greatest amount of information on the biological 

integrity of an aquatic system, while at the same time permitting the examination of 

individual taxa that occur within the benthic invertebrate community. Communities are 

assessed from either a structural or functional perspective. Community structure is the 

measurement of biotic characteristics (e.g., abundance, diversity, and species composition) 

at a point in time; whereas, community function is the measurement of rate processes (e.g., 

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HAZMAT 93-1–Benthic Community Studies 

species colonization rates) of the ecosystem. The use of biological communities in 

environmental monitoring is normally done from a structural perspective because structural 

studies normally take less time, are more conventional, and permit comparisons to be easily 

made with data from other studies (Mattews et al., 1982). However, it must be kept in mind 

that contamination is not the only factor capable of changing community structure. 

Changes in salinity, temperature, sediment texture, and shading, to name a few, can all 

effect community structure. 

This chapter reviews three of the four main elements of a benthic invertebrate study: study 

objective, sampling methods, and data analysis. The fourth element, study design, will be 

covered in Chapter 7. The last section of this chapter reviews the use of EPA’s Rapid 

Bioassessment Protocols for benthic invertebrates in streams. 

STUDY OBJECTIVES 

The most important component of a benthic invertebrate study is a well-defined objective. 

Numerous studies conducted at CERCLA sites have included a benthic invertebrate 

component, but have commonly lacked an overall study design discussing the need or 

appropriateness of benthic invertebrate community assessments and how the information 

will be used. The objective(s) of a study must be determined before an appropriate sampling 

program is designed. 

Benthic invertebrate communities can be used at CERCLA sites to determine: 

• Benthic resources using the habitats of interest and potential risks to these 

communities associated with the release of site-related contaminants, 

• Appropriate organisms for bioassays or bioaccumulation studies, 

• Whether the stream of interest has a benthic community that can be efficiently used 

to monitor a site, 

• If benthic invertebrate communities in the vicinity of contaminated sediments 

and/or water are statistically different from non-impacted (reference) areas. 

• Effectiveness of remediation activities. 

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HAZMAT 93-1–Benthic Community Studies 

SAMPLING METHODS 

This section presents information on qualitative, semiquantitative, and quantitative 

sampling, along with a review of sampling devices used for collecting benthic invertebrate 

samples. 

Quantitative vs Non-Quantitative Sampling 

There are numerous sampling methods that can be used to assess benthic invertebrate 

communities. The major considerations prior to sampling are whether one needs 

quantitative or qualitative data and what sampling device will best collect the information 

required. Quantitative sampling is time consuming and therefore more expensive, but is 

required when one needs to determine statistical differences among stations;, whereas 

qualitative sampling is more rapid, less expensive, and typically used for surveys or to 

supplement quantitative sampling. 

The concepts of qualitative, semi-quantitative, and quantitative sampling are well illustrated 

in an example presented by Holme and McIntyre (1984): 

qualitative: Collecting samples from different benthic habitats to 

produce a list of taxa or species in the area. 

quantitative: Estimating the number of individuals or biomass per unit 

area, using grabs, corers or other quantitative samplers. 

semi-quantitative: (Qualitative): determining the relative abundance of 

species using a dredge. 

In this example, qualitative sampling is used for preliminary surveys, which may be 

followed by more complex qualitative sampling (semi-quantitative), and by quantitative 

studies. 

Qualitative Sampling: The objective of simple qualitative sampling is to compare sites 

using the presence or absence of benthic invertebrates. Because statistical analysis is not 

conducted on qualitative data, there are no set guidelines for how or where the samples 

should be collected. Samples can be collected using most any type of method and gear. 

Commonly used equipment includes dip, kick or push nets, grab samplers, dredges, trawls, 

and artificial substrate samplers. For comparability, the same approach and sampling 

equipment should be used at each station. While this type of method is acceptable for 

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HAZMAT 93-1–Benthic Community Studies 

general surveys, it is highly subjective and therefore will limit the degree of confidence 

associated with statements made about the community of interest. 

Quantitative Sampling: Quantitative sampling provides an estimate of the abundance 

and/or biomass (standing crop) of various components of the benthic invertebrate 

community per unit area, volume, or sampling unit. It needs to be conducted using 

statistically appropriate methods, paying particular attention to sample randomization. 

Quantitative sampling also provides information on the composition of the community and 

the distribution of the various taxonomic groups. There are a wide variety of sampling 

devices (e.g., stream net, grab, and core samplers) that can be used for sampling benthic 

invertebrate communities quantitatively. It is critical that the sampling device be 

appropriate for the habitat of interest. 

Semi-quantitative Sampling: The rather nebulous term 'semi-quantitative' sampling is 

often used to refer to sampling that involves collecting benthic invertebrates by level of 

effort, as in time expended per habitat rather than by area or volume sampled. It can also 

refer to sampling involving the use of quantitative sampling devices in a non-random 

manner. The principal difference between quantitative and semi-quantitative sampling is in 

the degree of confidence that the replicate samples are comparable and free from 

investigator-induced bias. By comparable is meant that each replicate is sampling identical 

portions of the population, e.g., the portion present in 1 m2 of bottom sediment. The 

nonrandom use of quantitative sampling devices (e.g., grabs, cores) introduces bias into the 

sampling. The lack of comparable replicates or sampling bias both preclude the statistical 

analysis of the data. Semi-quantitative sampling is basically just qualitative sampling with 

more rules. 

Examples of commonly used semi-quantitative freshwater sampling methods include the 

traveling kick method (Hornig and Pollard, 1978; Pollard, 1981) and the U.S. EPA Rapid 

Bioassessment Protocols II and III (Plafkin et al., 1989). Estuarine and marine semiquantitative methods involve the use of push nets (intertidal) or trawls (subtidal) operated 

for specified units of time at specified speeds (Eleftheriou and Holme, 1984). The results 

from these sampling methods are based on unit of effort as opposed to area or volume 

sampled. These sampling methods provide only semi-quantitative results because the 

investigator cannot be sure that equal units of effort sample equal proportions of the desired 

population. For example, during a 20-minute bottom trawl, the trawl may not actually be 

on the bottom for the entire time (e.g., it may bounce on and off the bottom) and each 

replicate trawl may be on the bottom for different proportions of the 20 minutes. 

November 1992 6-4 
HAZMAT 93-1–Benthic Community Studies 

Whether sampling is quantitative or qualitative, habitats sampled should be as similar in 

relation to physical and chemical parameters as possible. In streams this means more than 

just comparing riffles with riffles and pools with pools; other factors need to be taken into 

account such as substrate, water velocity, grain size, depth, pH, hardness, and degree of 

shading from adjacent terrestrial plants. An important additional parameter effecting 

benthic communities in estuarine and marine environments is salinity. Intertidal 

community sampling needs to take into account tidal elevation because duration of 

submergence will effect the composition of the community. Submergence time will also 

effect the duration of exposure to waterborne contaminants. Intertidal benthic communities 

are also effected by their degree of exposure to wave action; for example, a sheltered rocky 

intertidal community will be different from an exposed intertidal community. The physical 

and chemical parameters of the sampled sites need to be taken into account when 

comparing sites for degree of exposure to contaminants so you can be reasonably confident 

that differences between sites are due to differences in contamination and not just 

differences in normal environmental variables. 

Sampling Devices 

Benthic macroinvertebrates can be collected from either natural or artificial substrate with 

each type offering advantages, depending on site-specific conditions. Natural substrates 

should be sampled wherever possible. Artificial substrates should be used when natural 

substrates cannot physically be sampled or when the substrate is so highly variable that 

heterogeneity needs to be removed as a variable. For example, when upstream stations are 

dominated by sand and downstream stations are dominated by gravel, the use of artificial 

substrates would permit the sampling of uniform substrates in both areas. More detailed 

information on sampling design, sampling devices and their use are reviewed extensively in 

Klemm et al. (1990), Mudroch and MacKnight (1991), and Holme and McIntyre (1984). 

Stream-Net Samplers: Stream-net samplers are fitted with a fine mesh net (210 to 500 μ) 

and collect benthic invertebrates from flowing water as it passes through the sampler. A 

horizontal, rectangular frame upstream of the vertical net mouth marks off the area to be 

sample. Generally, the procedure involves stirring up the substrate within the rectangle and 

scraping stones to remove clinging organisms, the current then sweeps the organisms into 

the net. These samplers are typically used in shallow waters (< 0.5 meters) with coarse 

substrate. In the majority of cases this means shallow riffle habitats. The dominant streamnet samplers include the Surber, Invertebrate Box Sampler, and Hess Sampler (Table 6-1). 

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HAZMAT 93-1–Benthic Community Studies 

Table 6-1. Stream-net samplers used to assess benthic communities in freshwater 

streams.

SAMPLER SIZE HABITAT EFFECTIVENESS ADVANTAGES LIMITATIONS

Surber 1 ft2 Shallow, flowing

streams, less than 

32 cm deep with 

good current; 

rubble substrate, 

mud, sand and

gravel. 

Relatively quantitative 

when used by 

experienced biologist; 

performance depends 

on current and 

substrate. 

Encloses area 

sampled; easily 

transported or 

constructed; samples a 

unit area. 

Difficult to set in 

some substrate types,

that is, large rubble;

cannot be used 

efficiently in still,

slow-moving waters. 

Hess 0.09 m2 Shallow, flowing

streams, less than 

32 cm deep with 

good current; 

rubble substrate, 

mud, sand and

gravel. 

Relatively quantitative 

when used by 

experienced biologist; 

performance depends 

on current and 

substrate. 

Completely encloses 

area sampled; easily 

transported or 

constructed; samples a 

unit area; can be used

in weed beds.

Difficult to set in 

some substrate types,

that is, large rubble;

cannot be used 

efficiently in still,

slow-moving waters. 

Intertidal Samplers: The intertidal zone is unique among those aquatic environments that 

are routinely sampled for environmental assessments. It can be sampled while exposed to 

air. On sandy or muddy shores a square sheet-metal frame (e.g., 0.1 or 0.25 m2) is driven 

into the substrate and the sediments within the frame are excavated to the desired depth 

and sieved (Eleftheriou and Holme, 1984). On rocky shores, a square frame of heavy gauge 

wire is laid on the substrate and the animals within the frame are counted, weighed, or 

estimated in terms of percent cover (Eleftheriou and Holme, 1984). This latter operation can 

either be done in situ or by taking a photograph.

Grab Samplers: A grab sampler is any device that is lowered vertically to collect a sample 

by penetrating the substrate and obtaining a discrete quantity of bottom sediment (Table 6-

2). All grab samplers have some type of jaw mechanism that closes upon impact with the 

sediments, and usually cover a surface area of 0.1 or 0.2 m2. Grab samplers are most 

commonly used in medium to fine sediments and in deeper waters, i.e. nonwadable. There 

are numerous types of grabs, with the more common ones including the Ponar, Ekman, 

November 1992 6-6 
HAZMAT 93-1–Benthic Community Studies 

Peterson, van Veen, and Smith-McIntyre (Table 6-2). The Ponar is the most commonly used 

grab sampler for pools and lakes, while the van Veen is generally the grab of choice in 

estuaries and sheltered marine waters. The van Veen has been adopted as the standard 

sampler in Puget Sound (Simenstad et al., 1991) and the Baltic Sea (Eleftheriou and Holme, 

1984). For more exposed marine waters the more stable Smith-McIntyre or Day samplers 

are preferred. 

Core samplers: Core samplers are tubes that are vertically inserted into the substrate and 

when withdrawn contain the enclosed material and associated fauna. They are best suited 

for sampling soft homogeneous substrates and are commonly used for assessing benthic 

invertebrates in lake, estuarine, and marine environments; however, they are occasionally 

used in streams. Their major advantage over grabs is they sample to a deeper, more 

uniform depth. There are numerous types of cores ranging from small homemade, handheld cores for sampling low-order streams to large complex cores for deep-water, openocean sampling. 

Air-lift samplers: Air-lift samplers use compressed air to scour the substrate and raise 

water, lighter substrate material, and fauna as the air ascends in a delivery pipe. The 

material is passed through a net to collect the benthic invertebrates. The air-lift sampler is 

most efficient in static or slowly moving water. While this type of sampler is not used 

extensively due to cost and complexity, they are beginning to receive more use in larger 

rivers and estuaries with fine substrate. 

Trawl and Dredge Samplers: Trawl and dredge samplers are towed horizontally across 

the substrate and their use is generally restricted to lake, estuarine, and marine 

environments. Trawls consist of nets with their mouths held open by a frame, towed across 

the surface of the benthic substrate with little if any penetration. Dredges, like trawls, are 

towed horizontally across the benthic substrate, but they are of heavy construction, often 

with metal mesh nets or metal collection boxes, and are designed to break off pieces of rock, 

scrape organisms off hard surfaces, or for limited penetration into the substrate (Eleftheriou 

and Holme, 1984). Both samplers are often towed for a specified period of time at a uniform 

speed to permit a better qualitative comparison of sites. Trawls and dredges are good 

supplements to cores and grabs because they sample a larger area of the benthos. 

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HAZMAT 93-1–Benthic Community Studies 

Table 6-2. Dominant grab samplers used in assessing benthic communities. 

SAMPLER SIZE HABITAT EFFECTIVENESS ADVANTAGES LIMITATIONS

van Veen 0.1 m2 

&0.2 m2

Estuaries, bays, oceans, and other open water habitats with sand, gravel, mud, or clay. 

More effective than Peterson grab or Ponar grab in deeper water. 

Long arms give increased stability and better digging efficiency; adopted as standard sampler by Puget Sound Protocols. 

A very heavy grab that requires use of a boat with winch and cable; stones, pebbles and other debris can hold jaws open causing loss of sample.

SmithMcIntyre 0.1 m2 Estuaries, bays, oceans, and other open-water habitats with sand, 

gravel, mud, or clay. 

More effective than van Veen under rough conditions.

Grab is contained in a frame giving greater stability; trigger plates insure grab is not activated before resting on the bottom; activated by heavy springs giving more uniform penetration. 

A very heavy grab that requires use of a boat with winch and cable; stones, pebbles, and other debris can hold jaws open causing loss of sample;considered by some as complicated and dangerous because of the spring operation. 

Standard Ponar 523 cm2 Freshwater lakes, rivers, estuaries, 

and reservoirs with hard and soft sediments such as clay, hard pan,sand, gravel, and muck; somewhat less efficient in softer sediments.

Not entirely adequate for deep burrowing organisms in soft sediments; very efficient for hard sediments; collects both qualitative and quantitative samples. 

Better penetration than other grabs; side plates and screens reduce washout, shock waves, and substrate disturbance; best quantitative grab sampler for freshwater use. 

A very heavy grab that requires use of a boat with winch and cable; stones, pebbles, and other debris can hold jaws open causing loss of sample.

Petite Ponar 232 cm2 Freshwater lakes, rivers, estuaries, 

and reservoirs with moderately hard sediments such as sand, silt,and mud; will not penetrate clay;somewhat less efficient in soft sediments and coarse gravel. 

Not entirely adequate for deep burrowing organisms in soft sediments; not useful in clay. 

Good penetration for such a small grab; side plates and screens reduce washout, shock waves, and substrate disturbance; can be operated by hand without boat or winch. 

Jaws can be blocked by stones, sticks, and other debris causing loss of part of the sample; not efficient in swiftly flowing water of over one meter per second velocity.

Ekman Freshwater rivers, lakes ,and 

reservoirs where there is little current; soft sediments such as muck and silt.

Efficient only in soft sediments but weights can be added for deeper penetration in fine sand; collects both qualitative and quantitative samples.

Easy to operate by hand without winch, can be pushed into substrate in shallow water; hinged doors at top reduce washout, shock waves, and disturbance of the substrate; comes in a range of sizes.

Light weight so that jaw will not penetrate hard substrates; jaws often do not close completely due to blocking of jaws or failure of closing mechanism; inefficient in deep water or where there is even moderate current. 

Peterson 0.6

to 

0.099 m2

Freshwater lakes, reservoirs, rivers, and estuaries with sand, gravel, clay, and hard pan substrates. 

Less effective in most substrates than the Ponar. 

Can give quantitative samples if used properly; ranges of sizes available. 

Standard grab is heavy and requires boat with winch; can cause washout if dropped rapidly to the bottom; shallow bite by jaws; jaws are easily blocked by debris causing loss of sample; of questionable value as a quantitative sampler. 

November 1992 6-8 
HAZMAT 93-1–Benthic Community Studies 

Cameras: Cameras are coming into increasingly wider use for analyzing benthic 

communities. They can either be hand or remotely operated. One type of camera analysis, 

REMOTS® (Remote Ecological Monitoring of the Seafloor), couples photographs of the 

benthos with a video digitizer and computer image analysis system (Science Applications 

International Corporation (SAIC), 1985). The camera is located in a wedged shape prism 

mounted on a frame that is lowered to the bottom. When the frame rests on the bottom, the 

prism is lowered several centimeters into the sediment and a photograph is taken through 

the plexiglass window of the prism. The resulting negative is directly subjected to computer 

imaging analysis producing information on grain size, sediment surface boundary 

roughness, erosional and deposition criteria, depth of apparent redox potential 

discontinuity, and infaunal successional stage. REMOTS® data has been used by EPA as 

part of a risk assessment study for a site in Narragansett Bay (Munns et al., 1991). 

Miscellaneous Qualitative Devices: Any sampling equipment presented in this report 

can be used for qualitative sampling; however, there are a few devices that are used 

explicitly for qualitative sampling. These include hand-held screens, dip nets, sweep nets, 

push nets, kick nets, etc. These sampling devices can be used in most any type of shallow 

water habitat and, if used by an experienced biologist, can provide an adequate assessment 

of the relative health of a stream. To allow for a better qualitative comparison of the sites 

with these devices, they can be used to sample a predetermined area or for a predetermined 

time interval. 

Artificial Substrates: An artificial substrate is defined as any device used to mimic specific 

features of the aquatic environment into which it is placed. Artificial substrates are typically 

used to sample aquatic habitats that can not be sampled effectively using conventional 

devices due to physical limitations or substrate heterogeneity (Table 6-3). Artificial 

substrates remove substrate heterogeneity as a variable by providing identical substrates for 

organisms to settle on at each station sampled. The two most commonly used artificial 

substrates include rock-filled wire baskets and Hester-Dendy multiplate samplers. Both 

effectively sample 'drift' communities but are poor for sampling infauna. NOAA’s Marine 

Ecosystem Analysis Project used horizontal artificial substrates successfully in Puget Sound 

to assess pollution effects in Commencement Bay, Elliott Bay, and Dyes Inlet in the mid1980s (Long, personal communication). It should be noted that the purpose of artificial 

substrates is to monitor changes in invertebrate communities over time and space and do 

not necessarily reflect the benthic invertebrate community that resides on the natural 

 6-9 November 1992 
HAZMAT 93-1–Benthic Community Studies 

substrate. However, this does not negate their usefulness under specific conditions. There 

are numerous advantages and disadvantages to artificial substrates. 

The overall sampling design for artificial substrates is similar to the design for natural 

substrates described above. Stations selected should be as similar as possible to reduce 

variability. Artificial substrates can either be anchored to a float near the water surface, 

suspended within the water column, or set on the stream bottom. Substrates should be 

positioned at all stations in the same manner. The artificial substrates should remain in the 

stream for a six- to eight-week colonization period. 

Table 6-3. Advantages and disadvantages of artificial substrates. 

Advantages Disadvantages

Allows collection of data from locations 

that cannot be sampled effectively by 

other means. 

Permits standardized sampling. 

Reduces variability compared with 

other types of sampling. 

Requires less operator skill than other 

methods. 

Convenient to use. 

Permits nondestructive sampling of an 

environment. 

Permits greater flexibility in sampling 

programs. 

Provides effects data for a specific time period 

(period of deployment). 

Colonization dynamics not fully known. 

Nonrepresentative sampling under either 

natural or polluted conditions. 

Artificial substrates require long 

exposure time (6-8 weeks) to obtain a

sample. 

Loss of fauna on retrieval of samplers. 

Unforeseen losses of artificial substrates. 

Inconvenient to use and logistically 

awkward. 

DATA ANALYSIS 

This section presents a review of commonly used ecological and statistical approaches for 

assessing benthic invertebrate communities. Analysis of biological data should focus on 

approaches that enhance biological interpretation. Statistical techniques should not be the 

primary assessment approach, but should be used in conjunction with other methods to 

November 1992 6-10 
HAZMAT 93-1–Benthic Community Studies 

obtain a clearer understanding of the biological communities and factors controlling 

community structure. Statistical analyses may indicate a significant difference in a 

particular community parameter, but that difference may have little, if any, ecological 

meaning or relevance. 

Ecological Analysis 

There are numerous approaches to assess the ecological health of benthic invertebrate 

communities, including community structure and function. Measurements of community 

structure include total abundance, diversity, and species composition. Measurements of 

community function include species colonization rates and feeding guilds. The use of 

biological communities in environmental monitoring is normally assessed from a structural 

perspective (Matthews et al., 1982). 

The most common ecological analyses used to assess benthic invertebrate communities are 

listed below. There are several community measures that should always be part of any 

benthic invertebrate study, including total abundance, taxa richness, percent contribution of 

the dominant taxa, community similarity, and a listing of all taxa collected by station. 

Species diversity indices, based on total number of taxa present and relative abundance of 

each taxa, have been used in the past to characterize sites, but they can be very deceiving 

depending on community structure and should no longer be used. Furthermore, reports 

should include all raw data by replicate and a description of the physical habitat at each 

station. Other analyses can be used on a site by site basis. The ecological metrics selected to 

assess benthic invertebrate communities in the vicinity of a CERLCA site should be realistic 

for the type of habitat and the natural resident community. This is particularly important in 

low-gradient coastal streams since they can naturally have a substrate dominated by fine 

materials that may support taxonomic groups typically considered stress-tolerant (e.g., 

Oligochaeta and Chironomidae). Therefore, when interpreting data from aquatic systems it 

is important to consider the natural status of the stream and not assume that a community 

dominated by stress-tolerant groups has been negatively impacted by contaminants. 

Abundance: Abundance is the number of individuals collected in a specific area, typically 

1 m2. Abundance can be assessed in relation to total number of invertebrates or number of 

individuals of specific taxonomic groups, usually ranging from order to species. In general, 

variability between samples increases from total abundance down the taxonomic scale to 

species. Therefore, four replicate samples may be sufficient for assessing total abundance, 

whereas, up to fifty samples could be required to have any accuracy on a particular species. 

 6-11 November 1992 
HAZMAT 93-1–Benthic Community Studies 

Taxa Richness: Taxa richness is the total number of taxa or taxonomic units recognizable 

as individual species, whether or not identified to the species level, collected from a station. 

This is the most basic measure of community diversity, and is the strongest measurement 

available for assessing community health. Generally, richness increases with increasing 

water quality and/or habitat diversity, but not always. 

Percent Contribution of Dominant Taxa: This provides an indication of community 

balance. A community dominated by a few taxa is considered to indicate environmental 

stress, whereas a community with a more even distribution of taxa is generally indicative of 

better water quality. Percent contribution of dominant taxa should always be part of any 

benthic invertebrate study. 

Community Similarity Indices: These indices are used to determine the similarity in 

benthic invertebrate communities among stations. Community similarity indices provide a 

measure of how similar two benthic communities (stations) are in relation to taxa present at 

both stations, or by the taxa present and their relative abundance. Community similarity 

indices are widely used and are extremely useful in assessing benthic invertebrate 

communities in polluted rivers. The similarity matrix is then used to perform a cluster 

analysis that groups stations based upon their similarity. 

EPT Index; This is the total number of distinct taxa within the orders Ephemeroptera 

(mayflies), Plecoptera (stoneflies) and Trichoptera (caddisflies) which are orders of 

freshwater insects, classified as pollution sensitive. 

Ratio of EPT to Chironomidae: This index uses the abundances of these indicator groups 

as a measure of community balance. Ephemeroptera, Plecoptera, and Trichoptera are 

pollution-sensitive indicators; whereas, Chironomidae (midges) are pollution-tolerant 

indicators. A healthy community has an even distribution among all four groups; whereas, 

an unhealthy system can have a community dominated by the Chironomidae. 

Hilsenhoff Biotic Index (HBI): This index summarizes overall pollution tolerance of a 

benthic invertebrate community with a single value. The index was originally developed to 

detect organic pollution in riffle/run habitats; therefore, it is uncertain at this point how 

sensitive the index is to other forms of contamination. 

November 1992 6-12 
HAZMAT 93-1–Benthic Community Studies 

Statistical Approaches 

Statistical techniques should be used to help examine and evaluate data, but, there are 

situations where a nonstatistical approach will provide a better answer to a particular 

question for far less money and time. Statistical analyses used to assess invertebrate 

communities can include graphical presentation of data, descriptive statistics, hypothesis 

testing, correlation and regression statistics, and classification and grouping methods. 

Graphing raw data is useful for a cursory review of the data and can provide insight to the 

types of analyses that may be most useful. 

The most common statistical approach to assessing benthic invertebrate communities are 

descriptive statistics, which can include mean, variance (S2

), confidence intervals (CI) (95 

percent CI), and range of values. For a survey study, this level of data analysis may be 

sufficient for assessing status and trends of benthic communities. However, more specific 

questions, such as, “Is there a statistically significant difference among stations in relation 

to parameter x ?,” require more sophisticated analyses such as analysis of variance 

(ANOVA). Power analysis may be required to determine the proper sample size for these 

types of statistical tests. Selection of the statistical analyses to be applied to the data set 

must be done at the design stage of the study, not after the data is collected (see Chapter 7). 

A correlation analysis can be used to determine if there is a relationship between two 

variables (e.g., taxa richness and Cu) near a site. Regression analysis can be used to 

demonstrate that there is a dependency of one variable on another. 

There are a number of multivariate approaches that can be used for pattern analysis. Some 

of the more common approaches include cluster analysis, principal component analysis, and 

discriminate analysis. For more detail on these and other multivariate methods see Green 

(1979), Johnson and Wichern (1982), Gauch (1982), and Pielou (1984). 

Chapter 7 of this document has a more detailed discussion of statistical concerns with 

regards to bioassessment studies. 

EPA’S RAPID BIOASSESSMENT PROTOCOLS 

In 1987 the EPA released a report entitled "Surface Water Monitoring: A Framework for 

Change" that concluded that the present monitoring programs be restructured to address 

more current environmental problems. In response to this emphasis, the Assessment and 

Watershed Protection Division of the EPA developed and published a report entitled "Rapid 

 6-13 November 1992 
HAZMAT 93-1–Benthic Community Studies 

Bioassessment Protocols for Use in Streams and River: Benthic Macroinvertebrates and Fish" 

(EPA/444/4-89-001). These protocols (Protocols I through V) are to be used for planning 

and management purposes as screening, site ranking, and trend monitoring. 

Protocols I through III use benthic invertebrate communities with each higher level 

involving a more complex sampling method and analysis. Protocol I is a qualitative survey 

method that can be used to establish the general condition of a stream using the presence 

and/or absence of particular invertebrates; taxonomic identification is done in the field with 

organisms identified to the order or family level. Protocol II is a more detailed program that 

includes more habitats and the assessment of the community using a variety of community 

metrics; taxonomic identification is done in the field with organisms identified to the order 

or family level. Protocol III is the most complex of the benthic invertebrate protocols and 

includes taking all invertebrates collected to the laboratory for identification of 

genus/species. This method also involves more complex community analysis. Protocols IV 

and V involve fish communities. Protocol IV is based on a questionnaire survey and 

published literature on the area of interest. Protocol V involves field sampling fish 

communities with a more complex analysis of the information. All five protocols involve 

assessing physical and chemical parameters for a more integrated analysis. 

The EPA Rapid Bioassessment Protocol Manual is an extremely useful document. However, 

there are a number of things one must remember when using it. All five protocols require a 

biologist to collect and analyze the data, preferably one that has extensive training in stream 

ecology. Furthermore, it is important to remember that these protocols and the metrics used 

to analyze the data have not been tested in all the ecoregions of the United States, and 

therefore may not be the correct approach in every situation. One needs to select the 

protocol and/or metrics best suited for the type of habitat of interest. 

The EPA Rapid Bioassessment Protocol is useful at CERCLA sites for preliminary 

assessment, low priority sites, screening and prioritization of numerous habitats at a 

CERCLA site, and monitoring success of site remediation. In all cases the method should 

only be used in wadable freshwater streams that have adequate reference stations. The 

Rapid Bioassessment Protocol should not be used for other types of habitats or for sites 

where one needs a high degree of confidence in whether there has been a statistically 

significant change in the benthic community. 

November 1992 6-14 
HAZMAT 93-1–Benthic Community Studies 

SUMMARY 

Study Objective 

The first step in designing a benthic invertebrate study is to determine the objective(s) of the 

study. Field studies at CERCLA sites are observational and not experimental; therefore, 

while statistical techniques can be used, there are limitations in the interpretation of results 

from some tests. Observational studies cannot determine cause and effect, but only 

differences among stations, spatial patterns, and correlations among variables. For example, 

if there is a statistically significant difference among stations in a particular community 

parameter (e.g., taxa richness), it is acceptable to state that there was a difference among 

stations. It would be inappropriate to state that the difference was due to contaminants 

released from a CERCLA site. However, benthic studies used in conjunction with sediment 

chemistry and toxicity tests, as part of a sediment quality triad approach (Long and 

Chapman, 1985), can provide a preponderance of evidence that strongly infers that the 

CERCLA site is responsible for the differences. 

Sampling Methods 

• Quantitative sampling should be used for statistically based programs. 

• Qualitative sampling should be used for a general survey of a site. 

Sampling Devices 

• Natural substrates should be sampled whenever possible, with artificial substrates 

used when natural substrates cannot physically be sampled or when high substrate 

variability needs to be controlled as a factor. 

• Stream-net samplers (e.g., Hess or Surber) should be used for assessing benthic 

invertebrate communities in riffle habitats; whereas, a grab sampler (e.g., Ponar) 

should be used for assessing benthic invertebrate communities in pool habitats. 

• Qualitative sampling can be done with most any device available. 

• Artificial substrates can either be anchored to a float near the water surface, 

suspended within the water column, or set on the bottom. 

 6-15 November 1992 
HAZMAT 93-1–Benthic Community Studies 

• Artificial substrates should remain in the water for a six- to eight-week 

colonization period. 

Data Analysis 

• All benthic invertebrate studies should at a minimum include total abundance, taxa 

richness, percent contribution of dominant taxa, and community similarity indices. 

Additional ecological analysis would depend on the nature of the community and 

aquatic system. 

• Statistical analysis used to assess benthic invertebrate communities should be based 

upon the objective of the study. 

REFERENCES 

Eleftheriou A. and N.A. Holme. 1984. Macrofauna techniques. In: N.A. Holme and A.D. 

McIntyre (eds). Methods for the Study of Marine Benthos (2nd Ed.) London. pp 1-26. 

Gauch, H.G. 1982. Multivariate Analysis in Community Ecology. Cambridge Studies in 

Ecology. New York, Cambridge University Press. 298 pp. 

Green, R.H. 1979. Sampling Design and Statistical Methods for Environmental Biologists. 

New York, John Wiley and Sons. 257 pp. 

Herricks, E.E. and J.Cairns, Jr. 1982. Biological Monitoring. Part III - Receiving system 

methodology based on community structure. Water Resources 16:141-153. 

Holme, N.A. and A.D. McIntyre (eds.). 1984. Methods for the Study of Marine Benthos (2nd 

Ed.). London, Blackwell Scientific Publications. 387 pp. 

Hornig, C.E. and J.E. Pollard. 1978. Macroinvertebrate sampling techniques for streams in 

semi-arid regions. Comparisons of the Surber method and a unit-effort traveling kick 

method. EPA-600/4-78-040. Las Vegas, NV. U.S. Environmental Protection Agency. 

Hunsaker, C.T. and D.E. Carpenter, eds. 1990. Ecological indicators for the Environmental 

Monitoring and Assessment Program. EPA 600/3-90/060. Triangle Park, NC, U.S. 

Environmental Protection Agency, Office of Research and Development, Research. 

Johnson, R.A. and D.W. Wichern. 1982. Applied Multivariate Statistical Analysis. New 

Jersey, Prentice-Hall. 594 pp. 

Klemm, D.J., P.A. Lewis, F. Fulk, and J.M. Lazorchak. 1990. Macroinvertebrate field and 

laboratory methods for evaluating the biological integrity of surface waters. EPA 600/4-

90/030. Cincinnati, OH, U.S. Environmental Protection Agency, Environmental Monitoring 

Systems Laboratory. 

November 1992 6-16 
HAZMAT 93-1–Benthic Community Studies 

Long, E. R. and P. M. Chapman. 1985. A sediment quality triad: Measures of sediment 

contamination, toxicity and infaunal community composition in Puget Sound. Marine 

Pollution Bulletin (16):10. pp 405-416 

Long, E.R. 1992. personal communication. Coastal Monitoring and Bioeffects Division, 

Office of Ocean Resources Conservation and Assessment, National Oceanic and 

Atmospheric Administration. Seattle, WA. 

Mason, C.F. 1981. Biology of Freshwater Pollution. New York, Longman Inc. 250 pp. 

Matthews, R.A., A.L. Buikema Jr., J.Cairns Jr., and J.H. Rogers Jr. 1982. Biological 

monitoring: Part IIA. receiving system functional methods, relationships and indices. Water 

Resources. 16:129-139. 

McIntyre, A.D., J.M. Elliott, and D.V. Ellis. 1984. Introduction: Design of sampling 

programmes. In: N.A. Holme and A.D. McIntyre.(eds). Methods for the Study of Marine 

Benthos (2nd Ed.) London. pp 1-26. 

Mudroch, A. and S.D. MacKnight. 1991. Handbook of Techniques for Aquatic Sediment 

Sampling. Boca Raton, FL. CRC Press. 210 pp. 

Munns, W.R., Jr., C. Mueller, D.J. Cobb, T.R. Gleason, G.G. Pesch, and R.K. Johnston. 1991. 

Marine Ecological Risk Assessment at Naval Construction Battalion Center, Technical 

Report 1437. Davisville, RI. Naval Ocean Systems Center. 129 pp+. 

Pielou, E.C. 1984. The interpretation of ecological data: A primer on classification and 

ordination. New York, John Wiley and Sons.. . 263 pp. 

Plafkin, J.L., M.T. Barbour, K.D. Porter, S.K. Gross, and R.M Hughes. 1989. Rapid 

bioassessment protocols for use in streams and rivers: Benthic macroinvertebrates and fish. 

EPA/440/4-89-001. Washington, DC, U.S. Environmental Protection Agency, Assessment 

and Watershed Protection Division. 135 pp+ appendices. 

Pollard, J.E. 1981. Investigator differences associated with a kicking method for sampling 

macroinvertebrates. Journal. Freshwater Ecology 1 (2) 215-224. 

Scientific Applications International Corporation (SAIC). 1985. REMOTS® Survey of Zones 

of Siting Feasibility Puget Sound, Washington. Newport, RI, SAIC pp 154. 

Simenstad, C.A., C.D. Tanner, R.M. Thom, and L.L. Conquest. 1991. Estuarine habitat 

assessment protocol. U.S. EPA 910/9-91-037. 

Wilhm, J.L. 1975. Biological indicators of pollution. In: B.A. Whitton (ed) River Ecology 

Berkeley CA, University of California Press. .pp. 375-402. 

 6-17 November 1992 
HAZMAT 93-1–Benthic Community Studies 

November 1992 6-18 
HAZMAT 93-1–Study Design and Statistical Analysis 

CHAPTER 7 

STUDY DESIGN AND STATISTICAL ANALYSIS 

INTRODUCTION 

This chapter discusses the major elements of designing a bioassessment study to assess 

conditions at a hazardous waste site, including the statistical analysis of the resulting data. 

Before beginning to design an environmental study at a hazardous waste site it is necessary 

to understand the general limitations of this type of study. There are basically two types of 

studies, experimental and observational1. Experimental studies permit the researcher to 

control all the variables and to replicate the treatments of concern (e.g., contaminant 

concentrations). With observational studies the only variables the researcher can control are 

time and space (location). While statistical analysis can be applied to both types of studies, 

the experimental study permits conclusions regarding cause and effect, but the 

observational study can only determine spatial and temporal patterns and correlations 

among variables. Environmental studies at hazardous waste sites are observational studies, 

therefore, they do not permit the drawing of absolute conclusions with regards to the causes 

of any bioeffects that might be observed. For example, in a benthic community study, if 

there is a statistically significant difference among stations in a particular community 

parameter (e.g., taxa richness), it is acceptable to state that there was a difference among 

stations. It would be inappropriate to state that the difference was due to contaminants 

released from the hazardous waste site. However, a properly designed bioassessment study 

can provide evidence that strongly suggests a cause for the observed bioeffects. Waste site 

studies are further limited by the fact that contamination has already occurred so there is no 

opportunity for before- and after-contamination sampling. 

If properly misused, statistical analysis can prove anything you wish to prove, regardless of 

the facts. If properly used, statistics become a very helpful decision-making tool. However, 

even when properly used, statistics can give unwarranted credence to biological data sets, 

especially in the eyes of the statistically naive. Therefore, whether applying or reviewing 

statistical analyses you need to know that the data analyzed is in an appropriate form and 

 

1 Hurlbert, 1984 uses the terms manipulative and mensurative experiments, while Eberhardt and 

Thomas, 1991 talk about controlled and uncontrolled events. 

 7-1 November 1992 
HAZMAT 93-1–Study Design and Statistical Analysis 

that you understand exactly what the statistical results mean. The purpose of this chapter is 

not to make the reader a statistician, but to familiarize the reader with some of the 

requirements and problems of statistical analysis. This will be accomplished by defining the 

more commonly encountered statistical terms, briefly discussing the differences between 

descriptive and inductive statistics, as well as parametric and nonparametric methodologies 

for both comparative and correlative analyses. Other topics of discussion include 

hypothesis formulation, probability, and replication and pseudoreplication. 

Before continuing, some terms as they apply to bioassessment studies and statistical 

analysis, in particular, need to be defined. A statistical population consists of the totality 

of all possible observations of the variable with which we are concerned. While a 

biological population consists of a group of individuals of the same species between 

which genetic material freely flows. Since the terms 'statistical' and 'biological' rarely 

precede the term 'population,’ the context in which the term 'population' is found must be 

used to determine the term's exact meaning. If we are concerned with the mercury 

concentration in the mussels in the Bay of Belfalas, then the 'statistical' population would 

consist of the measurements of mercury concentration in each of the five million mussels 

making up the 'biological' population in the bay. Since we would not want to analyze all 

five million mussels, thus wiping out the mussel population in the bay, we would take a 

sample, which is a subset of the population. In this case, the sample would consist of 

something less than five million observations. In environmental studies the term sample 

often refers to a single observation and in discussions of the statistical analysis of these 

studies, the term often refers to single observations as well as to population subsets 

consisting of several observations. In the context of this chapter, individual observations 

making up a sample will be referred to as sample replicates or simply replicates. The 

characteristics of a statistical population, such as the population mean or variance, are 

referred to as parameters; the characteristics of the sample (e.g., sample mean, sample 

variance) are referred to as statistics. 

STUDY DESIGN 

Question Formulation 

The first step in designing any environmental study is to determine the question or 

questions you wish the study to answer. The more concisely you formulate your 

November 1992 7-2 
HAZMAT 93-1–Study Design and Statistical Analysis 

question(s), the more precisely you can design a study that will provide appropriate 

answers. At hazardous waste sites the general questions of concern to NOAA are: 

1. Are contaminants in or entering the aquatic environment? 

2. Are contaminants bioavailable? 

3. Are contaminants causing or have the potential to cause bioeffects? 

The first question also includes concern over what contaminants are present and what is the 

extent of the contamination (i.e., how far away from the site does contamination extend). 

No single study methodology is capable of answering all of these questions. Therefore, an 

environmental study at a hazardous waste site should consist of a multiplicity of methods. 

The use of a multiplicity of study methods helps relate environmental contamination and 

bioeffects to the waste site through a preponderance of evidence. 

The presence of contaminants in the environment can be determined by chemical analysis of 

various media (e.g., water, sediment). Samples taken near the hazardous waste site can be 

statistically compared with samples from an area that represents a similar habitat but is not 

influenced by the site (i.e., a reference site). Such comparisons can show that higher 

concentrations of contaminants exist near the waste site than at the reference site, however, 

they cannot show that the waste site is the source of the contaminants. If the study design 

correlates decreasing contaminant concentrations with increasing distance from the waste 

site, then it would be reasonable to infer that the waste site is the source of the 

contaminants. 

The presence of contaminants in the environment does not mean that they are bioavailable 

(i.e., able to be taken up by biological organisms). Therefore, the next step is to determine 

the bioavailability of the contaminants. Currently no acceptable method of chemical 

analysis can determine contaminant bioavailability. Therefore, chemical analysis can 

answer question number one, but supplies little if any information with regard to the other 

two questions. 

The most direct way to determine if contaminants are bioavailable is by performing tissue 

analysis on indigenous organisms to determine if contaminants or metabolites of 

contaminants are bioaccumulating in these organisms. Bioaccumulation studies can also be 

conducted with transplanted or artificial organisms as well as by collecting contaminated 

media and exposing laboratory organisms to it. To determine whether or not the 

bioaccumulation is related to the waste site, it needs to be correlated to media contamination 

 7-3 November 1992 
HAZMAT 93-1–Study Design and Statistical Analysis 

levels. This is accomplished by collecting indigenous organisms from or placing 

transplanted or artificial organisms in the same locations from which the chemically 

analyzed media samples were taken. For conducting correlation analyses, transplanted and 

artificial organisms have an advantage in that the contaminant levels measured in these 

organisms are solely due to current levels of media contamination. Contaminant levels in 

indigenous organisms may reflect past media contamination and therefore may not show a 

correlation with current media contamination levels. Laboratory bioaccumulation studies 

could also be conducted, but the media samples to be used should be split, with one portion 

being chemically analyzed and the other used in the bioaccumulation study. 

While bioaccumulation of contaminants implies their presence in the environment, it does 

not necessarily mean that there are any bioeffects related to the contamination. Determining 

whether contaminants from a hazardous waste site have caused or have the potential to 

cause bioeffects can be the most difficult question to answer. Hazardous waste sites can fall 

into three basic categories with regard to contamination of aquatic environments: 

• Sites that do not release contaminants into aquatic environments. 

• Sites that release such high concentrations of contaminants into aquatic 

environments that no biological organisms exist near the site. 

 • Sites that release some intermediate concentrations of contaminants. 

Sites in the first category are of no concern to NOAA. At sites in the second category the 

effects are obvious. Sites in the third category, which make up the majority of sites with 

which NOAA is concerned, present the biggest challenge with regard to determining the 

existence of or potential for bioeffects and relating the effects to the waste site. 

Toxicity testing and benthic community studies are the two most commonly used 

bioassessment methods for determining bioeffects or the potential for bioeffects at 

hazardous waste sites. A third approach for determining bioeffects is the use of biomarkers. 

These methods by themselves cannot tell you that the waste site is causing bioeffects; they 

can only tell you that conditions at the waste and reference sites are different. However, 

when these methods are used in conjunction with media chemical analysis and 

bioaccumulation studies, a body of evidence can be built up from which a cause and effect 

can be inferred. For example, suppose a bioassessment study, using bioaccumulation, 

toxicity testing, benthic community studies, and metallothionein analysis (a biomarker test 

indicating a response to metal exposure), was conducted at a riverine hazardous waste site 

in conjunction with sediment chemistry, and the following results were obtained: 

November 1992 7-4 
HAZMAT 93-1–Study Design and Statistical Analysis 

• Sediment chemistry showed high concentrations of metals near the site 

with a gradient of decreasing concentrations moving downstream from the 

site. 

• Tissue analysis of clams showed elevated levels of metals in areas of high 

sediment metals concentrations. 

• Toxicity tests correlated increased mortality of waterfleas with high 

sediment metals concentrations. 

• Benthic communities near the site had reduced biomass when compared to 

more distant and upstream sites. 

• MT levels were elevated in crayfish. 

By themselves each of these bioassessment techniques indicate only a difference between 

sampling stations near the waste site and reference sites. Taken together and in conjunction 

with the sediment data, they present strong evidence that metals are being released from the 

waste site and are causing bioeffects in the adjacent river. This approach is often referred to 

as a 'preponderance of evidence.’ 

So far this discussion has only dealt with general questions that could relate to any waste 

site, but before a study design can be completely developed, questions specifically related to 

the waste site of concern and the techniques to be employed must be formulated. It is at this 

early stage of question formulation that decisions should be made with regard to the type of 

statistical analysis to be performed on the collected data. Statistical analysis selection 

should be driven by the biological concerns at the site, not the other way around (Skellam, 

1969; Green, 1979). However, the precise design of a study must be appropriate for the 

desired statistical analysis. More information on question specificity and statistical analysis 

will follow in the section on statistical analysis. 

Reconnaissance Survey 

Before finalizing a study design, a reconnaissance survey, which should include some 

preliminary sampling, should be conducted. A reconnaissance survey provides information 

on the types of habitats present, sources of pollution (point and/or nonpoint), access points, 

and any other factors that may influence either the natural community or the ability to 

sample those communities. A reconnaissance survey can also be used to determine the best 

sampling device for the habitats of concern. In addition, if the objective of the study is to 

determine if there is a significant difference among stations, then preliminary samples can 

be collected for determining the sample size required for a particular level of statistical 

 7-5 November 1992 
HAZMAT 93-1–Study Design and Statistical Analysis 

power (Gilbert, 1987; Green, 1979). While a reconnaissance survey may appear to add time 

and expense to the overall study, it more often than not will save both (Green, 1979). It 

would be exceedingly costly, in both time and money, to conduct a full-scale bioassessment 

sampling program only to find out the organism you were trying to sample did not exist at 

the site or you were in the tidally influenced section of a river and your upstream reference 

station was actually impacted by the site and could not be used as a reference station. Either 

of these problems, as well as many others, could cause you to completely redo your 

sampling and could have been avoided by conducting a reconnaissance survey. 

Station Selection 

The selection of inappropriate stations can reduce the efficiency of the sampling devices and 

the data analysis and interpretation, thereby leading to misinformation and poor decisions 

about the health of an aquatic ecosystem and future actions to be taken at a site. The 

selection of appropriate stations will minimize the influences of the natural variability that 

occurs in aquatic ecosystems and increase the confidence for making specific statements 

about biological integrity. 

A wide variety of non-contaminant related abiotic factors, which can influence the results of 

bioassessment studies, should be considered when designing a study. The combination of 

physical and chemical factors can determine whether a particular taxa will successfully 

inhabit a given area or whether an organism will survive a toxicity test irrespective of 

human influences, i.e., anthropogenic contamination. All sampling stations to be compared 

should be as similar as possible with respect to all physical and chemical parameters, (e.g., 

sediment grain size, TOC, salinity, current velocity, and depth) excluding contamination. 

Physical and chemical factors of concern should be measured at each station to determine 

any abiotic differences among stations. This is necessary to determine whether biological 

differences between stations are caused by differences in contaminant levels or by some 

other abiotic factor. 

The number and locations of stations will depend on the objective of the study, the nature of 

the aquatic system, and the funds available for the assessment. Stations should be 

positioned in similar habitats with a spatial distribution that will permit an assessment of 

background conditions as well as cover a gradient from the most-to the least-contaminated 

areas. A minimum of one station should be located where contamination is expected to be 

the highest, for example, in a stream one station location should be just downstream of the 

hazardous waste site. Several stations may be required to identify the area of highest 

November 1992 7-6 
HAZMAT 93-1–Study Design and Statistical Analysis 

contamination, because of high spatial variability of contamination resulting from 

incomplete mixing of contaminants and water or sediments. Therefore a haphazardly 

placed station may fall outside the contaminant plume. In attempting to determine a 

gradient, stations should be spaced farther apart as one moves away from the site. This 

spacing will permit a clearer delineation of the extent of biological effects and identification 

of “recovery” zones. Particularly in the case of benthic community studies, stations should 

not be located in areas influenced by atypical conditions or structures, such as bridges, 

channelized areas, dredging activities, or culverts. To facilitate interpretation of 

relationships among the data, stations used for bioassessment methodologies should be the 

same as those used for water or sediment chemistry. 

When selecting a habitat for establishing stations, it is important to consider whether the 

benthic invertebrate community being sampled is being exposed to the contaminant of 

concern. One of the major areas of disagreement in station location, related to benthic 

community studies in streams, is whether they should be located in a riffle (erosional 

habitat) or pool (depositional habitat). Traditionally, the majority of benthic invertebrate 

monitoring has focused on riffle communities due to ease of sampling, increased sampling 

precision, higher species diversity, presence of pollution-sensitive taxa, and knowledge of 

riffle communities and how they respond to stress. This bias towards sampling riffle 

communities may have inherent problems in the field of contaminant assessment. Because 

contaminants can be transported over a riffle habitat to settle out in a depositional area, 

invertebrates in riffle habitats may not be exposed to contaminants in the same way that 

invertebrates in pool habitats are exposed. For bioassessments at hazardous waste sites, 

pool habitats will have a higher concentration of contaminants and benthic community data 

would be more easily correlated with sediment chemistry than in riffles. Therefore, pool 

habitats would be the preferred habitats to sample, however, an optimal design would be to 

sample benthic invertebrate communities in both riffle and pool habitats. 

Reference stations are critical to assess the biological condition of aquatic systems in the 

vicinity of a hazardous waste site. An optimal sampling design would be to have the 

number of reference stations equal the number of treatment stations (i.e., potentially 

impacted stations). As this is not practical for most studies, a minimum design should 

include two reference stations. The reference stations should be located in an area not 

influenced by the site or any additional sources of contamination and should be as similar to 

treatment stations in all other respects as possible. A single reference station should not be 

used since it can be misleading if it does not truly represent background conditions. If 

 7-7 November 1992 
HAZMAT 93-1–Study Design and Statistical Analysis 

reference stations are not available in the same aquatic system (i.e., same stream or estuary), 

select stations from a reference system that is physically and biologically similar to the 

system of interest. This could be a stream in an adjacent watershed or one in the same 

ecoregion. 

Random versus Non-random Designs 

Sample locations can be determined by either random or nonrandom means. Random 

sampling is used when quantitative data is required for statistical analysis; whereas 

nonrandom sampling is sufficient for qualitative or semiquantitative sampling. 

Two types of random sampling designs are simple random and stratified random sampling. 

Simple random sampling is when every unit of the population has an equal chance of being 

sampled. This is done by using random numbers to select sample locations. Simple 

random designs are not often used in waste site studies because contamination at the site is 

invariably heterogeneously distributed throughout the environment, therefore the relatively 

small number of samples or replicates taken at most sites might miss the areas of highest 

contamination. This heterogeneity of spatial distribution also applies to benthic organisms 

whose presence at any one location is dependent on physical factors such as substrate, 

current, etc. Therefore, the results of simple random sampling at a hazardous waste site 

may not be representative of conditions at the site. To characterize a Superfund site based 

on simple random sampling, the sample size must be greatly increased above what is 

currently the norm at these sites. 

Stratified random sampling is preferred in bioassessment studies at hazardous waste sites 

because it increases sampling efficiency by dividing the habitat into similar strata thus 

reducing natural variability between stations. Physical features commonly used to stratify 

an aquatic system are substrate, depth, flow, and duration of submergence (in tidally 

influenced areas). Several substrata can also be used to ensure a complete assessment of the 

entire aquatic system of interest, but only similar strata should be compared to determine 

differences that might be caused by contamination. After the system is stratified, the 

number of samples can be proportioned among the various strata based upon areal 

coverage or taken from a single strata. 

If quantitative data are not required for a study, then a nonrandom sampling program can 

be used. A nonrandom design should not be equated with an inadequate sampling design. 

The design used should depend on the question of interest, which may or may not require a 

statistical design. Nonrandom designs can either consist of systematic or nonstructured 

November 1992 7-8 
HAZMAT 93-1–Study Design and Statistical Analysis 

sampling. Systematic sampling involves using transects or grids with samples collected at 

predetermined or random intervals across the transect/grid. This type of sampling can be 

used to determine if there is a gradient of change away from a source of contamination; 

however, some consider it nonrandom and therefore there are inherent limitations on the 

type of data analysis that can be performed. 

Samples can also be collected using a nonstructured sampling program where there is no 

predetermined pattern. This approach provides strictly qualitative data.. The Rapid 

Bioassessment Protocol is technically a nonrandom design, the method states that samples 

should be collected from riffle and run habitats only, therefore there is a type of 

stratification to the design (see Chapter 6, EPA’s Rapid Bioassessment Protocol). 

Sample Replication 

For quantitative bioassessments, replicate samples need to be taken for each time and 

location sampled. Because, to determine that the differences between sampling stations or 

areas of concern are real, the within station or area variability needs to be determined. 

Within station variability is determined by the variability of the replicate samples. For the 

simplest and most straight forward statistical analysis, an equal number of randomly 

allocated replicate samples should be taken for each time and location sampled. 

One commonly occurring problem in environmental studies is pseudoreplication. 

Pseudoreplicates are not independent replicates of the population they are claimed to 

represent. The easiest way to understand the concept is through an example. A simple 

example is the case of subsampling. Suppose you took a single grab sample from each of 

two sites (X and Z) and then took three random subsamples from each grab for analysis. 

The sampling methodology in and of itself is not in error, but now suppose you try to 

statistically compare the two sites based on the analysis of these subsamples. Statistical 

comparison tests assume that the variability between the replicates of the samples being 

compared are representative of the within population variability (i.e., between replicate 

variability in sample X is representative of within population variability at site X) of the 

populations being compared. For this to be true, the replicate samples must be independent 

of each other, i.e., the location of any one replicate sample must not influence the location of 

any other replicate sample However, the subsamples that were analyzed actually only 

represent the variability within the individual grabs, not within the entire population at the 

site. The subsamples of the grabs are true sample replicates for the individual grabs, i.e., 

they are random independent observations comprising samples of the statistical 

 7-9 November 1992 
HAZMAT 93-1–Study Design and Statistical Analysis 

populations consisting of all possible observations of the individual grabs. However, they 

become pseudoreplicates if used to compare the two sites because they lack independence 

with respect to the site population; they came from the same grab. Therefore you can 

statistically compare grab X to grab Z but you cannot statistically compare site X to site Z 

based on the subsamples. 

As a second example of pseudoreplication, assume you wish to determine whether or not 

submergence time affects Pb levels in mussels from the Bay of Belfalas where the average 

tidal range is three meters. You decide to sample mussels from one meter below mean high 

tide (BMHT) and from two meters BMHT. The hypothesis you wish to test would be: there 

is no significant difference in lead concentrations in mussels from the two depths. You then 

go out to Mussel Rock and with the aid of a random number table you take eight replicates 

of mussels from one meter BMHT; then you proceed to Bird Rock and in the same manner 

take eight replicates of mussels from two meters BMHT. After performing an appropriate 

statistical analysis you declare that lead concentrations in mussels from the Bay of Belfalas 

are higher at two meters BMHT than at one meter BMHT with α=0.05. 

By stating that the mussels from the one-meter BMHT on Mussel Rock were representative 

of the entire population of one-meter mussels in the bay and that those from Bird Rock were 

representative of the entire population of two-meter mussels in the bay you have committed 

the error of basing your conclusions on pseudoreplicates. The replicates taken from one 

meter BMHT were independent representatives of the population of one-meter mussels on 

Mussel Rock, but were not independent representatives of the baywide one-meter mussel 

population. Likewise the two-meter Bird Rock replicates represented the two-meter 

population on Bird Rock, not the two-meter baywide population. You can validly conclude 

from your data that mussels from two meters BMHT on Bird Rock have higher 

concentrations of lead than do mussels from one meter BMHT on Mussel Rock. You can't 

conclude that depth is a factor in the difference because location may be a factor; Bird Rock 

may be located near a point source and all the mussels there may have elevated Pb levels. If 

both samples were taken from Mussel Rock you could relate the difference to depth, but 

you still couldn't relate the results to baywide conditions. In order to draw baywide 

conclusions, the replicates for the two depth samples must be randomly selected from 

throughout the bay not from just one or two locations. As Hurlbert (1984) states: 

"Pseudoreplication thus refers not to a problem in experimental design . . . per se but rather 

to a particular combination of experimental design . . . and statistical analysis which is 

inappropriate for testing the hypothesis of interest." 

November 1992 7-10 
HAZMAT 93-1–Study Design and Statistical Analysis 

The previous example on pseudoreplication raises another problem, which is really more a 

sampling design problem than an actual statistical analysis problem, although the solution 

does effect the type of statistical analysis used. The example suggested that Bird Rock 

might be near a point source and that this was the cause of the difference between samples. 

When using relatively small sample sizes, completely independent random selection of the 

two sets of sample replicates could result in the sample being unduly influenced by a single 

replicate being located near a point source. If Pb concentrations really do increase with 

depth and the replicate influenced by the point source was from the shallower sample, then 

no difference might be indicated by the test results. Probably the easiest solution in this case 

would be to pair the replicates; randomly select sites throughout the bay then take a onemeter and two-meter sample at each site. The statistical analysis used would be designed 

for paired replicates. 

STATISTICAL ANALYSIS 

In the past many environmental biologists have had a fear and distrust of statistics; this was 

in large part probably due to the backwards approach to statistical analysis that was often 

taken in environmental studies. These biologists would too often conduct a study, 

compiling quantities of data only to find that it could not be properly analyzed statistically. 

Because a data set cannot be statistically analyzed does not mean that it contains no useful 

information, but if data is to be statistically analyzed, determine the type of analysis to be 

performed during the early planning stages of the study not upon the completion of data 

collection. 

The first step in designing a scientific study is to determine what question or questions you 

want the study to answer. The questions you want answered will determine whether 

statistical analysis is necessary, and, if so, the appropriate type of statistical analysis needed. 

If statistical analysis is desired then the sampling plan must insure that the data collected is 

quantitative. To answer questions regarding the existing conditions at a site, descriptive 

statistics would be sufficient. Descriptive statistics include things like: means, medians, 

standard deviations, and ranges. However, to make predictions or inferences about 

conditions at the site, inductive statistics are required. Inductive statistics may use 

descriptive statistics to permit the determination of whether conditions at a waste site are 

different from those at a control site with a known probability (as in the case of t-tests). 

Inductive statistics may utilize the raw data to determine the probability of relationships or 

dependencies between different parameters, as in correlation and regression analyses. The 

 7-11 November 1992 
HAZMAT 93-1–Study Design and Statistical Analysis 

key word with regard to inductive statistics is probability. Inductive statistics tell the 

probability of something being the case; they do not tell whether or not it is the case. 

There are some instances when descriptive statistics are sufficient for the task at hand. For 

example, if the mean concentration of Hg in lobster muscle at a site is two parts per million 

(ppm) and the U. S. Food and Drug Administration limit for seafood is one ppm, you know 

that lobsters at the site represent a potential human health risk. Most of the time 

bioassessment studies will be concerned with whether or not a hazardous waste site has 

produced a significant adverse impact on the environment and to what extent this impact 

exists. Inductive statistics will permit you to answer these questions with a specified 

probability of being right. 

Hypothesis Formulation and Testing 

Before determining the type of inductive statistics best suited to answer your question, you 

need to make your question as specific as possible. Your general question may be, 'does the 

hazardous waste have any potential impact on the local aquatic environment?' You may 

decide to test for impact by conducting a bioaccumulation study with mussels. Your 

question would then become, 'do mussels at the site have higher levels of contaminants than 

do mussels not influenced by the site (e.g., at a reference site)?' Once you have posed the 

question specifically, including the type of test or study to be conducted, it needs to be put 

in the form of a null hypothesis (H0). A H0 is one we hope to reject in favor of an

alternative hypothesis (Ha) with a known probability of being correct. However, failure 

to reject H0 only implies you have no statistically valid evidence to believe otherwise. A H0

can be disproven with a known probability but cannot be proven. 

In the above Bay of Belfalas mussel example the H0 would be: there is no difference between 

contaminant levels in mussels at the site and the control site. A statistical way of phasing 

the H0 would be that the samples are from the same parent population or are from different 

parent populations with the same population mean. A possible Ha would be there is a 

difference, or statistically speaking, the samples are not from the same parent population or 

the parent populations have different means. The most common H0 in bioassessment 

studies is a no difference or no effect hypothesis. 

Testing a H0 is generally accomplished by using one of two types of statistical methods: 

parametric or nonparametric. Parametric methods are concerned with the 

characteristics of population parameters (e.g., population mean and variance), while 

nonparametric methods are not dependent on the characteristics of population parameters 

November 1992 7-12 
HAZMAT 93-1–Study Design and Statistical Analysis 

for their validity. Parametric tests require that certain assumptions pertaining to the 

population parameters be met for the tests to be valid. These assumptions include that the 

samples are drawn from a normally distributed population(s), the parent populations have 

the same variance, and the mean and variances are independent (i.e., the size of the variance 

is independent of the size of the mean). These assumptions can (if sample size is sufficiently 

large) and should be tested prior to applying any statistical tests to your data. Green (1979) 

describes in detail an appropriate approach for such testing, and today, many computer 

statistical software packages will test for these assumptions. 

Populations in environmental studies rarely meet these conditions. One alternative is to 

transform the data in such a manner that the conditions are met and then perform a 

parametric test on the transformed data. The most common transformations are logarithmic 

or natural logarithmic. However, transformations are not a cure all and it must be kept in 

mind that if the sample statistics calculated from the transformed data are transformed 

back, the resulting sample statistics will be different than those calculated from the 

untransformed data. For example: Hg concentrations in three mussels from the Bay of 

Belfalas were 2.5, 3.7, and 1.2 with a mean concentration of 2.47; if the concentrations are log 

transformed they become 0.398, 0.568 and 0.079 with a mean of 0.348. When the antilog of 

0.348 is calculated it is 2.23, not the 2.47 arithmetically derived from the original data. 

Transformations are one more step removed from reality and must be used with caution. 

The parametric tests most commonly used in environmental studies are the Student's t-test 

for the comparison of two samples and ANOVA for the comparison of multiple samples 

(Table 7-1). 

If the parametric test conditions are not met and transformation is undesirable, then 

nonparametric tests can be considered. Nonparametric tests are essentially independent of 

the population parameters. While nonparametric tests are not quite as precise as parametric 

tests when parametric test conditions are met, they lose little precision when these 

conditions are not met, and may therefore be preferable to parametric tests of transformed 

data. Nonparametric tests involve less complex calculations than do parametric tests and 

are also available in commercial software packages. It is perfectly legitimate to apply 

nonparametric tests to data that meet parametric test conditions as long as it is kept in mind 

that the results are slightly less precise. Commonly used nonparametric tests for 

environmental studies are the Mann-Whitney U-test and Kolmogorov-Smirnov two-sample 

test for comparing two samples and the Kruskal-Wallis test for multiple samples (Simenstad 

et al., 1991) (Table 7-1). 

 7-13 November 1992 
HAZMAT 93-1–Study Design and Statistical Analysis 

Because you are dealing with probabilities and not absolutes, there is still the possibility that 

you might reject the H0 when in fact it was true, a type I error; or you might fail to reject 

H0 when it is false, a type II error. At hazardous waste sites the most common form of H0

is: there is no difference between conditions (e.g., contaminant concentrations, benthic 

Table 7-1. Examples of parametric and nonparametric tests for the comparison of samples 

adapted from Elliott (1977) (see Elliott for examples of applications of the tests). 

Conditions Parametric Test 

Transformatio

n necessary 

Nonparametric Test 

Comparison of two samples 

 A. Means of large samples (n>50) Normal deviate (d) no U-test 

Kolmogorov-Smirnov 

 Small samples (n<50) from a: 

 1. random distribution 

 2. contagious distribution 

d for Poisson 

t-test 

no 

yes 

U-test 

Kolmogorov-Smirnov 

U-test 

Kolmogorov-Smirnov 

 B. Variances of large samples (n>50) 

 Small samples (n<50) 

F-test 

F-test 

no 

yes 

Comparison of three+ samples from 

random or contagious distributions 

ANOVA Yes Quenouille 

Kruskal-Wallis 

Friedman 

Correlation between two variables 

(dependent variable y and independent 

variable x) 

Correlation coefficient

Regression analysis 

Yes (x & y) 

Yes (y only) 

Rank correlation 

 coefficient 

Correlation between three+ variables: 

 Variables analyzed in pairs 

 Variables analyzed simultaneously 

Regression analysis 

Multiple regression 

Yes (y only) 

Yes 

November 1992 7-14 
HAZMAT 93-1–Study Design and Statistical Analysis 

community structure, etc.) at the waste site compared with those at a clean reference site. If 

this H0 is true then the waste site is clean and no remediation needs to be performed; to the 

contrary, if it is proven to be false then possibly very expensive remediation would have to 

be perform. As trustee for certain natural resources, NOAA would like the H0 to be true 

(i.e., the site is clean and no threat to NOAA's resources). However, NOAA would prefer to 

err on the conservative side and commit a type I error, saying that a clean site is 

contaminated and needs to be cleaned, rather than commit a type II error, saying that a 

contaminated site is clean thus leaving the contamination in place. Polluters would prefer 

to commit a type II error because it would mean that they would not have to spend any 

money on remediation even though the site was actually contaminated. 

The results of both parametric and nonparametric tests are generally reported with a 

probability value (α or p), usually 0.1, 0.05, or 0.01. These values represent the probability of 

committing a type I error, rejecting the H0 when it is true. If the statistical test indicates that 

the samples are significantly different (the H0 is rejected) with α=0.05 then based on the 

available evidence there is still a 1 in 20 or 5 percent chance that the samples are not 

significantly different (the H0 is true). If a 5 percent chance of being wrong in rejecting the 

H0 is too great, then α can be lowered to 0.01. However, by reducing the probability of 

committing a type I error, without changing the sampling design, you increase the 

probability of committing a type II error, accepting the H0 when it is false. The probability 

of committing a type II error is expressed as 'β'. The power of a test is the probability of 

rejecting the H0 and accepting the Ha when the H0 is in fact false; it is represented as '1-β'. 

Power is an important concept, however, it can only be calculated for a specific Ha (e.g., Hg 

concentrations at Site 1 are twice as high as at the control site) not a general one (i.e., there is 

a difference between the sites). NOAA would prefer a powerful test with a concurrently 

high α and low β. 

Whether you use parametric or nonparametric tests, any comparison of samples is 

dependent on a knowledge of the variability within the individual samples. To accomplish 

this, each sample must consist of a minimum of three and preferably more sample 

replicates. The more replicates per sample the smaller the detectable difference for a given 

value of α. Increasing the number of replicates is also one way of reducing the probability 

of both type I and type II errors. Since unlimited sampling is extremely impractical with the 

limited resources available for bioassessment studies, you need to determine the most 

efficient allocation of your sampling resources (e.g., ten sampling sites with three replicates 

or three sites with ten replicates per site). Formulas for determining the ideal sample size 

 7-15 November 1992 
HAZMAT 93-1–Study Design and Statistical Analysis 

based on the desired type of analysis, degree of precision and costs can be found in texts 

(e.g., in Gilbert, 1987) or by consulting a statistician. 

Randomization 

Precision is a measure of the closeness of agreement of individual measures of the same 

quantity (closeness of replicate values), while accuracy is the closeness of a measured or 

computed value to its true value (closeness of sample mean to the true population mean) 

(Sokal and Rohlf, 1983). It is possible to have precision without accuracy with a biased 

sampling methodology, but it would only be pure chance to have accuracy without 

precision. If your sampling methodology called for a four-centimeter mesh size for 

determining the size of a fish population based on the number of fish caught per unit of 

effort, then your sample would be biased for fish too large to pass through four-centimeter 

mesh. While your sampling may be precise, i.e., you catch roughly the same number of fish 

per unit effort in each replicate, your conclusions about the size of the fish population 

would not be accurate because your sampling methodology systematically excluded the 

smaller fish from your calculations. 

Statistical analyses take into account random error but not systematic error. Systematic 

error, or bias, is the systematic distortion of a statistic due to sampling methodology. 

Therefore, for a given value of α to be an accurate indication of potential error, the samples 

must be unbiased representations of their respective populations. To fulfill this 

requirement, the selection of any particular replicate must be independent of the selection of 

any other replicate; in other words, the population must be randomly sampled. Random 

selection does not mean haphazard selection; each sample replicate must be selected 

"...using a consistent, standardized technique" (Simenstad et al., 1991). While the selection 

of each replicate must be random, the treatment (e.g., how it is taken and how it is 

measured) of each replicate must be identical. The importance of replication and 

randomization are summed up by Hurlbert (1984): "Replication reduces the effects of 'noise' 

or random variation or error, thereby increasing the precision of estimates. . . Randomization 

eliminates possible bias on the part of the experimenter, thereby increasing the accuracy of 

such estimates. 

November 1992 7-16 
HAZMAT 93-1–Study Design and Statistical Analysis 

Correlation and Regression Analysis 

So far we have been discussing how to compare populations to determine whether or not 

they are different. Suppose the question you wish to ask is, “does the concentration of lead 

in mussels from the Bay of Belfalas decrease with increasing distance from the Mordor, Inc. 

lead smelter?” There are two approaches you can take to this problem: you can take 

samples of n replicates each at various distances from the smelter and use some comparison 

test to test for differences, or you could perform either a correlation or regression analysis 

on the individual replicates or the sample means with respect to distance from the smelter. 

A correlation analysis determines whether changes in two or more variables are related. 

A regression analysis determines whether changes in a dependent variable are the result 

of changes in an independent variable. Correlation or regression analyses do not require 

multiple replicates at each distance nor do they require random selection of sampling 

locations. At the same time they do not prove that a relationship exists; they only suggest 

that it does. The easiest approach to determine an environmental gradient is to establish a 

transect in the direction of concern (e.g., downstream in rivers and streams) and select 

sampling locations increasingly farther apart as they move away from the site (e.g., 10, 25, 

50, 100, 200, 500, and 1000 meters). Then take samples consisting of one random replicate at 

each distance and perform a regression analysis of Pb concentrations with distance from the 

smelter. As with the comparison tests, the larger the sample size the smaller the detectable 

gradient. 

SUMMARY 

Here are a few final thoughts on the place of statistical analysis in bioassessment studies. 

The design of bioassessment studies should be driven by the question or questions you 

want to answer, not by the statistical test you want to perform. Statistical analysis is a tool 

to help you properly interpret collected data. It is not a magic wand to turn bad data into 

good answers. Hypothesis testing only tells you the probability of the difference between 

samples being real or being due to the natural variability of the populations sampled. It 

cannot tell you whether or not the statistical difference is ecologically significant. Finally, if 

you have to design a bioassessment study and you are not comfortable with statistical 

analysis, consult a biostatistician before you conduct your study, not after. 

 7-17 November 1992 
HAZMAT 93-1–Study Design and Statistical Analysis 

Table 7-2 presents ten principles in the design of an environmental study as set forth by 

Green (1979). These are the same principals, expressed in a slightly different format as have 

been covered in the preceding discussion of bioassessment study design. 

Table 7-2. Principals of environmental study design from Green (1979).

1. Be able to state concisely to someone else what question you are asking. Your results 

will be as coherent and as comprehensible as your initial conception of the problem.

2. Take replicate samples within each combination of time, location, and any other 

controlled variable. Differences among can only be demonstrated by comparison to 

differences within. 

3. Take an equal (preferably) number of randomly allocated replicate samples for each 

combination of controlled variables. Putting samples in "representative" or "typical" 

places is not random sampling. 

4. To test whether a condition has an effect, collect samples both where the condition is 

present and where the condition is absent but all else is the same. An effect can only be 

demonstrated by comparison with a control. 

5. Carry out some preliminary sampling to provide a basis for evaluation of sampling 

design and statistical analysis options. Those who skip this step because they do not 

have enough time, usually end up losing time. 

6. Verify that your sampling device or method is sampling the population you think you 

are sampling, and with equal and adequate efficiency over the entire range of sampling 

conditions to be encountered. Variation in efficiency of sampling from area to area 

biases among-area comparisons. 

7. If the area to be sampled has a large-scale environmental pattern, break the area up into 

relatively homogeneous subareas and allocate samples to each in proportion to the size 

of the subarea. If it is an estimate of total abundance over the entire area that is desired, 

make the allocation proportional to the number of organisms in the subarea. 

8. Verify that your sample unit size is appropriate to the size, densities, and spatial 

distributions of the organisms you are sampling. Then estimate the number of replicate 

samples required to obtain the precision you want. 

9. Test your data to determine whether the error variation is homogeneous, normally 

distributed, and independent of the mean. If it is not, as will be the case for most field 

data, then (a) appropriately transform the data, (b) use a distribution-free 

(nonparametric) procedure, (c) use an appropriate sequential sampling design, or (d) 

test against simulated H0 data. 

10. Having chosen the best statistical method to test your hypothesis, stick with the result. 

An unexpected or undesired result is not a valid reason for rejecting the method and 

hunting for a "better" one. 

November 1992 7-18 
HAZMAT 93-1–Study Design and Statistical Analysis 

REFERENCES 

Eberhardt, L.L. and J.M. Thomas. 1991. Designing environmental field studies. Ecological 

Monographs 61(1). pp 53-73. 

Elliott, J.M. 1977. Statistical analysis of samples of benthic invertebrates. Freshwater 

Biological Association Scientific Publication No.25. 2nd ed. Kendal, Titus Wilson & Son Ltd. 

160 pp. 

Gilbert. R.O. 1987. Statistical Methods for Environmental Pollution Monitoring. New York, 

Van Nostrand Reinhold Co. 330 pp. 

Green, R.H. 1979. Sampling Design and Statistical Methods for Environmental Biologists. 

New York, J. Wiley & Sons. 257 pp. 

Hurlbert, S.H. 1984. Pseudoreplication and the design of ecological field experiments. 

Ecological Monographs, 54(2), pp 187-211. 

Simenstad, C.A., C.D. Tanner, R.M. Thom, and L.L. Conquest. 1991. Estuarine habitat 

assessment protocol. Seattle, U.S. Environmental Protection Agency Region 10, Office of 

Puget Sound. 212 pp. 

Skellam, J.G. 1969. Models, inferences, and strategy. Biometrics 25. 457-475. 

Sokal, R.R. and F.J. Rohlf. 1983. Biometry. 2nd ed. New York. W.H. Freeman and Co. 859 

pp. 

 7-19 November 1992 
HAZMAT 93-1–Study Design and Statistical Analysis 

November 1992 7-20 
HAZMAT 93-1–Protocols 

CHAPTER 8 

SUMMARY OF RECOMMENDED 

BIOASSAY PROTOCOLS 

INTRODUCTION 

Summaries of recommended bioassay protocols and protocol documents are presented 

in this chapter to provide a brief overview of each test procedure. Table 8-1 presents the 

various protocols by media tested (soil, water, or sediment) and habitat of origin 

(freshwater, brackish water, or marine). As can be seen from Table 8-1, several of the 

protocols are applicable to more than one combination of media and habitat. Because of this 

multiple use of the various protocols, the arrangement of the summaries below into the 

categories of soil, water, and sediment is only approximate. 

SOIL 

Lettuce Seed (Latuca sativa) Germination Bioassay (Thomas and Cline, 

1985). 

The lettuce seed germination test is both rapid and economical and has been the 

preferred test to determine and evaluate phytotoxicity of contaminants in upland soils. 

However, seed germination is relatively insensitive to chemical insult because it is 

essentially a self-contained unit that doesn't need external nutrients for germination 

(Kaputska, personal communication). Growth after germination is far more sensitive to 

contamination. The lettuce seed germination and initial plant growth test protocol consist 

of taking 100 grams of air-dried test soil mixed with 50 grams of washed 16 mesh screened 

silica sand. Place the mixture in the bottom half of a 150-mm plastic petri dish and seeded 

with 40 lettuce (Latuca sativa) seeds. Place 35 milliliters of distilled water onto the soil-sand 

mixture and spread 50 grams of silica sand evenly over the dampened soil and seed. Place 

the petri dish ion the bottom of a 0.1-mm polyethylene bag expanded to contain air, closed 

at the top with a rubber band, and incubated in a controlled-environment chamber. Seed 

germination is determined visually by counting the shoot spikes when they extend 1 cm 

above the soil’s surface. Stem lengths are determined by measuring each plant with a ruler 

after 10 days. In most cases, overall germination appears to be the most sensitive, but there 

are contaminants that will permit germination but do not allow shoot growth. 

 8-1 August 1997
HAZMAT 93-1–Protocols 

Table 8-1. Bioassay tests by habitat and media. Media Habitat 

Test Organism Species 

Indigenous Region 

for Species Protocol References 

Contaminant 

Sensitivity 

Mtl Pest. PCB PAH 

Acute 

vs 

Chronic 

Lab time & 

analysis 

duration Endpoints Availability Comments

SOILS Upland 

Eisenia foetida[C. Oligochaeta-worm] 

Common in U.S. Soils 

w/considerable organic 

matter; sewage beds, 

trickling filters 

Porcella. 1983 (19) 7,14D=LT

AT= 

Survival Cocoons commercially

purchased; can be cultured in lab 

Worms naturally occur in soils rich in organic materials. Worms may be protected from toxic chemicals due to adsorption of toxins to soil particles and organic matter (19). 

Latuca sativa[Lettuce] 

Thomas and Cline. 1985 (25) 

10D=LT

AT= 

Germination In most cases, overall germination is most sensitive, but there are 

contaminants that will still permit germination but do not allow shoot growth (25). Seed germination least sensitive stage of plantdevelopment (9). 

WATER Fresh 

Ceriodaphnia dubia[O. Cladocera-water flea] 

Abundant in lakes, 

ponds, and quiescent sections of streams and rivers throughout N. 

America 

Horning, W. B. and C. I. Weber. 1985. (7) 

Y Y Y C 7D=LT

AT=2-3wks 

Survival/ Reproduction 

Purchase brood stocks from Newton, OH. (22) 

Elevated turbidity and dissolved substances problem (22); avoid sudden pH and temperature changes. 

Daphnia magna[O. Cladocera-water flea] 

Occur in nearly all types 

of freshwater habitats; lake & pond dweller, restricted to waters in N. & W N. America 

Porcella. 1983. (19) Y Y Y 48H=LT

AT=1-1.5wk 

Immobilization Cultures available from

commercial supply

houses & from government labs 

Pimephales promelas[O. CypriniformesFathead minnow] 

Warm-water ponds, lakes,& sluggish streams 

Horning, W. B. and C. I. Weber. 1985. (7) 

Y Y Y C 7D=-LT

AT=2-3wks 

Survival/ Growth Acquire from Labs that 

is known to have disease-free fish; US EPA Environ. Research Lab @ Duluth, Minn. 

Selenastrum capricornutum Ubiquitous in most 

freshwaters in N. America 

Porcella. 1983. (19) Y Y 96H=LT

AT=2-3wks 

Growth (direct counting or mass) 

Strains recommended are ATCC 22662, UTEX1648, UTCC 37; commercial suppliersAmerican Type Culture 

Collection & Culture Collection of Algae 

August 1997 8-2
HAZMAT 93-1–Protocols 

Media Habitat 

Test Organism Species 

Indigenous Region 

for Species Protocol References 

Contaminant 

Sensitivity 

Mtl Pest. PCB PAH 

Acute 

vs 

Chronic 

Lab time & 

analysis 

duration Endpoints Availability Comments

SOILS Upland Brackish 

Daphnia magna[O. Cladocera-water flea] 

Occur in nearly all types 

of freshwater habitats; lake & pond dweller, restricted to waters in N. & W N. America 

Porcella. 1983. (19) Y Y Y 48H=LT

AT=1-1.5wk 

Immobilization Cultures available from

commercial supply

houses & from government labs 

Photobacterium phosphoreum[Luminescent bacterium] 

Marine bacterium Puget Sound Estuary 

Program. 1991. (20) 

N Y Y A 15,30min

AT=2-3wks 

Decreased luminescence 

Purchase from commercial sources; Strain # NRRL B-11177 deposited w/ Northern Regional Research 

Laboratory in Peoria, 

Illinois, USA 

Organic extraction method–specific for neutral, nonionic organic compounds (PAH, PCB); not for metals and highly acidic or basic organic materials. Saline extraction method specific for water-soluble fraction of sediment-adsorbed trace metals and organic pollutants fromsediments (20). 

Selenastrum capricornutum Ubiquitous in most 

freshwaters in N. America 

Porcella. 1983. (19) Y Y 96H=LT

AT=2-3wks 

Growth (direct counting or mass) 

EC90 , EC50 , SC20

Strains recommended are ATCC 22662, UTEX1648, UTCC 37; commercial suppliersAmerican Type Culture 

Collection & Culture Collection of Algae 

Microscope counting recommended.(19) 

Marine 

Crassostrea gigasMytilus edulis[C. Pelecypoda-clam;mussel]

Puget Sound region; bays, 

inlets, etc. of the Pacific coast; oyster range-Morro 

Bay, CA to N. British 

Columbia; mussel rangeArctic, N. Pacific, N. 

Atlantic 

Puget Sound Estuary Program. 1991. (20) 

Y Y Y A 48H=LT

AT=3wks 

Survival/ Abnormal larval development 

Field collection Not recommended for sediments with interstitial salinity less than

10ppt C. gigas spawns during summer; M. edulis during the spring 

and summer; recovery of larvae from sediments difficult (20). 

Photobacterium phosphoreum[Luminescent bacterium] 

Marine bacterium Puget Sound Estuary 

Program. 1991. (20) 

N Y Y A 15,30min

AT=2-3wks 

Decreased luminescence 

Purchase from commercial sources; Strain # NRRL B-11177 deposited w/ 

Northern Regional 

Research Laboratory in 

Peoria, Illinois, USA 

Organic extraction method specific for neutral, nonionic organic compounds (PAH, PCB); not for metals and highly acidic or basic organic materials. Saline extraction method specific for water-soluble fraction of sediment-adsorbed trace metals and organic pollutants from sediments (20). 

 8-3 August 1997
HAZMAT 93-1–Protocols 

Media Habitat 

Test Organism Species 

Indigenous Region 

for Species Protocol References 

Contaminant 

Sensitivity 

Mtl Pest. PCB PAH 

Acute 

vs 

Chronic 

Lab time & 

analysis 

duration Endpoints Availability Comments

SOILS Upland SEDIMENT Fresh 

Chironomus riparius[O. Diptera--midge] 

Distribution world-wide; thermophilous; standing 

water most spp.; 

eutrophic lakes, ponds, & streams; aquatic during

larval & pupal stages 

ASTM Designation E 

1706-95b (3) 

Y Y A 10D=LT

AT=2-3wks 

Survival and 

growth 

Lab stock culture or commercial source 

Ammonia accumulation (8). Avoid H2S2 if possible; over feeding 

leads to fungus contamination (3). 

Chironomus tentans[O. Diptera--midge] 

Holarctic distribution; mid-continental N. America; eutrophic lakes & ponds 

ASTM Designation E 

1706-95b (3) U.S. EPA 1994 TestMethod 100.2 (26) 

Y A 10D=LT

AT=2-3wks 

Survival, growth 

and emergence 

Lab stock culture or commercial source 

Affected by H2S2 > 0.3mg/l (ASTM 1706-95b); over feeding leads to

fungus contamination (3). 

Daphnia magna[O. Cladocera-water flea] 

Occur in nearly all types 

of freshwater habitats 

Nebeker et al., 1984 (15) Y 48H=LT

AT=1-1.5wk 10D=LTAT=1-1.5wk 

Survival Survival/ Reproduction 

Purchase brood stocksNewton, OH (5) and 

EPA Western Fish Tox. Station, Corvallis OR. (13) 

Hyalella azteca[O. Amphipoda] 

Entire American continent; permanent meso or eutrophic lakes supporting aquatic plants,

ponds, streams 

ASTM Designation E 

1706-95b (3) U.S. EPA 1994 TestMethod 100.1 (26) 

Y Y Y Y A

C 

10D=LTAT=2-3wks 30D=LTAT=2-3wks 

Survival/# of 

young/Growth 

Reproduction/ Behavior/Sexual development. 

Field: collected in freshwater lakes, 

ponds, and streams 

Recovery of young difficult (3). Caution in feeding to avoid bacterial and fungal growth on sediments (3). 

Photobacterium phosphoreum[Luminescent bacterium] 

Marine bacterium Puget Sound Estuary

Program, 1991. (20) 

N Y Y A 15,30min

AT=2-3wks 

Decreased luminescence 

Purchase from commercial sources; Strain # NRRL B-11177 deposited w/ Northern Regional Research 

Laboratory in Peoria, 

Illinois, USA 

Organic extraction method specific for neutral, nonionic organic compounds (PAH, PCB); not for metals and highly acidic or basic organic materials. Saline extraction method specific for water-soluble fraction of sediment-adsorbed trace metals and organic pollutants from sediments. (20) 

August 1997 8-4
HAZMAT 93-1–Protocols 

Media Habitat 

Test Organism Species 

Indigenous Region 

for Species Protocol References 

Contaminant 

Sensitivity 

Mtl Pest. PCB PAH 

Acute 

vs 

Chronic 

Lab time & 

analysis 

duration Endpoints Availability Comments

SOILS Upland 

Pimephales promelas[O. CypriniformesFathead minnow] 

Central part of the US, touches northern Mexico; 

not native west of the 

Rocky Mountains, nor the 

three most easterly

Atlantic provinces 

Horning, W. B , and C. 

I. Weber, 1985. (7) 

Y Y Y C 7D=-LT

AT=2-3wks 

Survival/ Growth EPA Fish Tox. Lab 

Newton, OH (24); ponds, lakes, ditches, & slow muddy streams 

Food added during tests may affect results; highly volatile/degradable toxins may not be detected (7); spawning in May or June in northern part of their range when average water temperatures are as low as 13 to 17°C

Brackish 

Ampelisca abdita[O. Amphipoda] 

Range from Maine to 

south-central Florida & eastern Gulf of Mexico; low intertidal zone to 60 m; San Francisco Bay; 

euryhaline 

ASTM Designation E 1367-92 (1) 

Y Y Y Y 10D=LT

AT=2-3wks 

Survival/ Growth 

Field: East Coast; Gulf of Mexico; SF Bay. 

Commercial: John Brezina, San Rafael, CA; SAIC Narragansett, 

RI 

Chronic tests best indicators of environmental impacts (6); Sensitive to coarse grain sediments; exposed to pore water also; flow-through tests recommended and very sensitive to O2 stress (4); aeration 

recommended to break up surface contamination (29) 

Crassostrea gigas Mytilus edulis[C. Pelecypoda-clam;mussel]

Puget Sound region; bays, 

inlets, etc. of the Pacific coast; oyster range-Morro 

Bay, CA to N. British 

Columbia; mussel rangeArctic, N. Pacific, N. 

Atlantic 

Puget Sound Estuary

Program, 1991. (20) 

Y Y Y A 48H=LT

AT=3wks 

Survival/ Abnormal larval development 

Purchase from commercial sources 

Not recommended for sediments with interstitial salinity less than10ppt C. gigas spawns during summer; M. edulis during the spring 

and summer; recovery of larvae from sediments difficult. Unknown high mortalities sometimes in controls (20). 

Hyalella azteca[O. Amphipoda] 

Entire American continent; permanent lakes, ponds, streams 

ASTM Designation E 

1706-95b (3) 

Y Y Y Y A

C 

10D=LTAT=2-3wks 30D=LTAT=2-3wks 

Survival/# of 

young/Growth 

Reproduction/ Behavior/ Sexual development 

Field: collected in freshwater lakes, 

ponds, and streams 

Feed cautiously to avoid bacterial and fungal growth on sediments (3). 

 8-5 August 1997
HAZMAT 93-1–Protocols 

Media Habitat 

Test Organism Species 

Indigenous Region 

for Species Protocol References 

Contaminant 

Sensitivity 

Mtl Pest. PCB PAH 

Acute 

vs 

Chronic 

Lab time & 

analysis 

duration Endpoints Availability Comments

SOILS Upland 

Leptocheirus plumulosus[O. Amphipoda] 

Chesapeake Bay & other 

East Coast estuaries; found in both oligohaline 

& mesohaline portions; Cape Cod to N. Florida;fine sand to very fine 

mud 

ASTM Designation 

E 1367-92 (1) Schlekat, C. E. et al. 1992. (23) McGee, B. L. et al. 1993. (14) 

Y A 10-d, 20-d, & 

28 or 30-d LTAT = 

Survival Field collectedChesapeake Bay, 

Chester River 

Tests--static (10-d) & static renewal (20-28-d). It appears that this amphipod is a generalist; able to thrive in sediments differing in particle size and organic content. Absence of salinity-related effects on the survivorship of L. plumulosus allows for testing of sediments at ambient salinities. Two broods--early to midspring & in the fall; timing may vary. Limitations: •pH needs to be monitored/adjusted by adding spring water (average pH, hardness, & alkalinity are 7.7, 110 ppm as CaCO3, and 100 ppm as CaCO3 respectively) to distilled water at a ratio of 1:4 (v/v). •Effects on numbers of young per female indicate the potential for the development of endpoints more sensitive than adult mortality. •Temperature should be @ 20°C; potential effects on toxicity. •Juveniles should be feed during long-term exposures; potential effects on toxicity. •Sediment organic carbon needs investigation-may have supplemented the amphipod nourishment. 

Photobacterium phosphoreum[Luminescent bacterium] 

Marine bacterium Puget Sound Estuary 

Program, 1991. (20) 

N Y Y A 15,30min

AT=2-3wks 

Decreased luminescence 

Purchase from commercial sources; Strain # NRRL B-11177 deposited w/ Northern Regional Research 

Laboratory in Peoria, 

Illinois, USA 

Organic extraction method specific for neutral, nonionic organic compounds (PAH, PCB); not for metals and highly acidic or basic organic materials. Saline extraction method specific for water-soluble fraction of sediment-adsorbed trace metals and organic pollutants from sediments. (20) 

Rhepoxynius abronius[O. Amphipoda] 

West coast N. Americafrom central California to Puget Sound, WA; clean, 

fine, sandy sediments; 

lower intertidal to 274 m

ASTM Designation 

E 1367-92 (1) 

Y Y Y 10D=LT

AT=2-3wks 

Survival/ Emergence/ Reburial 

Field: West Coast, central California to Puget Sound 

Fine sediments adversely affect; less than 10D tests not recommended (24); do not freeze sediments (18); aeration recommended to break upsurface contamination (29). Emergence and reburial not very sensitive (13) 

August 1997 8-6
HAZMAT 93-1–Protocols 

Media Habitat 

Test Organism Species 

Indigenous Region 

for Species Protocol References 

Contaminant 

Sensitivity 

Mtl Pest. PCB PAH 

Acute 

vs 

Chronic 

Lab time & 

analysis 

duration Endpoints Availability Comments

SOILS Upland 

Rhepoxynius abronius[O. Amphipoda] 

Same as ASTM 1367; fine sands low intertidal to 60 m 

Puget Sound Estuary

Program, 1991. (20) 

Y Y Y 10D=LT

AT=2-3wks 

Survival/ Emergence/ Nonreburial 

Field: West Beach, 

Whidbey Is., WA. 

Comm.: Yaquina Bay, 

OR (10) 

Interstitial salinity @ • 25ppt; grain size affects negative @ extremes of fine and coarse material. Sensitivity varies with seasons (20). Emergence and reburial not very sensitive (13) 

Crassostrea gigas Mytilus edulis[C. Pelecypoda-clam;mussel]

Puget Sound region; bays, 

inlets, etc. of the Pacific coast; oyster range-Morro 

Bay, CA to N. British 

Columbia; mussel rangeArctic, N. Pacific, N. 

Atlantic 

Puget Sound Estuary

Program, 1991. (20) 

Y Y Y A 48H=LT

AT=3wks 

Survival/ Abnormal larval development 

Field collection Not recommended for sediments with interstitial salinity less than

10ppt C. gigas spawns during summer; M. edulis during the spring 

and summer; recovery of larvae from sediments difficult (20). 

Dendraster excentricus[C. Echinoidea-sand dollar] 

Puget Sound region; 

sandy beaches @ low tide 

Puget Sound Estuary

Program, 1991. (20) 

Y Y Y A 48H=LT

AT=3wks 

Survival/ Abnormal larval development/ % fertilization 

Field collected on Puget 

Sound beaches during

low tide 

Spawn from April to October; recovery of larvae from sediments difficult. Not recommended for sediments with < 10ppt; unknown 

high mortalities might occur in controls (20). 

Strongylocentrotus drobaciensis[C. Echinoidea-green sea urchin] 

Puget Sound region; 

shallow subtidal zone 

Puget Sound Estuary 

Program, 1991. (20) 

Y Y Y A 48H=LT

AT=3wks 

Survival/ Abnormal larval development/ % fertilization 

Field collected on coast of Washington 

Spawn from December to April; recovery of larvae from sediments difficult. Not recommended for sediments with interstitial salinity less than 10ppt ; unknown high mortalities might occur in controls (20). 

Strongylocentrotus purpuratus[C. Echinoidea-purple sea urchin] 

Puget Sound region; 

intertidal zone 

Puget Sound Estuary

Program, 1991. (20) 

Y Y Y A 48H=LT

AT=3wks 

Survival/ Abnormal larval development/ % fertilization 

Field collected on coast of Washington 

Spawn from December to April; recovery of larvae from sediments difficult. Not recommended for sediments with interstitial salinity less than 10ppt ; unknown high mortalities might occur in controls (20). 

Eohaustorius estuarius[O. Amphipoda] 

N. American west coastfrom British Columbia to central California; low tide zone to 10 m; fine sand 

Puget Sound Estuary

Program, 19 91. (20) 

Y Y Y Y 10D=LT

AT=2-3wks 

Survival/ Emergence/ Nonreburial 

Field collected Interstitial salinities from 2-28ppt. Do not freeze sediments (6); 

seasonal factors (20). 

 8-7 August 1997
HAZMAT 93-1–Protocols 

Media Habitat 

Test Organism Species 

Indigenous Region 

for Species Protocol References 

Contaminant 

Sensitivity 

Mtl Pest. PCB PAH 

Acute 

vs 

Chronic 

Lab time & 

analysis 

duration Endpoints Availability Comments

SOILS Upland 

Eohaustorius estuarius[O. Amphipoda] 

N. American west coastfrom British Columbia to central California; low tide zone to 10 m; fine sand 

ASTM Designation E 

1367-92 (1) 

Y Y Y Y 10D=LT

AT=2-3wks 

Survival Field collected BC 

(Canada) to Central California 

Less sensitive than R. abronius (1). 

Grandidierella japonica[O. Amphipoda] 

San Francisco Bay & other

N. Calif. bays; sand, silt, 

or clay sediments 

ASTM Designation E 

1367-92 (1) 

Y Y Y 10D=LT

AT=2-3wks 

Survival/ Emergence/ Reburial 

Field collected northern California bays, SF Bay, 

and southern California 

bays. (16) 

Females carrying embryos should not be used in tests. Aeration recommended to break up surface contamination and abnormal swimming behavior affect results (18); grain size affected growth and molting affects survival results (16). Most reliable source, upper Newport Bay CA, is highly polluted reducing sensitivity of test (10) 

Rhepoxynius abronius[O. Amphipoda] 

West coast N. Americafrom central California to Puget Sound, WA; clean, 

fine, sandy sediments; 

lower intertidal to 274 m

ASTM Designation E 

1367-92 (1) 

Y Y Y 10D=LT

AT=2-3wks 

Survival/ Emergence/ Reburial 

Field: west coast, central California to Puget Sound 

Fine sediments adversely affect; less than 10D tests not recommended (24); do not freeze sediments (18); aeration recommended to break upsurface contamination (29). Emergence and reburial not very sensitive (12) 

Rhepoxynius abronius[O. Amphipoda] 

Same as ASTM 1367; fine sands low intertidal to 60 m 

Puget Sound Estuary

Program. 1991. (20) 

Y Y Y 10D=LT

AT=2-3wks 

Survival/ Emergence/ Nonreburial 

Field: West Beach, 

Whidbey Is., WA. 

Comm.: Yaquina Bay, 

OR (10) 

Interstitial salinity @ • 25ppt; grain size affects negative @ extremes of fine and coarse material. Sensitivity varies with seasons (20) Emergence and reburial not very sensitive (12) 

Photobacterium phosphoreum[Luminescent bacterium] 

Marine bacterium Puget Sound Estuary

Program. 1991. (20) 

N Y Y A 15,30min

AT=2-3wks 

Decreased luminescence Decreased luminescence 

Purchase from commercial sources; Strain # NRRL B-11177 deposited w/ Northern Regional Research 

Laboratory in Peoria, 

Illinois, USA 

Organic extraction method specific for neutral, nonionic organic compounds (PAH, PCB); not for metals and highly acidic or basic organic materials. Saline extraction method specific for water-soluble fraction of sediment-adsorbed trace metals and organic pollutants from sediments. (20) 

Neanthes arenaceodentata[C. Polychaeta-polychaete worm] 

Marine; world-wide distribution; N. England, Florida, Calif. Europe, & central Pacific ocean; Conn. & Calif. 

populations; estuarine intertidal sand or muddy

sand beaches, bays, 

harbors 

Puget Sound Estuary

Program. 1991. (20) 

ASTM Designation E 

1611-94 (2) 

Y Y 

Y Y Y A 20D=LT

AT=2wks 10D=LTAT= 

Survival/ Biomass & Avg. individual biomass/ Fecundity Survival (10-d) 

California species fromcommercial source; Reish (21). Environ. Division, U.S. 

Army Corp of 

Engineers, Vicksburg, 

MI 

Adjust for tests below salinities @ less than 20 ppt. Size of juveniles critical to test; do not freeze sediment (17) Survival and growth aboutequal sensitivity, reproductive fecundity (# and size of eggs in coelom) more sensitive (10) Chromosome numbers differ in populations of worms from California, Connecticut, and Hawaii--thus far, only the California worms have been used as test organisms; watch for avoidance to the sediments-replace worms if occurs. 

August 1997 8-8
HAZMAT 93-1–Protocols 

Media Habitat 

Test Organism Species 

Indigenous Region 

for Species Protocol References 

Contaminant 

Sensitivity 

Mtl Pest. PCB PAH 

Acute 

vs 

Chronic 

Lab time & 

analysis 

duration Endpoints Availability Comments

SOILS Upland 

Neanthes virens[C. Polychaeta- polychaete worm-sand- or clamworm] 

N. Atlantic Ocean fromArctic to Virginia, 

Iceland, Europe from British Isles, N. Sea, & France; shallow intertidal; fine sands, clay, peat & 

water soaked wood 

ASTM Designation E 

1611-94 (2) 

10D=LT

AT= 

Survival Field collection @ low 

tides from intertidalsand-mud flats 

Worm also known as Nereis virens can not be cultured in lab because 

sexual maturity is only a short period of time (hours) & their life-cycle is long (1-2 years). The protocols state that the mortality endpoint for N. virens is rather 

insensitive and thus makes the organism suitable for bioaccumulation studies. 

Oncorhyunchus mykiss[O. SalmoniformesRainbow trout] 

Gonad cells of rainbow trout; steelhead range-N. Baja, Bering Sea, Japan; 

steelhead trout rangecentral Calif. to coastal 

streams of Alaska 

Puget Sound Estuary

Program. 1991. (20) 

48H=LT

AT=3wks 

Anaphase aberration 

Media for culture from

commercial sources; Cells from trout gonads 

Specific for neutral, nonionic organic compounds (PAH, PCB); not for metals and highly acidic or basic organic materials. Natural genotoxicity may influence results (20). 

Key: LT = lab time (time for the actual protocols to be completed) 

AT = analysis time (time to complete the analysis and 

interpretation of data) (Times estimated by EVS, C. A. McPherson, Lab Supervisor 2/7/92) 

H = hour(s) 

D = day(s) Min = minutes Y = Yes, implying that test is sensitive to at least some

contaminants in this category 

N = No S = Some (#) indicates number of reference. 

 8-9 August 1997
HAZMAT 93-1–Protocols 

Earthworm (Eisenia foetida) Lethality Bioassay (Porcella, 1983). 

The earthworm lethality bioassay is used to determine toxicity in upland soils. 

Earthworms were selected as an indicator species because they are representative of the 

terrestrial environment and are of considerable importance in improving soil aeration, 

drainage, and fertility. Eisenia foetida is a species that generally occurs in soils with very 

high organic content. It is also a species with a short life cycle and can breed readily in a 

wide range of organic wastes. This means that a standard strain can be used because 

laboratories could easily breed their own stock if supplied with cocoons from a central 

source. In this test, Eisenia foetida must be at least two months old, with a clitellum, and 

weigh 400 to 800 milligrams. Worm survival is evaluated after a 7- or 14-day exposure to a 

mixture (3:1) of an artificial soil and the test soil. Since soil is such a variable medium, the 

addition of a carefully defined artificial loam soil provides a suitable growth medium for the 

earthworm. For each test, 400 grams of this test mixture containing ten worms is placed in a 

500-ml crystalizing dish and kept from dehydrating. Mortality is determined at the end of 

the test by emptying the soil into a tray, sorting out the worms and testing their reaction to 

mechanical anterior stimulus. 

WATER COLUMN 

Selenastrum capricornutum Algal Lethality Bioassay (Porcella, 1983).

The algal Selenastrum capricornutum bioassay is used to test the toxicity of freshwater 

samples. S. capricornutum is a unicellular, non-motile chlorophyte that is readily available 

and easily maintained. Unicellular algae are important producers of oxygen and form the 

basis of the food web in aquatic ecosystems. The alga S. capricornutum is added to test 

solutions containing various concentrations of the test material and growth is measured at 

96 hours. Test material solutions are made up with the test material diluted to the proper 

concentration to which an algal assay growth medium of macro- and micro-nutrients is 

added. Between 0.1 and 1 ml of six- to eight-day old S. capricornutum culture stock is added 

to the test container to achieve a concentration of 10,000 cells per ml at the beginning of the 

test. The test mixture is incubated for 96 hours and algal growth is measured by any of the 

following methods: electronic particle counting, biomass (dry weight), absorbance, or direct 

microscopic counts. Because the algal test is designed to provide a comparative response to 

varying dilutions of sample, it is better to use absorbance or an electronic particle counter to 

measure growth. Results are expressed in terms of the EC50. It is also recommended that 

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reference controls with a known toxic substance to check the sensitivity of the organisms be 

used in the test. 

Water flea Daphnia magna EC50 Bioassay (Porcella, 1983). 

The Daphnia magna EC50 bioassay is the preferred test to determine the toxicity of 

freshwater samples. Daphnids occur in nearly all types of freshwater habitats and have 

been recommended as a bioassay organism by the Committee on Methods for Toxicity Tests 

with Aquatic Organisms (ASTM, 1975; ASTM, 1980) because of their wide geographic 

distribution, important role in the aquatic food web, wide temperature tolerance, wide pH 

tolerance, ready availability, and ease of culture. The test consists of placing five Daphnia 

magna (early instar stages 2-4) in a 250-ml container with 200 ml of the test solution and 

incubating 48 hours at 19°C using a photoperiod of 16 hours light to 8 hours of dark. The 

test material concentration, which effectively influences 50 percent of the population (EC50) 

within the 96 percent confidence limits, is determined after 24 and 48 hours. The EC50 is 

determined in this test because of the difficulty in ascertaining death (LC50) for the Daphnia. 

The principal criterion of effect on Daphnia is immobilization, defined as lack of movement 

except for minor activity of appendages. Reference controls with a known toxic substance 

are also recommended to check the sensitivity of the organisms used in the test. 

Ceriodaphnia dubia Survival and Reproduction Bioassay (EPA, 1985). 

The Ceriodaphnia bioassay can be used to determine toxicity in freshwater environments. 

Traditionally used to measure the chronic toxicity of industrial whole effluents and 

receiving water, this test takes into account 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 test requires that 

Ceriodaphnia be exposed in a static renewal system for seven days to different concentrations 

of test solution. Test results are based on survival and reproduction. 

One Ceriodaphnia (2 to 24 hours old, all within 4 hours of the same age) is placed in each 

of 10 replicate 30 ml beakers containing 15 ml of test solution. The minimum of five test 

concentrations should be used. Selection of these test concentrations should be based upon 

the particular objectives of the study. Two commonly used dilution factors are 0.3 and 0.5. 

If it is suspected that Ceriodaphnia predators are present in the test solution, dilution water, 

or culture media, water should be filtered through a plankton net with 30 μm mesh 

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openings. During the test, the Ceriodaphnia are fed digested trout chow, yeast, or 

CEROPHYLR at a rate of 0.1 ml food suspension per 15 ml of test solution. 

In the absence of toxic substances, young production may exceed 30 per adult, 10 to 15 

young are released every 36 to 48 hours. If toxic substances are present, young may develop 

in the brood pouch of adults, but may not be released during the exposure period. Adult 

survival and reproduction are recorded each day in the test chambers until the end of the 

test (seven days). Using several statistical techniques, the reproductive production and 

survival of Ceriodaphnia are evaluated with the various test concentrations and control to 

assess the chronic toxicity. 

Fathead Minnow (Pimephales promelas) Larval Survival and Growth 

Bioassay (EPA, 1985). 

The fathead minnow Pimephales promelas larval survival and growth bioassay can be 

used to determine toxicity in water and sediments in freshwater environments. 

Traditionally used as a test for estimating the chronic toxicity of industrial whole effluents 

and receiving water, this test takes into account 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. P. promelas larvae are 

exposed in a static renewal system for seven days to different concentrations of test solution. 

Test results are based on the survival and growth (increase in weight) of the larvae. 

Ten P. promelas larvae (preferably less than 24 hours old) are placed into each of two 

replicate one-liter beakers containing 500 ml of test solution. A minimum of five different 

concentrations of the test solution plus a control is needed to conduct the bioassay. The 

selection of the test concentrations should be based upon the particular objectives of the 

study. Two commonly used dilution factors are 0.3 and 0.5. The fish larvae in each test 

chamber are fed 0.1 ml of newly hatched (less than 24 hours old) brine shrimp nauplii, three 

times daily at four-hour intervals. The numbers of live and dead larvae in each test 

chamber are recorded daily and dead larvae are discarded. At the end of seven days, the 

larvae are counted, preserved in 4 percent formalin and dried. Dry weight analysis is 

conducted to determine growth rates of larvae in each of the test solutions, and chronic 

toxicity is evaluated using several statistical methods. 

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SEDIMENTS 

Daphnia magna Lethality and Partial Life Cycle Bioassay (Nebeker et al., 

1984). 

The Daphnia magna bioassay is the preferred protocol for determining toxicity in 

freshwater sediments. Daphnids occur in nearly all types of freshwater habitats and have 

been recommended as bioassay organisms by the Committee on Methods for Toxicity Tests 

with Aquatic Organisms (ASTM, 1980) because of their wide geographic distribution, 

important role in the aquatic food web, temperature requirements, wide pH tolerance, ready 

availability, and ease of culture. 

The 48-h D. magna lethality test consists of exposing water fleas to 200 ml sediments in a 

1000-ml beaker to which 800 ml of dilution water is added. Into each test beaker, 15 

organisms are placed and incubated for 48 hours. After 48 hours, the water and fine 

suspended sediment in each beaker (not the bulk of the sediment) is poured through a 0.5-

mm mesh screen to collect and count the live and dead organisms. Because death is not 

always easily determined with D. magna, a sublethal effect can be used as an endpoint. The 

principal criterion for effect on D. magna is immobilization, defined as lack of movement 

except for minor activity of appendages. Reference controls with known toxic substance are 

also recommended to check the sensitivity of the organisms used in the test. 

The 10-day D. magna partial life-cycle test is conducted using 20, five-day old D. magna

exposed in 2.5 liters of water to 500 ml of test sediment. Survival and reproductive success 

are assessed after 10 days (three broods) by screening the water and fine sediments to collect 

and count the live and dead adult and young D. magna. 

ASTM Designation: E 1367 - 92. 1996. Standard Guide for Conducting 

10-Day Static Sediment Toxicity Tests with Marine and Estuarine 

Amphipods. [Rhepoxynius abronius, Eohaustorius estuarius, Ampelisca 

abdita, Grandidierella japonica, Leptocheirus plumulosus.) 

General: 

This guide describes procedures for obtaining laboratory data concerning the short-term 

adverse effects of contaminated sediment, or test material added to contaminated or 

uncontaminated sediment on marine or estuarine infaunal amphipods during static 10-day 

exposures. Procedures for static sediment toxicity tests are described for the following 

species: Rhepoxynius abronius, Eohaustorius estuarius, Ampelisca abdita, Grandidierella japonica, 

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and Leptocheirus plumulosus. Methods outlined in this document should be useful for 

toxicity testing of other aquatic taxa, and are applicable to sediments containing most 

chemicals, commercial products, and known or unknown mixtures. These methods can also 

be used to conduct bioconcentration tests and in situ, tests. LC50 or EC50 values may be 

determined. Sections of this guide include: general considerations (referenced documents, 

terminology, summary, significance and use, interferences, safety precautions), apparatus, 

toxicity test water, test and control sediments, test organisms, experimental design, 

procedure, analytical methodology, acceptability of test, calculation of results, 

documentation, tables and appendices. 

Rhepoxynius abronius amphipod, emergence, mortality, and reburial bioassay: 

The free-burrowing amphipod, Rhepoxynius abronius, is used to test sediments from 

marine areas. Found along the West Coast from central California to Puget Sound, 

Washington (available year round), it is the most sensitive species of amphipod, (the first to 

disappear from polluted areas and somewhat adversely affected by very fine-grained 

sediments). Adults or large immature young (3 to 5 mm mixed sexes) should be used in the 

tests, 20 per test chamber, exposed for 10 days, monitored each day for emergence, and then 

separated from the test substrates for analysis. Amphipods in sorting trays are counted as 

live or dead. Survivors are transferred to dishes and allowed one hour to rebury. Numbers 

of survivors unable to rebury in clean sediment can be used to calculate an EC50. 

Some considerations pertaining to this test include sediment grain size, salinity above 25 

parts per thousand (ppt), size of test organisms, controls and sediments from non-toxic 

areas, and at least five laboratory replicates per test. Emergence from highly toxic sediment 

may occur, which should be observed and recorded (when the amphipod is completely or 

partially out of the sediment, on the sediment surface, swimming or floating). Mortality is 

determined at the end of the test. Total numbers of live and dead are recorded. Amphipods 

that are inactive but not obviously dead should be observed under a low-power microscope 

for neuromuscular pleopod twitch, with or without gentle prodding. Control survival is 

generally 95 percent or greater, and must be at least 90 percent for the toxicity test to be 

considered valid. Data on the ability of amphipods to rebury in clean sediment is used to 

detect sublethal effects. The numbers of organisms able to rebury within the time period 

specified should be recorded and an EC50 calculated. 

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Eohaustorius estuarius amphipod acute toxicity mortality bioassay: 

The free-burrowing sand dweller, Eohaustorius estuarius, is distributed from British 

Columbia south to central California (available year round). Salinity tolerance is from 2 to 

28 ppt. Twenty organisms per test chamber are used. The size of the animals should be 3- 

to 5-mm total length, with a caution not to use larger specimens, since they are senescent. A 

fine sediment control should be used if test sediments are predominantly silt or clays. 

Eohaustorius is only slightly less sensitive than Rhepoxynius to contaminants. 

The major consideration when interpreting the results of acute toxicity tests is to keep in 

mind that the reproductive ability or long-term survival may be affected by contaminants at 

concentrations lower than those that produce a lethal or sublethal response. Control 

survival should be at least 90 percent. 

Ampelisca abdita amphipod 10-day mortality growth and emergence bioassay: 

This amphipod is an infaunal tube dweller inhabiting the low intertidal zone from 

central Maine to north Florida, the Gulf of Mexico, and San Francisco Bay. Salinity tolerance 

is classified as fully marine to 10 ppt, and is found in fine sand and mud to silt habitats, 

generally with high organic content. Each replicate should test 20 to 30 amphipods. The 

endpoint for the 10-day test is mortality, and dead animals (if it does not respond to gentle 

probing) should be counted and removed daily. Any animals not accounted for when the 

sieved material is examined are presumed to have died during the test. Other observations 

include animals out of their tubes, those that only exhibit a muscular pleopod twitch, the 

presence of molts, and the condition of the tubes built. Emergence from the sediment and 

the inability to construct a proper tube are sublethal behavioral responses that would 

ultimately result in death. For the growth test, small juveniles should be selected. 

Additional organisms should be preserved for later comparisons since this group represents 

the initial size. Growth is measured by length from the base of the first antennae to the base 

of the telson. Measurements are done after preservation and counting of test survivors. 

Ampelisca is a good organism to test for PAHs, heavy metals, and PCBs. Because this 

amphipod is a particle feeder and will be exposed to contaminated particles in suspension, 

on the sediment surface, or through interstitial pore water, and since routes of exposure 

have not been fully examined, it is difficult to determine how toxicity occurs. 

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Grandidierella japonica amphipod emergence mortality bioassay: 

Some northern and southern California bays and San Francisco Bay are the habitats for 

these tube-dwelling amphipods. They are marine, with salinity tolerances from 30 to 35 ppt. 

Grandidierella japonica lives in a variety of sediments (sands, silts, or clays) in the intertidal 

zone. Immature animals, 3- to 6-mm long should be used in the tests. Use 20 organisms per 

test chamber and allow them to bury. Those that do not bury should be replaced, unless 

they repeatedly burrow into the sediment and immediately emerge in an apparent 

avoidance response to the test substrate. In that case amphipods are not replaced. No 

females carrying embryos in their marsupium should be used. At the termination of the 

test, the reburial data can be used to determine an EC50 for a sublethal measurement. 

Surviving animals should be allowed one hour to rebury in clean collection site sediment. 

When interpreting the data from acute toxicity tests, it should be kept in mind that the 

reproductive ability or long-term survival may be affected by contaminants at lower 

concentrations than those that produce a lethal or sublethal effect in a short-term test. 

Grandidierella japonica's ability to live in a burrow in a variety of sediment types gives broad 

application for its use in research and regulatory applications. 

Leptocheirus plumulosus amphipod, emergence, mortality, and reburial bioassay: 

The infaunal amphipod, Leptocheirus plumulosus, is indigenous to oligohaline and 

mesohaline regions thus it is used to test sediments from estuarine areas. The species builds 

U-shaped burrows and is found subtidally along the East Coast from central Cape Cod to 

Florida. L. plumulosus is considered to be as sensitive to contamination as Hyalella azteca. 

Adults or large juveniles (3 to 5 mm mixed sexes) should be used in the tests, 20 per test 

chamber, exposed for 10 days, monitored each day for emergence, and then separated from 

the test substrates for analysis. Amphipods in sorting trays are counted as live or dead. 

Survivors are transferred to dishes and allowed one hour to rebury. Numbers of survivors 

unable to rebury in clean sediment can be used to calculate a sublethal EC50.

While Leptocheirus plumulosus is tolerant of a wide range of sediment grain sizes, a grain 

size reference using coarse sediments should be included among the controls. Test-water 

salinity should preferably match the interstitial water salinity of the test sediments but must 

be between 2 to 32 parts per thousand (ppt). Emergence from highly toxic sediment may 

occur, which should be observed and recorded (when the amphipod is completely or 

partially out of the sediment, on the sediment surface, swimming or floating). Mortality is 

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determined at the end of the test. Total numbers of live and dead are recorded. Amphipods 

that are inactive but not obviously dead should be observed under a low-power microscope 

for neuromuscular pleopod twitch, with or without gentle prodding. Control survival must 

be at least 90 percent for the toxicity test to be considered valid. Partial life cycle tests (28 to 

30 days) have been conducted with L. plumulosus (Schlekat et al. 1992; McGee et al. 1993), 

and while no formal protocols exist for these tests, protocols are currently under 

development. 

ASTM Designation: E 1706 - 95b (Replaces 1383-90). 1996. Standard test 

methods for measuring the toxicity of sediment-associated contaminants 

with fresh water invertebrates. [Hyalella azteca, Chironomus tentans, 

Chironomus riparius]. 

General: 

This document covers procedures for obtaining laboratory data to evaluate adverse 

effects of contaminants associated with whole sediment on freshwater organisms. Another 

source for essentially the same procedures is the US EPA’s Methods for Measuring the 

Toxicity and Bioaccumulation of Sediment-associated Contaminants with Freshwater 

Invertebrates (U.S. EPA 1994). The methods are designed to assess the toxic effects on 

invertebrate survival, growth, or reproduction, for short-term (e.g., 10 days) or long-term 

tests, in static or flow-through water systems. Procedures are described for three species: 

Hyalella azteca, Chironomus tentans, and Chironomus riparius. With modifications, these 

procedures may be used for sediment tests with other aquatic species. These procedures are 

applicable to most sediments, chemicals, materials adhering to sediments, or interstitial 

water. They might also be used to conduct bioaccumulation tests. The results of these tests 

may be reported in terms of LC50s, EC50s, no observed ECs (NOEC), and low observed ECs 

(LOEC). 

Sections elaborated on in this document include: a referenced document list, 

terminology, summary guide, significance and use, interferences, hazards, apparatus, 

overlying water, sediment characterization, test organisms, experimental design, 

procedures, analytical methodology, acceptability of tests, calculation, report, and 

appendices. The appendices section describes the organisms used for sediment toxicity 

testing. 

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For each species, the significance, life history and cycle, collection, brood stock, 

handling, age, acclimation, toxicity test specifications (experimental design, type of tests, 

initiation of tests, feeding, and biological data) sections are included and described. 

Hyalella azteca amphipod survival, behavioral, growth, and reproductive bioassays: 

Naturally collected (they occur in permanent lakes, ponds, and streams throughout the 

entire American continent) or laboratory raised Hyalella azteca can be used in a wide range of 

sediment testing (particle size tolerance ranging from >90 percent silt and clay to 100 

percent sand-size particles). The life cycle is divided into three stages: immature (instars 1-

5), juvenile (instars 6-7), and the adult stages. It is an epibenthic burrowing detritivore, 

exhibiting sexual dimorphism. 

Tests should be started with organisms (second or third instar) about 2- to 3-mm long. 

Experimental designs (purpose, procedures, and calculations) dictate the number of 

treatments, test chambers, and amphipods per treatment. Nebeker et al. (1984) recommend 

two or more replicate aquaria per treatment with 100 organisms in each. Ingersoll and 

Nelson (1990) recommend four replicates per treatment with 20 organisms per replicate for 

a total of 80 amphipods per treatment. Duration can range from •10 days (short-term), 

continuing up to 30 days (long-term). Adult survival, the number of young, growth, and 

development can be used as endpoints. The 30-day tests can also measure reproductive 

capacity, behavior, and sexual development. Flow-through and static tests can be 

conducted. Feeding needs to be carefully monitored to avoid bacterial and fungal growth 

on sediments. Also, behavior should be monitored to check for floaters (sediment 

avoidance) and reproductive activities (amplexus). The amphipods are collected at the end 

of the test by screening methods; live amphipods should be counted or preserved for later 

examination using a low-power binocular microscope. Length of the body in millimeters 

(±0.01mm) from the base of the first antenna to the tip of the third uropod, or wet and dry 

measurements are used to quantify growth. An H. azteca sediment toxicity test is 

unacceptable if the average survival in any negative control chamber is less than 80 percent. 

Chironomus tentans midge larval survival, growth, adult emergence, reproduction 

bioassays: 

This is a fairly large midge with a short generation time, easily cultured in the 

laboratory. It also has holarctic distribution and is common in the mid-continental areas of 

North America. Brood stock can be obtained from the wild or a commercial source. The 

larvae of Chironomus tentans burrow into the sediment to build a case. They are collected 

August 1997 8-18
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from lotic (streams or running water), and lentic (standing ponds) habitats. The life cycle 

can be divided into three distinct stages: larval (4 instars), pupal, and adult. Sexual 

dimorphism is observed when the adults hatch. 

Tests with Chironomus tentans can be started with second instar larvae (10 days old). 

Experimental designs (purpose, procedures, and calculations) dictate the number of 

treatments, test chambers, and midges per treatment. Larval survival, growth (assessed at 

10 to 14 days when the larvae have reached the third or fourth instar), or adult emergence 

can be monitored as biological endpoints in static and flow-through tests. Growth 

determinations using dry weight are preferable to length. Growth measurements can also 

be estimated from head capsule width, and also used to determine instar development. 

Nebeker et al. suggests conducting adult emergence tests for 25 days when started with 

second instar larvae. Emergence begins on day 20 and lasts for five days. Percent 

emergence is generally less than 60 percent in these tests. Endpoints calculated include (1) 

percent emergence, (2) mean emergence time, or (3) day to first emergence. Egg hatching 

studies may also be conducted using egg masses to estimate effects of exposure on either the 

number of eggs produced or hatched. A C. tentans sediment toxicity test, independent of 

test duration, is unacceptable if the average survival in any negative control chamber is less 

than 70 percent. 

Chironomus riparius midge larval survival, growth, adult emergence, reproduction 

bioassays: 

This is a fairly large midge with a short generation time, easily cultured in the 

laboratory. The larvae have direct contact with the sediment by burrowing and building a 

case. The distribution is worldwide (most species are thermophilic and have adapted to 

living in standing water, but some occur in cold habitats and in running water). The 

tubiculous larvae frequently inhabit eutrophic lakes, ponds, and streams with a variety of 

substrates. They tolerate a wide range of grain sizes from <90 percent silt-and-clay particles 

to 100 percent sand-size particles. The life cycle can be divided into three distinct stages: the 

larval (4 instars), the pupal, and the adult. Sexual dimorphism is observed when the adults 

hatch. Brood stock can be obtained from the wild or from a commercial source, or they can 

be raised in the laboratory. 

Tests with C. riparius can be started with larvae less than 24 hours old or with 3-day-old 

larvae. Experimental designs (purpose, procedures, and calculations) dictate the number of 

treatments, test chambers, and amphipods per treatment. Ingersoll and Nelson (1990) 

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recommend using 50 specimens for flow-through testing. The duration of the tests can 

range from a •10-day test to >10 days and continuing up to 30 days. Larval survival, 

growth, or adult emergence can be monitored as biological endpoints in static or flowthrough tests. Larval survival and growth can be assessed by ending the tests on day 10 to 

day 14 (3d or 4th instar). At this time, the larvae are removed from the sediment. Growth 

determination using dry weight is preferable to length. Growth and instar development can 

be determined by measuring head capsule width. Endpoints in emergence tests include: (1) 

percent emergence, (2) mean emergence time, or (3) day of first emergence. Egg hatching 

studies may also be conducted, and egg masses can be used to estimate effects of exposure 

on either the number of eggs produced or hatched. A C. riparius sediment toxicity test is 

unacceptable if the average survival in control chamber is less than 70 percent. 

ASTM Designation: E 1611 - 94. 1996. Standard guide for conducting 

sediment toxicity tests with marine and estuarine polychaetous annelids. 

(Neanthes arenaceodentata, Neanthes virens)

General: This guide describes procedures for obtaining laboratory data concerning the 

short-term adverse effects of potentially contaminated sediment, or test material added to 

contaminated or uncontaminated sediment on marine or estuarine infaunal polychaetes 

during 10-day or 20- to 28-day exposures. Procedures for the 10-day static sediment toxicity 

tests are described for Neanthes arenaceodentata and Neanthes virens. Procedures also are 

described for the 20 to 28-day static-renewal sediment toxicity test for N. arenaceodentata. 

Methods outlined in this document could be used for toxicity testing with other aquatic 

infaunal taxa (e.g., other polychaetes, crustacea, bivalves), although modification of the 

procedures appropriate to the test species might be necessary. LC50 or EC50 values may 

also be determined. Sections of this guide include: general considerations (referenced 

documents, terminology, summary, significance and use, interferences, safety precautions), 

apparatus, test-water toxicity, test and control sediments, test organisms, experimental 

design, procedure, analytical methodology, acceptability of test, calculation of results, 

documentation, tables and appendices. 

Neanthes arenaceodentata survival and growth toxicity tests: N. arenaceodentata is 

distributed widely throughout the world in estuarine intertidal sand or muddy sand 

beaches. It grows up to 10 cm in length, resides in mucoid burrows and may be found at 

concentrations as high as 1000 /m2. Its ubiquitousness allows for easy collection and its size 

allows easy handling. It is readily cultured in the laboratory so, whether organisms are 

obtained through field sampling or laboratory culturing, they are always readily available 

August 1997 8-20
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for testing. The 10-day static test has survival as the sole endpoint, while the 20- and 28-day 

tests have both survival and growth as endpoints. Mean control survival must be at least 90 

percent and 80 percent or better in individual replicates. After the number of surviving 

worms are determined in the two longer tests, the worms from each replicate are placed in a 

clean petri dish, washed, then placed in clean pre-weighed aluminum pans, dried at 50° C to 

a constant weight and weighed to the nearest 0.1 mg. 

Neanthes virens survival toxicity test: N. virens is distributed widely in the Atlantic 

and is found from the Arctic to Virginia in North America. It is usually found in intertidal 

and shallow subtidal waters associated with a wide variety of sediment types including 

coarse and fine sands, clay, peat, and water soaked wood. Specimens measure up to 90 cm 

long and 4.3 cm wide. No mention is made of laboratory culturing so specimens for testing 

need to be field collected. Tests are usually run in small aquaria with test organisms readily 

collected at the end of the test with a small net or by simply sieving the sediments. Mean 

control survival must be at least 90 percent and 80 percent or better in individual replicates. 

The protocols note that the mortality endpoint is relatively insensitive to contaminants. This 

insensitivity along with its large size makes N. virens desirable for bioaccumulation studies. 

Protocols from Recommended Guidelines for Conducting Laboratory 

Bioassays on Puget Sound Sediments (Puget Sound Estuary Program, 

1991) 

Amphipod (Rhepoxynius abronius or Eohaustorius estuarius) sediment bioassay:

Sediment bioassays that test with the amphipods R. abronius or E. estuarius are used to 

characterize the toxicity of marine or estuarine sediments. This assay may be used alone as 

a screening tool in broad-scale sediment surveys, in combination with sediment chemistry 

and in situ biological indices, and in laboratory experiments. The endpoints include 

mortality, emergence, and nonreburial. For R. abronius, certain limitations should be noted 

including: an interstitial salinity requirement of • 25 ppt, poor survival at grain size 

extremes (clay at 50 percent and gravel at 35 percent), a temperature requirement of 15°C, 

and seasonal sensitivity. Seasonal sensitivity can be corrected for by the use of a positive 

control, e.g., CdCl2. Predator removal is also an important requirement for running 

successful tests. The limitations for E. estuarius include the same parameters, but sediments 

with interstitial water salinity of 2 to 28 ppt may be used. Both species may be collected 

using benthic grabs or small dredges. Approximately one-third more E. estuarius than R. 

abronius are required for the bioassay. Mature amphipods are used in the sediment bioassay 

and should be acclimated to laboratory conditions for two to ten days before testing. For 

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each field sample, five replicate tests are conducted. Approximately 175 ml of test sediment 

are placed in the bottom of 1-liter test beakers. The beakers are filled to 750 ml with 

seawater (28 ppt for R. abronius, and ambient salinity for E. estuarius). Constant illumination 

is provided and the water in the beakers is aerated without disturbing the sediments. 

Twenty amphipods are placed in each beaker and the seawater level is brought up to 950 

ml. The bioassay is terminated after 10 days of exposure. The primary endpoint is 

mortality (lack of pleopod twitch observed under magnification or response to gentle 

prodding) after 10 days exposure and the secondary endpoints of daily emergence and 

failure to rebury (within one hour) can also be measured. 

A typical sediment bioassay involves 50 to 60 beakers. All bioassays include five 

replicates of the collection-site control sediment. These beakers comprise a negative (clean) 

control. Mean mortality in this control should be •20 percent for the test to be considered 

valid. A positive (contaminated) control is also required for all testing. This involves 

determining 96-hour LC50 values (four to five logarithmic concentration series and a 

control) for organisms exposed to a reference toxicant (CdCl2) in clean, filtered seawater 

without sediment. The design of field surveys typically includes a reference sediment 

involving five replicate laboratory tests of samples from an area believed to be free from 

sediment contamination. The grain size and organic carbon content of the reference area 

sediment should be matched with the test sediment. This provides a site-specific basis for 

comparison of potentially toxic and nontoxic conditions while controlling the effects of 

exposing amphipods to non-native sediments. 

Bivalve larvae sediment bioassay: 

The bivalve larvae bioassay recommends Pacific oysters (Crassostrea gigas) and blue 

mussels (Mytilus edulis) for testing. The toxicity of marine sediments can be characterized 

by using these tests alone as a screening tool in broad-scale sediment surveys, in 

combination with sediment chemistry and in situ biological indices, and in laboratory 

experiments. During the first 48 hours of embryonic development, fertilized oyster and 

mussel eggs normally develop into a prodissoconch I, a free-swimming, fully shelled larval 

stage. Failure of the eggs to survive or the proportions of larvae developing in an abnormal 

manner have been used as the primary indicators of toxicity. A combined mortality and 

abnormality endpoint may also be calculated from the number of normal surviving larvae. 

Certain limitations should be noted when considering the use of this bioassay. Testing with 

bivalve larvae is not recommended for sediments that have an interstitial salinity of less 

than 10 ppt. This assay is primarily an indicator of the relative toxicity among different 

August 1997 8-22
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samples. Spawning of C. gigas occurs naturally in the Puget Sound area in the summer. The 

natural spawning period for M. edulis is late spring to early summer. Both of these bivalves 

can be induced to spawn at other times of the year, but may show decreased viability of 

gametes. A positive control is recommended (48-hour exposure to a reference toxicant in 

seawater only). Other problems that may affect the results include the lack of evaluation of 

the bivalves' sensitivities to natural physical and chemical factors, which may influence 

occasionally high mortalities in control or reference tests. The recovery of live abnormal 

larvae from the sediments is also difficult. 

The adult bivalves are induced to spawn. For each field sample, five replicate tests are 

conducted. Twenty grams (wet weight) of the appropriate sediment is added to each bottle 

and the volume is brought up to l liter with filtered or UV-treated seawater (28 ± 1 ppt 

salinity). The reference area sediment chambers each contain 20 grams of clean sediment. 

Negative and positive controls to determine LC50 and EC50 are also prepared consisting of 

clean seawater without sediment. The chambers are vigorously shaken for 10 seconds and 

allowed to settle for 4 hours. Each container is inoculated with 20,000 to 40,000 developing 

embryos. The containers are covered and incubated for 48 hours at 20 ± 1°C for oysters and 

16 ± 1°C for mussels under a 14-hour light: 10-hour dark photoperiod. The test chambers 

are not aerated unless the dissolved oxygen concentration declines below 60 percent of 

saturation. The mean embryo concentration at 0 hour should be determined by collecting 

five replicate 10-ml samples from control cultures. This is not a direct measurement of the 

embryos in the test chambers and the resulting density estimates have an unquantified error 

component associated with them. This error reduces the reliability of larval mortality 

estimates and may thereby influence the results of statistical analyses. However, it does not 

affect larval abnormality estimates. When the embryos in the duplicate seawater control 

have reached the prodissoconch I stage (approximately 48 to 60 hours), the bioassay is 

terminated. The water and sediment are stirred, and 10-ml aliquots of samples are removed 

by pipette and preserved in vials containing five-percent buffered formalin. 

Preserved samples (equal in volume to those containing 300 to 500 larvae in controls) are 

examined in Sedgewick-Rafter cells. Normal and abnormal larvae are enumerated to 

determine percentage survival and percentage abnormality. A minimum sample size of 20 

living larvae in each of five replicated bioassay chambers for test sediment and reference 

area sediment, and 100 larvae in each replicated chamber for the seawater control should be 

scored for abnormalities. Percentage survival is based on the number of larvae surviving 

relative to the mean number of survivors in the seawater controls. Larvae that fail to 

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HAZMAT 93-1–Protocols 

transform to the fully shelled, straight-hinged, D-shaped prodissoconch I stage are 

considered abnormal. Percent abnormal is based on the number of survivors that are 

abnormal. Five replicates of the seawater control are included in all bioassays. These 

comprise negative (clean) controls for comparisons among experiments and among 

laboratories (at least 70 percent of the larvae must survive the 48-hour exposure with 

seawater; of these, at least 90 percent must show no abnormalities). A positive control is 

also required (CdCl2 or sodium dodecyl sulfate). Bioassays to establish an LC50 or an EC50

involve a concentration series (four to five logarithmic) and a control. 

Echinoderm embryo sediment bioassay: 

The echinoderm embryo bioassay is a rapid and sensitive technique for assessing the 

toxicity of marine sediments. Purple sea urchins (Strongylocentrotus purpuratus), green sea 

urchins (S. droebachiensis), and sand dollars (Dendraster excentricus) are the recommended 

species for testing. During the first 48 to 96 hours of development, fertilized echinoderm 

eggs normally develop into the pluteus stage. Failure of the eggs to survive and the 

proportions of larvae developing in an abnormal manner are used as indicators of toxicity. 

These tests may be used alone for screening in broad-scale sediment surveys, in 

combination with sediment chemistry and in situ biological indices, and in laboratory 

experiments. Certain limitations should be noted when considering the use of this bioassay. 

Quantitative results for corresponding endpoints may not be strictly comparable since the 

three species show slightly different levels of sensitivity to various contaminants. This 

bioassay is not recommended for sediments that have an interstitial salinity of less than 10 

ppt. Echinoderm larvae normally reside in the water column and are not intimately 

associated with sediments. Therefore, this bioassay is primarily an indicator of the relative 

toxicity among different samples. Strongylocentrotus (spp.) spawning occurs naturally in the 

Puget Sound region from December to April. The natural spawning period for D. 

excentricus is from April to October. Echinoderms can be induced to spawn at other times of 

the year, but may show decreased viability of gametes. Care must be taken when 

transporting echinoderms due to epidemic spawning. A positive control is recommended 

(48-hour exposure to a reference toxicant in seawater only). Other problems that may affect 

the results include the lack of evaluation of the bivalves' sensitivities to natural physical and 

chemical factors, which may influence occasionally high mortalities in control or reference 

tests. The recovery of living abnormal larvae from the sediments is also difficult. 

All echinoderm species can be collected off the coast of Washington. Sand dollars can be 

collected by hand on many Puget Sound beaches during low tide. Adults are conditioned to 

August 1997 8-24
HAZMAT 93-1–Protocols 

the laboratory, and induced to spawn with chemical stimulation (1 ml of 0.5-molar KCl). 

Fertilization should be initiated within one hour of spawning by adding sperm to the beaker 

containing the eggs, at a sperm:egg ratio of approximately 2,000:1. Selected densities of 

embryos are exposed to the test or reference sediments for 48 to 96 hours. During this time, 

the embryos will normally develop into the four-armed pluteus stage. Data from tests with 

longer exposures (> 48 hours) may not be comparable to those from tests conducted using 

the standard 48-hour exposure. Toxicity test endpoints are based on abnormal shell 

development and larval death. Five replicate tests are conducted for each field sample. 

Twenty grams of reference or test sediment is added to each beaker. Filtered or UV-treated 

seawater (28 ppt salinity) is added to each beaker up to one liter to make a final 

concentration in all containers of 20 grams (wet weight) of sediment per liter of seawater. 

Each reference area sediment chamber also contains 20 grams per liter of clean sediment. In 

addition, two control series are prepared containing clean seawater without sediment (one 

series is used as a duplicate, sacrificial control to monitor embryo development). The 

sediments are vigorously shaken for ten seconds and allowed to settle for four hours. 

Within two hours of fertilization, a 1-ml aliquot of the solution of embryos (about 25,000) is 

added to each chamber by using an automatic pipette. The containers are covered and 

incubated for 48 hours at 15 ± 1°C under a 14-hour light: 10-hour dark photoperiod. The 

test chambers are not aerated unless the dissolved oxygen concentration declines below 60 

percent of saturation. The mean embryo concentration at 0 hour should be determined by 

collecting five replicate 10-ml samples from control cultures. This is not a direct 

measurement of the embryos in the test chambers, and the resulting density estimates have 

an unquantified error component associated with them. This error reduces the reliability of 

larval mortality estimates and may thereby influence the results of statistical analyses. 

However, it does not affect larval abnormality estimates. When the embryos in the 

duplicate seawater control have reached the four-armed pluteus stage (approximately 48 to 

96 hours), the bioassay is terminated. The water and sediment are stirred, and 10-ml 

aliquots of samples are removed by pipette and preserved in vials containing five-percent 

buffered formalin. 

Preserved samples (equal in volume to those containing 300 to 500 larvae in controls) are 

examined in Sedgewick-Rafter cells. Normal and abnormal larvae are enumerated to 

determine percentage survival and percentage abnormality. A minimum sample size of 20 

living larvae in each of five replicated bioassay chambers for test sediment and reference 

area sediment, and 100 larvae in each replicated chamber for the seawater control should be 

scored for abnormalities. Percentage survival is based on the number of larvae surviving 

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HAZMAT 93-1–Protocols 

relative to the mean number of survivors in the seawater controls. Embryos that fail to 

transform to the four-armed pluteus stage are considered abnormal. Percentage abnormal 

for each replicate is based on the number of survivors that are abnormal. Five replicates of 

the seawater control are included in all bioassays. These comprise negative (clean) controls 

for comparisons among experiments and among laboratories (at least 70 percent of the 

larvae must survive the 48-hour exposure with seawater; and of these, at least 90 percent 

must show no abnormalities). A positive control is also required (CdCl2 or sodium dodecyl 

sulfate). Bioassays to establish an LC50 or an EC50 involve a concentration series (four to 

five logarithmic) and a control. 

Anaphase aberration sediment bioassay: 

This sediment bioassay is used to characterize the genotoxicity of marine sediments. 

These tests may be used alone for screening in broad-scale sediment surveys, in 

combination with sediment chemistry and in situ biological indices, and in laboratory 

experiments. This assay can be used with any type of sediment regardless of the interstitial 

salinity or grain size characteristics. Rainbow trout gonad cells (RTG-2) are recommended 

(although any cell type can be used). Certain limitations should be noted when considering 

the use of this bioassay. The assay depends on a chemical extraction procedure that is 

specific for neutral, nonionic organic compounds (aromatic and chlorinated hydrocarbons). 

Other contaminants such as metals and highly acidic and basic organic materials are not 

efficiently extracted. Natural genotoxicity may occur in the marine sediments and may 

cause positive genotoxic responses. Extractions are prepared by evaporating a volume of 

the reagent equivalent to that which would be used for the actual extraction. This is added 

to the cultures in varying amounts dissolved in the solvent (e.g., dimethyl sulfoxide 

[DMSO]). Once the extracts have been made, gravimetric determinations of their absolute 

organic content must be made so that comparable organic concentrations from each site can 

be used in cell cultures exposed to extracts from different locations. RTG-2 cells grow in a 

variety of commercially available culture media. The Leibovitz L-15 medium was found to 

be the most consistent in terms of ease of preparation. Generally, heat-deactivated fetal calf 

serum is added to the culture medium at 10-percent concentrations to ensure proper growth 

factors are present. 

The cells are grown and tested at 18°C on standard, clean microscope slides or on 1- by 

5-cm coverslips in Leighton tubes. The cells are placed into the culture one day before the 

exposure to insure attachment to the substrate and to begin growing. After 18 to 24 hours, 

the culture medium is removed and the test material is added (consisting of the L-15 

August 1997 8-26
HAZMAT 93-1–Protocols 

medium dissolved in DMSO to which the extract has been added). Exposure time should be 

48 hours (maximum exposure) from the time of addition of the treated medium until 

fixation. An initial screening test must be conducted to determine the actual extract 

dilutions to be used for the bioassay. Ideally, dilutions tested for anaphase aberrations 

comprise the highest concentration of extract (ml/L) that permits continued cell 

proliferation (i.e., is nontoxic) and a second concentration one dilution lower. Previous 

experience in Puget Sound has shown that the following six extract dilutions should be 

prepared: 50, 25, 15, 5, 2, and 1 μg/ml. Cells are first exposed to these concentrations for 

each sediment tested, and then the concentrations that inhibit mitosis are determined. All 

results are normalized to organic content that has been previously determined. 

To determine mitotic effects and anaphase aberrations, the slides or coverslips 

containing the cells are removed from the culture medium and fixed in methanol:acetic acid 

(3:1). Following 15 to 60 minutes in the fixative, the slides are air dried and placed in threepercent Gurr's R66 Geimsa stain for 15 to 30 minutes. The slides are observed with a 

microscope to determine optimum staining time. The staining is selective for the condensed 

chromosomes undergoing mitosis. Three replicate slides are made of each exposure 

concentration with two concentrations for each sediment extract. Each slide is then 

examined at 500X to 1,000X until a minimum of 100 anaphase cells is observed and scored. 

In this way, there will be three replicates per dose with 100 anaphase cells per replicate. The 

numbers and percents of normal and abnormal anaphase stages are recorded. Cells are 

scored as abnormal if they contain any of the described chromosomal lesions reported for 

this test (see protocols for list of references). 

Controls consist of 1) untreated cultures (used as negative controls), 2) a solvent blank, 

and 3) a positive control consisting of several concentrations of known genotoxic agents 

(one should be 0.25 μg/ml benzo(a)pyrene resulting in an anaphase aberration frequency of 

50 to 65 percent). 

MicrotoxTM sediment bioassay–organic extract: 

The Microtox organic extract bioassay is a rapid, sensitive method of toxicity testing 

based on light emission by the luminescent bacterium Photobacterium phosphoreum in the 

presence and absence of aqueous toxicants. The emitted light is a product of the bacterial 

electron transport system and thus directly reflects the metabolic state of the cells. 

Decreased luminescence provides a quantitative measure of toxicity. These tests may be 

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HAZMAT 93-1–Protocols 

used alone for screening in broad-scale sediment surveys, in combination with sediment 

chemistry and in situ biological indices, and in laboratory experiments. This assay can be 

used with any type of sediment regardless of the interstitial salinity or grain size 

characteristics. Certain limitations should be noted when considering the use of this 

bioassay. The assay depends on a chemical extraction procedure that is specific for neutral, 

nonionic organic compounds (aromatic and chlorinated hydrocarbons). Other contaminants 

such as metals and highly acidic and basic organic materials are not efficiently extracted. 

Extraction by an organic solvent results in exposure of test organisms to concentrations 

much higher than those expected to occur in sediment interstitial water. There may also be 

naturally occurring toxic substances present in the marine sediments that might affect the 

results of the assay. Luminescence may increase rather than decrease. For now, these 

samples are considered nontoxic. 

The bioassay is performed using a Microtox toxicity analyzer system (a temperatureregulated photometer equipped with a photomultiplier). Freeze-dried bacteria, 

reconstitution solution, dilutent, and other necessary materials can be purchased from 

commercial suppliers. The approach to testing organic extracts uses the basic Microtox 

method described in the Microtox Operating Manual (see protocols for references). The dried 

bacteria are reconstituted with water and placed in a Microtox cuvette (4°C). The dilution 

concentrations are 5.0, 0.5, and 0.05-percent extract (v/v). Each primary dilution is adjusted 

to two-percent NaCl. These diluted extracts are used in a range-finding assay to determine 

an appropriate primary dilution (should cause 65 to 90 percent decrease in bioluminescence 

in 15 minutes) for the definitive assay. For the definitive assay, two-fold serial dilutions 

(e.g., 6.0, 2.5, 1.25, and 0.625 percent in two-percent NaCl) are prepared along with a blank 

(to measure spontaneous decay of light). In each of 10 test cuvettes, a 10 μL aliquot of 

bacterial suspension added to 500 μL of dilutent and incubated for 15 minutes in the 

incubation wells. After 15 minutes, initial levels of light emission are measured in each of 

the 10 test cuvettes. At 30-second intervals, 500 μL aliquots of each concentration extract are 

added to two of the cuvettes (i.e., two replicates each of the four extract dilutions and the 

saline blank). Exactly five minutes after addition of the sediment extract, light emission is 

measured at 30-second intervals and in the same sequence used for extract additions. Light 

emission is measured again at 15 minutes; additional measurements are sometimes made at 

30 minutes. An ethanol-only control is assayed using the same primary dilution sequence 

as the sediment test. This is used to adjust the sediment extract data for the contribution of 

the solvent vehicle. 

August 1997 8-28
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Ethanol, sodium lauryl sulfate, or other suitable reference toxicants should be used as 

positive controls to assess daily bioassay performance and to determine differences in 

response among many bacteria. Clean sediment can be evaluated as a negative control. 

Bioassay repeatability is evaluated by duplicate testing (i.e., extraction and analysis) of 10 

percent of the sediment extracts. Report the range-finding results, raw light emission data, 

and 15-minute EC50 values (at 95 percent CI). 

MicrotoxTM bioassay—saline extract: 

The Microtox saline extract bioassay is a rapid, sensitive method of toxicity testing based 

on light emission by the luminescent bacterium Photobacterium phosphoreum. The use of 

saline extracts of sediment for the Microtox bioassay has been described by Williams et al. 

(1986). The approach to testing saline extract uses the basic Microtox method described in 

the Microtox Operating Manual (see protocols for references). The major difference in the 

saline approach versus the organic approach is in the preparation of test samples. Each 

procedure is specific to the classes of contaminants tested for toxicity and, in general, the 

results for each approach can be viewed as complementary. The saline extracts Microtox 

bioassay procedure removes only the water-soluble fraction of sediment-adsorbed trace 

metals and organic pollutants from the sediments. Contaminants with extremely low water 

solubility (for example PCBs) will tend to be partitioned almost exclusively onto sediment 

particles and are unlikely to occur in high concentration in the saline extract. Certain 

limitations should be noted when considering the use of this bioassay. A correction factor 

needs to be established for changes in bacterial luminescence caused by variation among 

samples in sediment pore-water salinity. The 100-percent dilution specified in the protocol 

consists of 58-percent sediment and 42-percent Microtox dilutent, thereby limiting the 

sensitivity of the test. The use of a standardized dilution series limits the calculation of EC50

in some cases. A range-finding test could be conducted to correct this problem. The use of a 

saline extract may not mimic the actual pore water composition. Luminescence may 

increase rather than decrease. For now, these samples are considered nontoxic. 

The bioassay is performed by hydrating a vial of freeze-dried bacteria with 1.0 ml of 

reconstitution solution. Serial dilutions are prepared at 100, 50, 25, 12, 5, and 0 percent of 

sediment supernatant Microtox dilutent. The 0 percent is a reagent blank needed to 

measure spontaneous decay in bacterial luminescence. In each of 10 test cuvettes, 10 μL of 

the bacterial suspension is added to 350 μL of dilutent and incubated for 15 minutes at 15°C. 

After 15 minutes, initial luminescence in each of the 10 cuvettes is measured. At 30-second 

intervals, 500 μL aliquots of each supernatant dilution are added to two of the cuvettes (e.g., 

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HAZMAT 93-1–Protocols 

two replicates each of the four test dilutions and the saline blank). Exactly 15 minutes after 

addition of the sediment supernatants, luminescence is measured at 30-second intervals and 

in the same sequence used for supernatant additions. The percentage decrease in 

luminescence relative to the reagent blank is then calculated. 

Clean reference sediments are used as negative controls. A calibration curve to 

determine salinity-induced changes in bacterial luminescence is constructed. The use of 

reference toxicants (e.g., phenol, sodium arsenate) is needed to assess day-to-day 

performance and determine the differences in toxic response among lot number of bacterial. 

Percentage decrease in luminescence after 15-minute exposure for each concentration of 

supernatant tested is reported. 

Juvenile polychaete sediment bioassay: This protocol is for conducting a bioassay in 

which the survival and change in biomass of juvenile polychaetes (Neanthes spp.) are 

determined following a 20-day exposure to test sediments. Parameters measured to 

determine the effects of exposure include mortality, total biomass, and average individual 

biomass. Sediments can be naturally occurring (field collected), or sediments that have been 

experimentally modified by adding chemicals to the samples. This bioassay is conducted as 

a static renewal exposure, and food is provided to the test organisms during the exposure 

period to promote body tissue increase. Following the 20-day exposure period, all 

surviving worms are collected, dried to a constant weight, and total and average individual 

biomass is determined. This bioassay recommends the use of the California species of 

Neanthes. These worms are sensitive to metals, hydrocarbons, and multicontaminated 

media (e.g., sediments). Neanthes has not been collected from Puget Sound, but are 

distributed on the West Coast from Mexico to southern California. These tests may be used 

alone for screening in broad-scale sediment surveys, in combination with sediment 

chemistry and in situ biological indices, and in laboratory experiments. Certain limitations 

should be noted when considering the use of this bioassay. The bioassay should be 

conducted with laboratory-cultured juvenile Neanthes. Modification of the protocol may be 

required for tests conducted at salinities less than 20 ppt (interstitial and overlying water). 

In addition to exposure chambers containing test sediments, exposure chambers containing 

control sediment (sediment from where the worms were found or the substrate in which 

they were cultured) should also be tested. Reference sediments are also prepared (to 

observe the effects associated with physical and chemical characteristics) that should be 

similar to the test sediments in grain size and organic content. Sand is typically chosen as 

an appropriate control sediment. Bioassay seawater should be maintained at a salinity of 28 

August 1997 8-30
HAZMAT 93-1–Protocols 

± 2 ppt at a temperature of 20 ± 1°C. The chambers are 1-liter glass containers (diameter of 

10 cm) covered with lids. The gentle aeration rate should be at 150 and 300 ml per minute. 

A typical Neanthes bioassay for testing 10 sediment samples involves 50 to 60 exposure 

chambers. Five replicates are tested for each field sample. Each exposure chamber consists 

of a 1-liter jar containing 2 cm of sediment and seawater. The photoperiod during the 

testing should be continuous. Worms are collected from the holding tanks and placed in 

cups, five worms to a cup. Enough cups are used to equal three more than the number of 

exposure chambers. Worms from these cups are used to estimate initial total biomass. Five 

juvenile worms are randomly placed into each exposure chamber. The size of the worms 

used in the assay is a critical factor. They should be 0.5 to 1.0 mg dry weight, two to three 

weeks post-emergence to ensure that they are in a rapid growth phase during the exposure 

period. They are fed on an every-other-day basis. Seawater is exchanged on every third 

day. Following the exposure period, the contents of each replicate chamber are sieved and 

the number of living worms is recorded. The worms are then weighed to the nearest 0.1 mg 

dry weight. 

A control sediment and a reference sediment should be included as part of every test. A 

positive (toxic) control is also required. This involves determining 96-hour LC50 values for 

Neanthes juveniles exposed in clean, filtered seawater without sediment to reference 

toxicants (CdCl2). The positive control should be conducted with 10 juveniles per exposure 

chamber. Reporting must include acute lethality, survival, total biomass (dry weight), and 

average individual biomass (i.e., total biomass divided by the number of surviving worms). 

Each of these response criteria should be monitored in a "blind" fashion. This means the 

observer must have no knowledge of the treatment of the sediment in the beakers. 

TEST SELECTION 

One of the principle concerns in deciding which toxicity test to use for bioassessment is the 

sensitivity of the test organism and test endpoint(s). Table 8-2 is a brief summary of the 

relative sensitivity of various test organisms and endpoints; it is based on the review of 

some forty articles on comparative toxicity tests using different organisms and/or 

endpoints. The media used in the various studies included both water and sediment from 

fresh, brackish and marine environment (see Appendix C for a more complete table broken 

down by study and the list of articles reviewed). As Table 8-2 indicates, of those test 

organisms involved in more than one test series, no single organism or endpoint was 

consistently the most sensitive. For example, Chironomus tentans survival was sometimes 

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more sensitive, sometimes less sensitive and sometimes had the same sensitivity as Hyalella 

azteca survival. This variable sensitivity may be due to either the contaminants or some 

other parameter of the test medium. The bottom line is that no single toxicity test should be 

relied on to evaluate conditions at a particular site. 

Table 8-2. Relative sensitivities of selected test organisms and endpoints. Unless 

otherwise specified, endpoints for the compared species are the same as listed for them as 

a test species. When results are based on specific contaminants or groups of 

contaminants, the contaminants are indicated in parentheses (mtls = metals, org. = 

organics).

More Sensitive Species 

Test Species 

(endpoints)

Species of Equal Sensitivity

Less Sensitive Species 

Mytilus edulis 

Rhepoxynius abronius 

Strongylocentrotus purpuratus > Ampelisca abdita

(survival) > Eohaustorius estuarius 

Leptocheirus plumulosus 

Strongylocentrotus purpuratus

> Armandia brevis

(growth)

Rhepoxynius abronius

> Dendraster excentricus

> Ceriodaphnia dubia

(survival) > Daphnia magna 

Hyalella azteca

> Chironomus riparius 

(emergence)

Hyalella azteca

>

Hyalella azteca 

Lumbriculus variegatus (Cu) > Chironomus tentans

(survival) >Lumbriculus variegatus 

Hyalella azteca (dieldrin & 

chloropyrifos) 

Rhepoxynius abronius 

Photobacterium phosphoreum 

Neanthes spp. (growth) 

> Corophium volutator

(survival) > Macoma balthica 

Pseudopleuronectes americanus

Rhepoxynius abronius 

Photobacterium phosphoreum 

Mytilus galloprovincialis 

Dendraster excentricus (abn. devel.) 

> Crassostrea gigas

(survival/abn development)

Dendraster excentricus (survival)

> Neanthes spp. (survival) 

Photobacterium phosphoreum (org.) 

Ceriodaphnia dubia 

Daphnia pulex 

Microcystis aeruginosa 

Pimephales promelas (org.) 

> Daphnia magna

(survival) > Photobacterium phosphoreum

(mtls) 

Hyalella azteca 

Oncorhynchus mykiss 

Chironomus tentans (Cu) 

Gammarus lacustris (Cu) 

Pimephales promelas (mtls) 

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HAZMAT 93-1–Protocols 

More Sensitive Species 

Test Species 

(endpoints)

Species of Equal Sensitivity

Less Sensitive Species 

> Daphnia pulex

(survival) > Photobacterium phosphoreum

(mtls) 

Oncorhynchus mykiss 

Pimephales promelas (Mtls) 

Daphnia magna (Mtls) 

Armandia brevis 

Rhepoxynius abronius > Dendraster excentricus

(growth) >

Rhepoxynius abronius 

Dendraster excentricus (abn. devel.) > Dendraster excentricus (survival)

Crassostrea gigas (abn. devel.)

> Neanthes spp. (survival) 

> Dendraster excentricus

(abnormal development) 

Rhepoxynius abronius

> Dendraster excentricus (survival)

Neanthes spp. (survival) 

Crassostrea gigas (abn. devel.) 

Ampelisca abdita 

Leptocheirus plumulosus > Eohaustorius estuarius

(survival) 

Rhepoxynius abronius 

Photobacterium phosphoreum 

Leptocheirus plumulosus 

> Ampelisca abdita 

Neanthes (spp.) (Surv/biomass)

Hyalella azteca 

Chironomus tentans 

Daphnia magna

> Gammarus lacustris (Cu) 

(survival) >

Leptocheirus plumulosus (surv/grwth) 

Eohaustorius estuarius 

Daphnia magna 

Ceriodaphnia dubia 

Gammarus lacustris (Cu) 

> Hyalella azteca

(survival) 

Chironomus riparius (emergence) 

> Chironomus tentans (surv/grwth)

Lumbriculus variegatus 

Ampelisca abdita

Ampelisca abdita 

Hyalella azteca > Leptocheirus plumulosus 

(survival) 

Eohaustorius estuarius

>Leptocheirus plumulosus (growth)

Hyalella azteca

Hyalella azteca 

Chironomus tentans > Lumbriculus variegatus

(surv./growth/reproduction) > Chironomus tentans (Cu) 

Photobacterium phosphoreum 

Rhepoxynius abronius 

Corophium volutator 

Neanthes spp. (growth) 

> Macoma balthica

(bioaccumulation) > Pseudopleuronectes americanus

Rhepoxynius abronius > Mytilus edulis

(embryo surv./development)

Rhepoxynius abronius

> Ampelisca abdita 

Strongylocentrotus purpuratus

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More Sensitive Species 

Test Species 

(endpoints)

Species of Equal Sensitivity

Less Sensitive Species 

Rhepoxynius abronius > Neanthes spp. 

(growth) > Rhepoxynius abronius 

Macoma balthica 

Pseudopleuronectes americanus 

Corophium volutator

Rhepoxynius abronius 

Photobacterium phosphoreum 

Eohaustorius estuarius 

Crassostrea gigas 

Dendraster excentricus 

> Neanthes spp. 

(survival) >

Daphnia magna 

Daphnia pulex 

Photobacterium phosphoreum 

> Oncorhynchus mykiss

(survival) > Photobacterium phosphoreum

(mtls) 

Pimephales promelas 

Pimephales promelas (mtls) 

Daphnia magna (mtls) 

Oncorhynchus mykiss (mtls) 

Daphnia pulex (mtls) 

> Photobacterium 

phosphoreum/Microtox 

(bioluminesence) 

Rhepoxynius abronius (mtls/PAHs)

Eohaustorius estuarius (mtls/PAHs)

> Daphnia magna 

Rhepoxynius abronius 

Oncorhynchus mykiss 

Crassostrea gigas 

Neanthes spp. 

Pimephales promelas 

Macoma balthica 

Pseudopleuronectes americanus 

Corophium volutator

Daphnia magna 

Daphnia pulex 

Oncorhynchus mykiss

> Pimephales promelas

(survival) > Photobacterium phosphoreum 

Daphnia magna (effluent) 

Corophium volutator 

Neanthes (spp.) (growth) 

Macoma balthica 

Photobacterium phosphoreum 

Rhepoxynius abronius

> Pseudopleuronectes americanus

(histopathology) >

Neanthes spp. (growth) 

Photobacterium phosphoreum > Rhepoxynius abronius

(survival) 

Dendraster excentricus (abn. devel.)

Armandia brevis (growth) 

Eohaustorius estuarius

Mytilus edulis (abn. devel.) 

Photobacterium phosphoreum

> Crassostrea gigas 

Dendraster excentricus (surv/ 

growth) 

Neanthes spp. (surv/growth) 

Corophium volutator

Ampelisca abdita 

Photobacterium phosphoreum

(mtls/PAHs) 

Pseudopleuronectes americanus 

Strongylocentrotus purpuratus 

Macoma balthica 

Mytilus edulis (survival) 

Ampelisca abdita 

Mytilus edulis 

Rhepoxynius abronius

> Strongylocentrotus purpuratus

(cell changes/fertilization) > Ampelisca abdita

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Sensitivity is not the only, and may not be the most important, criteria on which test 

selection should be based. Long et al. (1990) suggest four additional criteria on which to 

judge the usefulness of toxicity tests. They are: (1) replicate variability/analytical precision, 

how consistent are the results in replicates of the same treatment; (2) discriminatory power, 

an indication of the range of responses with respect to the range of contamination levels; (3) 

correlation with other tests and endpoints, and (4) correlation between toxicity and 

chemistry. As an example of how these criteria may be applied, Long (1997) has found in 

his studies over the years that while Rhepoxynius abronius survival is more sensitive than 

Ampelisca abdita survival, A. abdita has better precision and is more readily correlated with 

sediment chemistry than is R. abronius . Therefore, he prefers using A. abdita survival tests. 

When it was decided to produce a manual of protocols for freshwater toxicity and 

bioaccumulation tests for sediment associated contaminants, the US EPA developed a list of 

criteria for toxicity tests that they used to select organisms and endpoints for test protocol 

development (US EPA 1994). Table 8-3, taken from the resulting manual, “Methods for 

Measuring the Toxicity and Bioaccumulation of Sediment-associated Contaminants with 

Freshwater Invertebrates”,(US EPA 1994) lists these criteria and their assigned ratings. 

Some of the additional criteria utilized in selecting the test organisms and endpoints for 

their manual included ecological relevance as well as practical concerns regarding the 

conductance and acceptability of the test. The test organisms selected were Chironomus 

tentans and Hyalella azteca for toxicity test protocols and Lumbriculus variegatus for 

bioaccumulation protocols.

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Table 8-3 Rating of selection criteria for freshwater sediment toxicity testing organisms. (US EPA, 1994) Criterion 

Hyalella 

azteca 

Diporeia 

spp. 

Chironomu

s tentans 

Chironomus 

riparius 

Lumbriculus 

variegatus 

Tubifex 

tubifex 

Hexagenia

spp. Mollusks

Daphnia spp. & 

Ceriodaphnia spp.

Relative sensitivity toxicity database + - + - + - - - -Round-robin studies conducted + - + - - - - - -Contact with sediment + + + + + + + + -Laboratory culture + - + + + + - - +Taxonomic identification +/- +/- +/- +/- + + + + +Ecological importance + + + + + + + + +Geographical distribution + +/- + + + + + + +/-Sediment physicochemical tolerance + + +/- + + + - + NAResponse confirmed with benthos populations 

+ + + + + + + - +

Peer reviewed + + + + + + + - +/-Endpoints monitored S, G, M S, B, A S, G, E S, G, E B, S, R S, R S, G B S, G, R 

A “+” or a “-” rating indicates a positive or negative attribute. S = Survival, G = Growth, B = Bioaccumulation, A = Avoidance, R = Reproduction, M = Maturation, E = Emergence, NA = not applicable.August 1997 8-36
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The selection of a toxicity test, or some other bioassessment tool, should never be based 

on a single criterion, for example, sensitivity or the desire to use an indigenous test 

organism. The test(s) that will provide the most reliable, interpretable, relevant data, for the 

particular job at hand, should be selected, and this may not necessarily be the most sensitive 

test or utilize an indigenous organism. Finally, toxicity test were never meant as standalone tools, they should be conducted in concordance with other bioassessment tools and 

the physical and chemical analysis of the medium being tested. 

REFERENCES 

11 ASTM. 1996a. Standard guide for conducting 10-day static sediment toxicity tests 

with marine and estuarine amphipods. E 1367-92, 1996 Annual Book of Standards, 

Section 11 Water and Environmental Technology, Vol. 11.05, Biological Effects and 

Environmental Fate; Biotechnology; Pesticides. Philadelphia: ASTM, pp 769-794. 

2 ASTM. 1996b. Standard guide for conducting sediment toxicity tests with marine and 

estuarine polychaetous annelids. E 1611-94. 1996 Annual Book of Standards, Section 

11 Water and Environmental Technology, Vol. 11.05, Biological Effects and 

Environmental Fate; Biotechnology; Pesticides. Philadelphia: ASTM, pp 1046-1069. 

3 ASTM. 1996c. Standard test methods for measuring the toxicity of sedimentassociated contaminants with fresh water invertebrates. E 1706-95b, 1996 Annual 

Book of Standards, Section 11 Water and Environmental Technology, Vol. 11.05, 

Biological Effects and Environmental Fate; Biotechnology; Pesticides. Philadelphia: 

ASTM, pp 1176-1258 

4 Breteler, R. J., K. J. Scott, and S. P. Shepherd. 1989. Application of a new sediment 

toxicity test using the marine amphipod Ampelisca abdita to San Francisco Bay 

sediments. In: U. M Cowgill and L. R. Williams (eds). Aquatic Toxicology and Hazard 

Assessment: 12th Volume. ASTM STP 1027, Philadelphia: American Society for Testing 

and Materials pp. 304-314. 

5 Cairns, M. A., A. V. Nebeker, J. H. Gakstatter, and W. L. Griffis. 1984. Toxicity of 

copper-spiked sediments to freshwater invertebrates. Environmental Toxicology and 

Chemistry 3: 435-445. 

6 Gentile, J. H., K. H. Scott, S. M. Lussier, and M. S. Redmond. 1987. The assessment of 

Black Rock Harbor dredged material impacts on laboratory population responses. 

Report D-87-3. Prepared for: Department of the U.S. Army Corps of Engineers, 

Washington, D.C. and U.S. EPA, Washington D.C. by: Vicksburg, Mississippi: U.S. 

Army Engineer Waterways Experiment Station. 3 pp + Appendix 

 

1 Numbers correspond to reference numbers used in Table 8-1. 

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7 Horning. W. B., and C. I. Weber, (eds.). 1985. Short-term methods for estimating the 

chronic toxicity of effluents and receiving waters to freshwater organisms. Technical 

Support Document for Water Quality-based Toxics Control. EPA 440/4-85-032. 

Washington, D.C.: U.S. Environmental Protection Agency, pp 28-41. 

8 Ingersoll, C. G. and M. K. Nelson. 1990. Testing sediment toxicity with Hyalella azteca

(Amphipoda) and Chironomus riparius (Diptera). In: Landis, W. G. and W. H. van der 

Schalie (eds.). Aquatic Toxicology and Risk Assessment: Thirteenth Volume. Philadelphia: 

ASTM STP 1096, pp. 93-109. 

9 Kaputska, L. 1992. Personal communication. Ecological Planning and Toxicology, Inc. 

Corvallis, Oregon. 

10 Long, E.R. 1992. Personal communication. Coastal Monitoring and Bioeffects Division, 

Office of Ocean Resources Conservation and Assessment, Seattle: National Oceanic 

and Atmospheric Administration. 

11 Long, E.R. 1997. Personal communication. Coastal Monitoring and Bioeffects Division, 

Office of Ocean Resources Conservation and Assessment, Seattle: National Oceanic 

and Atmospheric Administration. 

12 Long, E. R., M. F. Buchman, S. M. Bay, R. J. Breteler, R. S. Carr, P. M. Chapman, J. E. 

Hose, A. L. Lissner, J. Scott, and D. A. Wolfe. 1990. Comparative evaluation of five 

toxicity tests with sediment from San Francisco Bay and Tomales Bay, California. 

Environmental Toxicology and Chemistry 9(9). 1193-1214. 

13 Malueg, K. W., G. S. Schuytema, D. F. Krawczyk, and J. H. Gakstatter. 1984. 

Laboratory sediment toxicity tests, sediment chemistry and distribution of benthos 

macroinvertebrates in sediments from the Keweenaw Waterway, Michigan. 

Environmental Toxicology and Chemistry 3: 233-242. 

14 McGee, B.L., C. E. Schlekat and E. Reinharz. 1993. Assessing sublethal levels of 

sediment contamination using the estuarine amphipod Leptocheirus plumulosus. 

Environmental Toxicology and Chemistry 12:??. pp 577-587. 

15 Nebeker, A. V., M. A. Cairns, J. H. Gakstatter, K. W. Malueg, G. S. Schuytema, and D. 

F. Krawczk. 1984. Biological methods for determining toxicity of contaminated 

sediments to freshwater invertebrates. Environmental Toxicology and Chemistry 3: 617-

630. 

16 Nipper, M. G., D. J. Greenstein, and S. M. Bay. 1989. Short- and long-term sediment 

toxicity test methods with the amphipod Grandidierella japonica. Environmental 

Toxicology and Chemistry 8: 1191-1989. 

17 Oakden, J. M., J. S. Oliver and A. R. Flegal. 1984. EDTA chelation and zinc 

antagonism with cadmium in sediment: Effects on the behavior and mortality of two 

infaunal amphipods. Marine Biology 84: 125-130. 

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18 Pastorok, R. A. and D. S. Becker. 1989. Comparison of bioassays for assessing 

sediment toxicity in Puget Sound. Bellevue, Washington: PTI Environmental Services, 

For U.S. EPA Region 10 Office of Puget Sound. Executive Summary + pp. 1-85. Tables 

D-1 to D-6. 

19 Porcella, D. B. 1983. Protocol for bioassessment of hazardous waste sites. EPA 600/2-

83-054. Corvallis, Oregon: U.S. Environmental Protection Agency, pp A-29–A-38. 

20 Puget Sound Estuary Program. 1991. Interim Final. Recommended guidelines for 

conducting laboratory bioassays on Puget Sound sediment. Prepared for: U.S. EPA 

Region 10, Seattle, July 1991. 81 pp. 

21 Rogerson. P. F., S. C. Schimmel, and G. Hoffman. 1985. Chemical and biological 

characterization of Black Rock Harbor dredged material. Technical Report D-85-9. 

Field Verification Program (Aquatic Disposal) Final Report. Washington, D.C.: 

Prepared for Department of the Army, U.S. COE, and U.S. EPA.

22 Sasson-Brickson, G. and G. A. Burton, Jr. 1991. In situ and laboratory sediment toxicity testing 

with Ceriodaphnia dubia. Environmental Toxicology and Chemistry Vol. 10: 201-207. 

23 Schlekat, C.E., B.L. McGee & E. Reinharz. 1992. Testing sediment toxicity in Chesapeake Bay 

with the amphipod Leptocheirus plumulosus: an evaluation. Environmental Toxicology & 

Chemistry 11:2. 225-236. 

24 Swartz, R. C., W. A. DeBen, J. K. P. Jones, O. J. Lamberson, and F. A. Cole. 1985. 

Phoxocephalid amphipod bioassay for marine sediment toxicity. ASTM STP 854: R. D. 

Cardwell, R. Purdy, and R. Comotto-Bahner (eds.). Aquatic Toxicology and Hazardous 

Assessment. Proceedings of the Seventh Annual Symposium. Milwaukee, Wisconsin, April 17-

19, 1983, Baltimore: American Society for Testing and Materials. pp 284-307. 

25 Thomas, J. M. and J. F. Cline. 1985. Modification of the Neubaurer technique to assess toxicity

of hazardous chemicals in soils. Environmental Toxicology and Chemistry 4: 201-207. 

26 U.S. Environmental Protection Agency. 1994. Methods for Measuring the Toxicity 

and Bioaccumulation of Sediment-associated Contaminants with Freshwater 

Invertebrates. EPA/600/R-94/024. Duluth, Minnesota: Office of Research and 

Development. 133 pp. 

27 Westerman, A. 1989. Yearly chronic toxicity comparisons of stream sediments and 

waters. In: G. W. Suter II and M. A. Lewis (eds) Aquatic Toxicology and Environmental 

Fate: Eleventh Volume, ASTM STP 1007. Philadelphia: American Society for Testing 

and Materials. pp. 204-214. 

28 Williams, L. G., P. M. Chapman, and T. C. Ginn. 1986. A comparative evaluation of 

marine sediment toxicity using bacterial luminescence, oyster embryo, amphipod 

sediment bioassays. Marine Environmental Research 19: 225-249. 

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August 1997 8-40

29 Word, J. Q., J. A. Ward, B. Brown, B. D. Walls, and S. Lemilich. 1989. Relative 

sensitivity and cost of amphipod bioassays. In: Oceans '89. An International 

Conference Addressing Methods for Understanding The Global Ocean, IEEE 

Publication Number 89CH2780-5. Sept. 18-21, 1989, Seattle: IEEE> pp. 467-473. 
HAZMAT 93-1–Protocols 

CHAPTER 9 

AQUATIC PHYSICO-CHEMICAL PARAMETERS 

DEFINITION 

Aquatic physico-chemical parameters are those characteristics of the aquatic 

environment that dictate the type of biological community that should be present under 

uncontaminated conditions. In order to properly interpret both biological and chemical 

contaminant data it is necessary to understand the physico-chemical characteristics of the 

environment being assessed. In this case, it is the aquatic environment, which includes both 

sediment and water. This is because the physico-chemical parameters (e.g., temperature, 

salinity, grain size, total organic carbon [TOC], dissolved organic carbon [DOC], pH, and 

redox potential) can affect all aspects of the biological community present, including the 

presence or absence of specific organisms, organismal behavior (e.g., burrowing and food 

selection), and the bioavailability of chemical contaminants. 

TEMPERATURE 

While temperature may be the easiest physico-chemical sediment parameters to 

measure, it is still a very important one. It influences the rates of both abiotic and biotic 

chemical reactions, and thus not only affects other physico-chemical parameters but 

contaminant (particularly metal and metalloid) bioavailability and toxicity as well (Burton, 

2000). An example of temperature’s influence on these various factors is that an increase in 

temperature will cause an increase in biological metabolism, which then results in an 

increase in oxygen consumption leading to reduced or anoxic sediment (lower Eh). The 

increase in reduced or anoxic sediment will cause an increase in acid volatile sulfides, which 

then bind with divalent metal ions making them less bioavailable. 

The temperature of sediments and associated interstitial water varies much more slowly 

over time than that of the overlying water. Sediment temperature generally varies on a 

seasonal scale, so the temperature taken at the time of sampling is usually a good 

representation of the sediment temperature for that season at that location. The exception to 

this assumption is tidally influenced sediment, which may show a diurnal variation in 

temperature. 

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The benthic community at a site will be adapted to the normal temperature variations 

occurring there. When evaluating the recorded temperature of sediment it is necessary to 

know if it falls within the normal range for the site. If it does not, it may cause stress to the 

biota in addition to that caused by any chemical contaminants present. Sediment toxicity 

tests need to be run at the appropriate temperature for the test organism; if it is different 

than the site temperature, the difference must be taken into account when the tests are 

evaluated. 

SALINITY 

Salinity is a parameter that defines whether an aquatic system is classified as fresh 

water, brackish, or salt water. Historically, salinity has been defined as “the total amount of 

solid material in grams contained in one kilogram of sea water when all the carbonate has 

been converted to oxide, the bromine and iodine replaced by chlorine, and all organic 

matter completely oxidized” (Sverdrup et al., 1942) and is reported as parts per thousand 

(ppt or ‰). In fresh water it is more simply defined as “the total concentration of ionic 

components” (Reid and Wood, 1976). As suggested by the definition’s complexity, the 

actual measuring of salinity is a complex and difficult process. One of the properties of 

seawater is described by the “rule of constant proportions”—that no matter how salinity 

may vary, the proportions of seawater’s major components remain constant (Anikouchine 

and Sternberg, 1973). Thus by measuring one component, one is able to calculate the 

concentrations of all components. Until the 1950s, salinity calculations usually took 

advantage of this rule: the concentration of chloride ions could easily and accurately be 

measured by titration, and salinity calculated from the result (salinity [‰] = 0.030 + 1.8050 ×

chlorinity [‰]). In the 1950s, scientists began to measure salinity using meters that utilized 

the electrical or magnetic properties of seawater that vary in proportion to its salinity, 

electrical conductance being the most common. 

In 1978, under the auspices of UNESCO, in order to increase interlaboratory uniformity 

and accuracy, a new internationally accepted definition of salinity was promulgated based 

on a potassium chloride (KCl) standard. Salinity (S) of a seawater sample is now defined in 

terms of the ratio (K) of the sample’s electrical conductivity (at 15°C and the pressure of one 

standard atmosphere) to a KCl solution (in which the mass fraction of KCl is 0.0324356, at 

the same temperature and pressure). The K value exactly equal to one corresponds, by 

definition, to a practical salinity equal to 35. Because the value is based on a ratio, it has no 

assigned units; while it is sometimes given the units “psu” (practical salinity units), they are 

considered inappropriate. 

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At freshwater and open ocean sites salinity will play only a minor role, if any, in site 

evaluation because it is either at insignificant concentration (freshwater sites), or it varies 

little both spatially and temporally across the study area (open ocean) even though it is 

present in significant amounts. However, at coastal sites such as embayments and estuaries, 

salinity will occur in significant amounts as well as vary significantly both spatially and 

temporally. It should be noted that high salinity inputs can occur in fresh water (e.g., from 

potash mines) and can be as toxic in their own right as any other contaminant. 

The salinity variability will affect both the local biotic community and 

contaminant/toxicant chemistry. Salinity changes may stress organisms, making them 

more susceptible to the additional stress imposed by toxic chemicals, or they may change 

the way an organism processes contaminants taken up. Salinity changes can affect 

bioavailability and toxicity of both organic and inorganic chemicals; the exact relationship 

depends on both the chemical and the organism involved. In general, as contaminated fresh 

water enters an estuary and mixes with seawater, the increase in salinity will cause metals 

to precipitate out of the water column, making them less bioavailable and less toxic. 

However, the same increase in salinity can cause the release of ammonia, resulting in 

ammonia toxicity (Rysgaard et al., 1999). 

A change in salinity also affects the toxicity of organic contaminants, but not consistently 

as it does with metal contaminants. Brecken-Folse et al. (1994) showed that the toxicity to 

sheepshead minnows (Cyprinodon variegatus) of both 4-nitrophenol and 2,4-dinitrophenol 

decreased with increasing salinity. However, in the same experiment they showed that 

while 4-nitrophenol toxicity to grass shrimp (Palaemonetes spp.) decreased with increasing 

salinity, the toxicity of 2,4-dinitrophenol to grass shrimp increased with increasing salinity. 

Even closely related species may respond differently to salinity toxicant interactions. 

Martello et al. (2000) found the effect of high-salinity stress plus pentachlorophenol (PCP) to 

be subadditive among red abalone, while high-salinity stress seemed to potentiate the effect 

of PCP on black abalone. 

In summary, when evaluating the risk posed by contaminants in coastal or estuarine 

environments, one must take into account that salinity will affect all bioassessment tools. 

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SEDIMENT GRAIN SIZE 

The grain-size of sediments is a fundamental characteristic of the sediment environment 

for three major reasons. First, the habitat for benthic organisms is determined in part by 

sediment grain size. For example, the feeding strategies of many organisms require suitable 

substrate: filter feeders need relatively coarser material and detrital consumers need finegrained sediments. As a result, different sediment textures support different "normal" 

communities of benthic organisms. Texture is thus vital when interpreting benthic 

community data (e.g., composition and diversity) to determine whether an area has been 

impacted. 

Second, grain-size is an important factor in the accumulation of toxic substances in 

sediments. Exposure of many dissolved substances to sediments and to particulate matter 

in the water column (which settles to become the sediments) results in the sorption of those 

substances by the particulate matter. This process is a surface phenomenon, so sorption 

increases with increasing available surface area. Therefore, because finer-grained particles 

have larger surface area per mass (dry weight), the finer particles have the potential to 

accumulate more toxic substances per dry weight than coarser particles. In response to 

similar inputs, coarse-grained sediments (sands and gravels) may exhibit minimal uptake of 

toxic substances, while fine sediments (silts and clays) in the same area may accumulate 

high concentrations. Thus chemical data obtained from coarse sediments at a site may 

provide low estimates of exposure and the threat to natural resources, while fine-grained 

sediments may contain much higher concentrations but not necessarily represent "hotter" 

areas (more contaminated). In addition, grain-size measurements are one way to account 

for the effects of differences in the accumulation associated with textural differences. 

Note that the differences in concentration among spatial areas that can be corrected for 

by normalizing to the sediment grain-size primarily reflect a response to the input and 

transport of toxic substances. The data are less clear as to how important the accumulation 

potential is in determining toxicity. It is considered likely that at least part of the 

bioavailability of many substances, particularly organic compounds, is basically the reverse 

of contaminant accumulation in the sediment; that is, the same contaminant concentrations 

are more bioavailable in coarse sediments than in fine. On the other hand, for those 

organisms that actually ingest the sediments, it is not clear whether sediment texture affects 

bio-uptake of contaminants. 

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Specific grain-size data can be used semi-quantitatively to determine, for example, that 

an area had too coarse a sediment to provide useful chemical data (the sediments would not 

be expected to significantly accumulate toxic substances), or that a finer-grained sediment 

area should have generally higher contaminant concentration. These data can also be used 

quantitatively, for example, by generating correlation plots of sediment texture versus the 

concentration of a particular contaminant. (In most systems distribution is a function of 

proximity to a source and to the effects of differential accumulation and settling.) In areas 

that are not close to the source, such correlations are usually quite linear. Such a correlation 

thus allows one to interpret the contamination’s spatial distribution as the result of either 

differential settling (coarse sediments with relatively low concentrations in one area, fine 

sediments with higher concentrations in another) or due to proximity to a source (much 

higher, non-linear relationship between concentration and sediment texture). 

The third major reason is that grain size is a good indicator of physical energy at the 

sampling site (i.e., sand ≈ high energy, silt ≈ low energy). When assessing contaminated 

sites it is recommended that sample locations be biased toward depositional areas (silty, 

low-energy areas) and away from sandy, high-energy areas if possible. Grain-size analysis is 

necessary to ensure proper site selection and to properly interpret chemical contaminant 

data. The preferred reporting method for such analysis is in percentages of sand (>0.063 – 2 

mm), silt (>2 – 63 μm), and clay (• 2 μm) (Mudroch and Bourbonniere, 1991). 

Another reason for knowing the sediment grain size is that it can affect the choice of test 

organism for sediment toxicity tests. DeWitt et al. (1988) found the percent mortality of the 

amphipod Rhepoxynius abronius was positively correlated with percent fine-grained 

sediment in unpolluted sediments. Specifically, in sediments with 80% or greater silt-clay 

content, survival can be reduced by 15%. They concluded that either the fine-grain size was 

directly causing amphipod mortality, (by such means as clogging the gills) or some other 

unmeasured covarying parameter was responsible. 

TOTAL SOLIDS/MOISTURE CONTENT 

The principal difference between soils and sediments is that, by weight, soils are 

composed almost exclusively of solids, with relatively little if any moisture content, while 

the major component of sediments by weight is water. Both total solids and moisture 

content are frequently reported as a percent, and as such they are the complement 

measurement of each other. The standard approach for both percent total solids and 

percent moisture is to weigh the wet sediment, dry it at between 50° and 105°C, and 

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reweigh the dried sediment. The dried weight of the sediment is the total solids content and 

the difference between the wet weight and the dry weight is the moisture content; to arrive 

at percent total solid and percent moisture, respectively, each amount is divided by the wet 

weight. 

Percentages of total solids and moisture are measured to allow conversion of wet-weight 

values of the samples taken for other chemical analyses to be converted to dry weight and to 

determine the appropriate quantity of sediment needed to ensure sufficient solids for 

chemical analysis. Very high moisture content (>70%) can actually reduce the reliability of 

the sediment chemical analysis. One approach to this problem is to accept the chemical 

analysis results unconditionally if moisture content is less than 70%; give a qualified 

acceptance of the results if the moisture content is between 70% and 90% and reject the 

results if the moisture content exceeds 90% (Finkelstein, 2002). Another approach, which 

has been tried, is to freeze-dry the sediments prior to analysis (Finkelstein, 2002). 

The degree of consolidation of sediments, which is important to the biota, also 

influences the assessment methods used to evaluate sediments. Sediments with low total 

solids and high moisture content will cause standard grab samplers to penetrate too deeply, 

and any biotic sampling devices placed on the sediment surface (e.g., bivalve sampling 

racks for bioaccumulation studies or biota traps) may actually sink into the sediment, 

rendering them useless. Knowing the degree of sediment consolidation is also useful for 

engineering and feasibility studies. 

As with all sediment parameters, total solids and moisture content affect the benthic 

community of the sampled sediments and may influence toxicity test results. For example, 

densely packed sediments (i.e., high total solids/low moisture content) may impede 

burrowing, tube building, or feeding by test organisms, thus modifying sediment toxicity by 

either reducing exposure (i.e., organism stays on sediment surface) or enhancing organism 

stress (i.e., fatigue from burrowing attempts) (Lamberson et al., 2000). 

TOTAL VOLATILE SOLIDS AND TOTAL ORGANIC CARBON 

Measurements of total volatile solids (TVS) and total organic carbon (TOC) provide data 

on slightly different aspects of sediments, as discussed below, but their primary intent is to 

provide a measure of the amount of organic matter in the sediments. TVS is specifically a 

measure of all volatile components of the sediment that are lost during high-temperature 

combustion (both organic and inorganic): TOC is specifically a measure of carbon that is 

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bound up in organic matter. Total organic matter (TOM) in water consists of thousands of 

components, including macroscopic particles, colloids, dissolved macromolecules, and 

specific compounds. In most cases, the total amount of organic matter in sediments is 

associated with plant debris, not with the toxic substances themselves. There are 

exceptions; for example, areas contaminated with coal tar will have high TOC, but it will be 

associated with the coal tar contamination. Organic content is also one of the fundamental 

characteristics of sediments and TVS/TOC values are used in a manner very analogous to 

that of grain size. 

In most areas, organic matter in sediment forms the food for many benthic organisms. 

Organic-poor sediments (TOC <0.25%) can be compared to infertile farm fields; they are not 

capable of supporting abundant benthic organisms. On the other hand, very organic-rich 

sediments (TOC >15%) may be inhospitable to many larger organisms because microbial 

activity consumes all of the available oxygen and may form natural toxic substances (e.g., 

ammonia and sulfides). 

The sorption energetics of some toxic substances, particularly some low-solubility 

organic compounds but also some metals, strongly favor accumulation on organic surfaces 

over inorganic particles. As a result, sediments with high TOC tend to accumulate higher 

concentrations of toxic substances than do low-TOC sediments from the same area. Thus 

TVS/TOC data help interpret the distribution of contamination and the threat to natural 

resources. The TOC/TVS data can be used in the same way as grain-size data (discussed 

above). It should also be noted that because some natural organic matter accumulates on 

sediment particles in the same way that toxic substances do, and because organic particles 

tend to be of small size and have a low settling velocity, they settle to the bottom only in 

quiet-water areas. Thus, high concentrations of TOC tend to correlate with areas of finegrained sediments. 

The equilibrium partitioning theory assumes that sediment organic carbon controls the 

sorption of contaminants onto sediment particles, and that these sorbed contaminants are 

not bioavailable. This assumption is not universally accepted because sediment organic 

carbon is also the principal food source for many benthic organisms. Boese et al. (1990), in 

their work with the clam Macoma nasuta and the contaminant hexachlorobenzene (HCB), 

concluded that 63% to 84% of the HCB tissue residues were derived by uptake through the 

gut from digested solids and only 11% to 12% were derived by uptake via the gills from 

interstitial water. Gunnarsson et al. (1999), working with the brittle star Amphiura filiformis

and five different sources of organic carbon, concluded that the type of organic carbon, 

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specifically its quality as a food source, was critical in the uptake of sorbed contaminants. 

They found bioaccumulation factors ranging from 2.7 for lignin to 14.6 for sea lettuce (Ulva 

lactuca) and directly correlated with the quality of the organic carbon as a food source. Selck 

et al. (1998), in studying the toxicity and toxicokinetics of cadmium in the polychaete 

Capitella sp., concluded that sediment-bound cadmium contributed 95% of the total 

cadmium taken up by the feeding worms. This conclusion agrees with that of Wang et al.

(1999), who concluded that most (>98%) of the cadmium (Cd), cobalt (Co), selenium (Se), 

and zinc (Zn) in polychaetes was derived from ingested sediment because of the high 

ingestion rates and the low uptake rates from pore water. 

Schlekat et al. (2000) also looked at the effect on contaminant uptake of different organic 

carbon sources. They determined the uptake efficiency of the amphipod Leptocheirus 

plumulosus for silver (Ag), cadmium, and zinc from seven sources: bacterial exopolymeric 

sediment coatings, polymeric coatings made from Spartina alterniflora extract, amorphous 

iron oxide coatings, the diatom Phaeodactylum tricornutum, the chlorophyte Dunaliella 

tertiolecta, processed estuarine sediment, and fresh estuarine sediment. The highest 

assimilation efficiencies for silver and cadmium were from the bacterial exopolymeric 

coatings, while for zinc the highest efficiencies were for phytoplankton and processed 

sediments. Ortego and Benson (1992) found that humic acid reduced the toxicity of the 

insecticide fenvalerate, but fulvic acid had no effect on its toxicity (as tested by the Microtox®

bioassay), while both reduced the toxicity of permethrin. Lee et al. (1993) found that the 

reduction of chemical toxicity due to the presence of organic matter (specifically, dissolved 

humic material [DHM]) was dependent on the specific chemical contaminant. DHM 

reduced the toxicity of 4-chloroanilin to Daphnia magna, but it failed to reduce the toxicity of 

both tetrabromobisphenol-A and pentachlorophenol. 

On the other side of the issue, DeWitt et al. (1992), studying the toxic responses of the 

amphipod Rhepoxynius abronius to fluoranthene, found that differences in organic matter did 

affect toxicity. But since the absolute range was small, equilibrium partitioning based 

sediment quality guidelines do not need to be corrected for organic matter quality. Ankley 

et al. (1994), working with Chironomus tentans, concluded that within the range of organic 

carbon tested (3% to 8%) the equilibrium-partitioning model based on organic carbon is 

appropriate for predicting the bioavailability of sediment-associated chlorpyrifos to benthic 

invertebrates. It should be noted that individual studies on either side of the issue looked 

only at a single species, whereas, the importance of type of organic matter may be species 

dependent. Also, those studies that looked at various types of organic matter did not 

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generally look at mixtures: in situ sediments, whether anthropogenically influenced or not, 

contain mixtures of organic matter, and the variability in in situ sediments may not be 

significant. Also, regardless of issues of uptake control by biota, as mentioned earlier, in 

areas contaminated by substances such as coal tar the TOC is actually associated with the 

contaminants. Any attempt to determine the toxicity of these sediments by normalizing to 

TOC will greatly underestimate the toxicity. Since there is currently no resolution of this 

issue, the best course is to evaluate the individual sediments at each site in order to 

determine the bioavailability of contaminants. 

TVS is measured by the loss of weight from high-temperature combustion of the 

sediments. This procedure results in the total oxidation of organic matter, oxidation of some 

inorganic substances, and dehydration of some minerals. TOC is measured by a number of 

techniques, usually less severe than TVS, and also with detection procedures specific for 

carbon. The U.S. Environmental Protection Agency’s preferred method is found in the 

Puget Sound Protocols and Guidelines (U.S.EPA, 2001). Depending on the technique, TOC 

may or may not measure all carbon if it is in large particles (wood fragments) or stable 

forms (e.g., coal fragments). In most sediments, TVS measurements will yield higher values 

than TOC, but the two are usually fairly well correlated. Because TOC appears more likely 

to measure organic components that have biological significance, it is generally the 

preferred measurement. 

OIL AND GREASE 

Oil and grease (O&G) measurements determine the concentrations of a specific class of 

organic substances, those soluble in a non-polar organic solvent. The substances recovered 

in an O&G extraction include a broad range of natural and anthropogenic compounds, but 

in most cases high O&G values reflect predominately human impacts. With regard to toxic 

substances, some of the target compound list substances would be included in O&G 

measurements, but would rarely be more than a small fraction of the total material 

recovered. There is some evidence that O&G distributions correlate with those of some 

toxic substances, probably a reflection of similar accumulation mechanisms and similar 

input sources. O&G has been used more as a general indicator of anthropogenic 

contamination and predates most modern analytical procedures, but its use at hazardous 

waste sites has been minimal. 

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TOTAL SULFIDE 

Sulfides are compounds containing one or more sulfur atoms connected directly to a 

carbon, metal, or other non-oxygen atoms. In sediments, sulfides exist as insoluble 

precipitates and as dissolved sulfide compounds. Hydrogen sulfide (H2S), the 

toxicologically important form of sulfide, is produced when bacteria reduce sulfates and 

putrefy proteins. Measurements of sediment sulfide content are relevant primarily in 

marine sediments, because the sulfate present in seawater provides a major reservoir of 

reducible material that supports the growth of microbial populations when available oxygen 

has been consumed. Total sulfide measurements provide an indication of how reducing an 

environment is (negative Eh values). Sulfides in pore water may be analyzed as total 

sulfides (TS), as dissolved sulfides (DS), and as hydrogen sulfide. TS consist of acid-soluble 

metallic sulfides in suspended matter plus dissolved hydrogen sulfide. DS remain after the 

suspended solids have been removed by flocculation and settling. Hydrogen sulfide may 

be analyzed directly or calculated from DS concentration, sample pH, and the H2S 

ionization constant (U.S. Army COE, 1995). 

A number of mineral phases that precipitate and sorb toxic trace metals under aerobic 

conditions, dissolve under anoxic conditions and may thus make sediment-bound metals 

much more available. On the other hand, most toxic metals form very insoluble sulfide 

minerals. In addition, because sulfide is toxic to most organisms and is formed when 

oxygen is absent, sediments with high sulfide levels are often devoid of any life save 

specialized microorganisms. 

When handling sediments to be analyzed for sulfides, care must be taken to prevent 

oxygenation of the sediments, which will rapidly convert the sulfides into sulfate. On the 

other hand, insufficient oxygenation of sediments during toxicity tests may result in a 

buildup of sulfides to toxic levels, thus confounding the test results. To avoid this problem, 

it is wise to monitor for sulfides while running toxicity tests. 

Interestingly, the human nose is more sensitive to hydrogen sulfide than any available 

chemical tests. Thus, field observations for the presence of sulfide are important. However, 

variables such as wind and temperature, as well as the fact that the nose rapidly loses its 

sensitivity to sulfide after repeated exposure, make it difficult to rely on field measurements 

for quantitative estimates. 

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ACID VOLATILE SULFIDES 

Acid volatile sulfides (AVS) are operationally defined metal sulfides that react with 

aqueous acids at room temperature to liberate gaseous hydrogen sulfide (Boothman et al., 

2001). In most sediments AVS are composed principally of amorphous ferrous sulfide (FeS) 

and manganese sulfide (MnS), while in contaminated sediments other divalent metal 

sulfides can make up a significant portion of the AVS present. It is the ability of these other 

divalent metals to displace the iron and manganese that make AVS a controlling factor in 

the concentration of dissolved metals in sediment interstitial water and thus their 

bioavailability and toxicity. The interpretation of AVS data requires the concurrent 

measurement of another operationally defined class of sediment components, 

simultaneously extracted metals (SEM). SEM are defined as those metals whose sulfides are 

less soluble than ferrous sulfide and are extracted from the sediments during the acidvolitization step of the analysis (Boothman et al., 2001). The divalent metals most commonly 

found as SEM are nickel, zinc, cadmium, lead, and copper. Silver is also sometimes found, 

and while mercury can also form an AVS, other factors are more important in determining 

mercury’s bioavailability and toxicity (U.S. EPA, 2000). 

The basic theory behind the use of AVS in sediment evaluation is as follows: when the 

molar concentration of AVS is greater than the combined molar concentration of SEM, the 

SEM will be bound up as sulfides, making them unavailable to biota and thus nontoxic. 

When there is more SEM than AVS, the metals are potentially bioavailable and toxic. The 

theory is based on the assumption that the dissolved divalent metal ion is the only 

bioavailable and toxic form of SEM and that bound metals are not bioavailable and 

therefore nontoxic. The earliest work on the AVS/SEM relationship involved cadmium 

only (Di Toro et al., 1990), and virtually as soon as the cadmium concentration exceeded 

AVS, toxicity was observed. When studies with other metals or metal combinations were 

performed, no toxicity was observed when AVS exceeded SEM; but, unlike the work with 

cadmium, toxicity was not always immediately observed when SEM exceeded AVS 

(Ankley et al., 1991, 1993; Besser et al., 1996). It was realized that, depending on the metal, 

other factors in addition to AVS were controlling its bioavailability, in particular organic 

carbon (Mahoney, 1995). It has been suggested that instead of trying to base sediment 

quality guidelines for metals on AVS alone, the guidelines can be based on both AVS and 

organic carbon (Ankley et al., 1996) 

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Other work has raised questions about the absolute reliability of the AVS/SEM 

paradigm. Hare et al. (1994) found in colonization and growth studies of cadmium 

contaminated test sediments that the abundance of only one taxa was related to the 

Cd/AVS ratio, while for the 17 other taxa there was no relationship between the Cd/AVS 

ratio and either abundance or growth. Lee and coworkers (B-G. Lee et al., 2000a,b) looked at 

bioaccumulation of metals in five marine benthic species and its relationship to AVS/SEM 

and porewater concentrations. They ran experiments in which metal concentrations 

(cadmium, nickel, and zinc) were varied while AVS concentrations remained fixed, and in 

which AVS concentrations varied while metals concentrations were fixed. They found that 

metal concentrations in animal tissue correlated with metal concentrations extracted from 

the sediments, not with AVS/SEM or porewater concentrations, in 13 out of 15 metal-animal 

combinations. They concluded that the influence of AVS on bioavailability of metals to 

benthic organisms depends on the organism and the metal. For a good review of these 

issues, see Mason (2000). 

Based on current knowledge, SEM/AVS data can best be used in conjunction with 

bioaccumulation and toxicity studies as a tool to explain the studies’ results, with the 

understanding that depending on the metals and organisms involved SEM/AVS may not be 

able to explain the results. SEM/AVS data by itself should not be used to predict the lack of 

adverse biological effects. One final important point when analyzing and evaluating 

SEM/AVS data is that AVS can vary both temporally and spatially (Besser et al., 1996, and JS. Lee et al., 2000). SEM/AVS data must be collected at the same time as the studies are 

performed and from the portion of sediment in contact with the biota. 

pH 

The pH value of sediments (a measure of hydrogen ion concentrations) indicates their 

acidity (low pH values) or basic/alkaline nature (high pH values) (more accurately, it is a 

measure of the pH of the interstitial water). Neutral sediments have a pH of 7, and "normal" 

pH values usually range from 6 to 8. This parameter is important to measure because many 

substances, particularly inorganic substances, may undergo a number of reactions in natural 

systems that are pH dependent. Some of these values are relatively simple, phase-change 

reactions. For example, iron/manganese oxyhydroxides form as solid phases, coating other 

sediment particles at neutral and high pH but dissolving at low pH. Many other metals 

form similar oxyhydroxides or are scavenged by the oxyhydroxides. As a result of these 

processes, the metals tend to be much more mobile (because they are dissolved) at low pH 

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(acid) and less mobile (because they precipitate) at high pH. Other more complex reactions 

are also possible. 

The solubility, hence mobility, of acidic and basic organic compounds is also affected by 

pH, although the importance of this effect is usually relatively minor compared to the 

elements. 

Finally, the toxicity of many substances is affected by pH, in part because of changes in 

mobility/bioavailability and in part because of additional stress on the organisms. 

Eh (ELECTRODE POTENTIAL) 

The Eh of the sediments (actually of the interstitial water) is a measure of 

oxidation/reduction potential. The latter indicates the direction of change for substances 

that can undergo oxidation and/or reduction reactions. Fundamentally, natural systems in 

which molecular oxygen is present (aerobic) will show high-position Eh values and 

represent oxidizing environments. As oxygen is consumed by chemical and biological 

reactions (all metabolism is primarily the oxidation of organic matter), the system becomes 

increasingly reducing. Systems in which all oxygen has been consumed (anoxic) will show 

negative Eh values. 

Similar to the effects of changes in pH, many substances, particularly the elements, 

undergo phase and/or speciation (form) changes depending on Eh. For example, at 

low/negative Eh (reducing environments) the iron/manganese oxyhydroxides are reduced, 

liberating the iron and manganese as dissolved metals. In addition, depending on the Eh 

value, iron may change from a valance state of +3 to +2. Other elements undergo similar 

changes. Further, a number of more dominant substances may also be altered. For 

example, nitrogen is predominantly oxidized to the nitrate ion in aerobic systems but 

reduced to ammonia in anoxic systems. Similarly sulfate is reduced to sulfide under anoxic 

conditions. The latter reaction is particularly important in seawater, where the relatively 

high concentration of sulfate can form high concentrations of sulfide. 

All of these changes are important for three reasons. 

1. As with pH, the mobility and availability of substances may be markedly different at 

different Eh values, but these changes may be complex. For example, the sulfides of many 

toxic metals are even less soluble than their oxidized form. 

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2. The form of the substances may change, generating more or less toxic forms. For 

example, the hexavalent form (i.e., oxidized chromium) is much more toxic than the 

trivalent form (i.e., reduced chromium). 

3. As with pH, low oxygen conditions add additional stress to most organisms and thus 

may make them more susceptible to the effects of toxic substances. 

SUMMARY 

When conducting a biological assessment of contaminated sediments it is necessary to 

understand their physico-chemical parameters and chemical contaminant concentrations in 

order to properly interpret the results. Many of these parameters (e.g., temperature, AVS, 

Eh) vary significantly by time scales (e.g., diurnally, seasonally), and it is necessary to 

understand how such variations affect contaminant bioavailability. This variability must be 

taken into account when making any conclusions based on the bioassessment. 

The parameters discussed in this chapter are not the only ones that can be used to 

evaluate sediment contamination and toxicity, but they are the ones that are either necessary 

or most helpful under virtually all conditions. Other parameters that can aid in sediment 

contamination and toxicity interpretation under certain conditions include ammonia, 

aluminum, and iron, to name a few. 

Finally, one of the best recent references for protocols for many of the measurements 

discussed above (grain size, total solids, TVS, TOC, O&G, and total sulfides) is the. 

Recommended Protocols for Measuring Conventional Sediment Variables in Puget Sound. (Tetra 

Tech, 1986). This reference, which is part of the Puget Sound Protocols and Guidelines, was 

based on modifications to standard procedures following formal review by scientists and 

laboratory personnel (academic, agency, and commercial) in the Puget Sound region and 

represents an attempt to provide the most useful methods. The protocols were developed 

specifically for work in Puget Sound, a marine/estuarine environment, but the protocols for 

these conventional variables should be usable anywhere. A current version of this document 

can be accessed on-line at: 

http://www.wa.gov/puget_sound/Publications/protocols/protocol.html 

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ADDITIONAL READING 

Buchanan, J.B. 1984. Sediment analysis. In: Methods for the Study of Marine Benthos, N.A. 

Holme and A.D. McIntyre, eds. Boston, Mass.: Blackwell Scientific Publications. pp. 41–65. 

Burgess, R.M. and R.A. McKinney. 1999. Importance of interstitial, overlying water and 

whole sediment exposures to bioaccumulation by marine bivalves. Environmental Pollution

104: pp 373–382. 

Di Toro, D.M., C.S. Zarba, D.J. Hansen, W.J. Berry, R.C. Swartz, C.E. Cowan, S.P. Pavlou, 

H.E. Allen, N.A. Thomas, and P.R. Paquin. 1991. Technical basis for establishing sediment 

quality criteria for nonionic organic chemicals using equilibrium partitioning. Environmental 

Toxicology and Chemistry 10(12): pp. 1541–1583. 

Folk, R.L. 1968. Petrology of Sedimentary Rocks. Austin, Tex.: Hemphill Publishing Co. 172 

pp. 

Harkey, G.A., M.J. Lydy, J. Kukkonen, and P.F. Landrum. 1994. Feeding selectivity and 

assimilation of PAH and PCB in Diporeia spp. Environmental Toxicology and Chemistry 13(9): 

pp. 1445–1455. 

Lee, H. II, B.L. Boese, and R.C. Randall. 1990. A method for determining cut uptake 

efficiencies of hydrophobic pollutants in a deposit-feeding clam. Environmental Toxicology 

and Chemistry 9(2): pp. 215–219. 

Lauenstein, G.G. and A.Y. Cantillo (eds.). 1993. Sampling and Analytical Methods of the 

National Status and Trends Program National Benthic Surveillance and Mussel Watch Projects 

1984-1992: Volume II Comprehensive Descriptions of Complementary Measurements. NOAA 

Technical Memorandum NOS ORCA 71. National Oceanic and Atmospheric 

Administration. Silver Spring, Maryland. 102 pp. 

Plumb, R.H. 1981. Procedures for Handling and Chemical Analysis of Sediment and Water 

Samples. Technical Report EPA/CE—81—1. Vicksburg, Miss.: U.S. Army Corps of 

Engineers. 

U.S. Environmental Protection Agency (EPA). 1983. Methods for Chemical Analysis of Water 

and Wastes. Technical Report EPA 600/4—79—020 (Revised 1983). Cincinnati, Oh.: 

Environmental Monitoring and Support Laboratory. 

REFERENCES 

Anikouchine, W.A. and R.W. Sternberg. 1973. The World Ocean An Introduction to 

Oceanography. Englewood Cliffs, N.J.: Prentice-Hall, Inc. 338 pp. 

Ankley, G.T., D.J. Call, J.S. Cox, M.D. Kahl, R.A. Hoke, and P.A. Kosian. 1994. Organic 

carbon partitioning as a basis for predicting the toxicity of chlorpyrifos in sediments. 

Environmental Toxicology and Chemistry 13(4): pp. 621–626. 

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Ankley, G.T., D.M. Di Toro, D.J. Hansen, and W.J. Berry. 1996. Technical basis and 

proposal for deriving sediment quality criteria for metals. Environmental Toxicology and 

Chemistry 15(12): pp. 2056–2066. 

Ankley, G.T., V.R. Mattson, E.N. Leonard, C.W. West, and J.L. Bennett. 1993. Predicting the 

acute toxicity of copper in freshwater sediments: Evaluation of the role of acid-volatile 

sulfide. Environmental Toxicology and Chemistry 12(2): pp. 315–320. 

Ankley, G.T., G.L. Phipps, E.N. Leonard, D.A. Benoit, V.R. Mattson, P.A. Kosian, A.M. 

Cotter, J.R. Dierkes, D.J. Hansen, and J.D. Mahoney. 1991. Acid-volatile sulfide as a factor 

mediating cadmium and nickel bioavailability in contaminated sediments. Environmental 

Toxicology and Chemistry 10(10): pp. 1299–1307. 

Besser, J.M., C.G. Ingersoll, and J.P. Giesy. 1996. Effects of spatial and temporal variation of 

acid-volatile sulfide on the bioavailability of copper and zinc in freshwater sediments. 

Environmental Toxicology and Chemistry 15(3): pp. 286–293. 

Boese, B.L., H. Lee II, D.T. Specht, R.C. Randall, and M.H. Winsor. 1990. Comparison of 

aqueous and solid-phase uptake for hexachlorobenzene in the tellinid clam Macoma nasuta

(Conrad): A mass balance approach. Environmental Science and Technology 9(2): pp. 221–231. 

Boothman, W.S., D.J. Hansen, W.J. Berry, D.L. Robson, A. Helmstetter, J.M. Corbin, and S.D. 

Pratt. 2001. Biological response to variation of acid-volatile sulfides and metals in fieldexposed spiked sediments. Environmental Toxicology and Chemistry 20:2, pp. 264-272. 

Brecken-Folse, J.A., F.L. Mayer, L.E. Pedigo, and L.L. Marking. 1994. Acute toxicity of 4-

nitrophenol, 2,4-dinitrophenol, terbufos and trichlorfon to grass shrimp (Palaemonetes spp.) 

and sheepshead minnows (Cyprinodon variegatus) as affected by salinity and temperature. 

Environmental Toxicology and Chemistry 13(1): pp. 67–77. 

Burton, G.A., Jr. 2000. Sediment collection and processing: Factors affecting realism. In: 

Sediment Toxicity Assessment, G.A. Burton, Jr., ed. Chelsea, Mich.: Lewis Publishers, Inc. pp. 

183-211. 

Dewitt, T.H., G.R. Ditsworth, and R.C. Swartz. 1988. Effects of natural sediment features on 

survival of the phoxocephalid amphipod, Rhepoxynius abronius. Marine Environmental 

Research 25: pp. 99–124. 

Dewitt, T.H., R.J. Ozretich, R.C. Swartz, J.O. Lamberson, D.W. Schults, G.R. Ditsworth, J.K.P. 

Jones, L. Hoselton, and L.M. Smithe. 1992. The influence of organic matter quality on the 

toxicity and partitioning of sediment-associated fluoranthene. Environmental Toxicology and 

Chemistry 11(2): pp. 197–208. 

Di Toro, D.M., J.D. Mahony, D.J. Hansen, K.J. Scott, M.B. Hicks, S.M. Mayr, and M.S. 

Redmond. 1990. Toxicity of cadmium in sediments: The role of acid volatile sulfide. 

Environmental Toxicity and Chemistry 9(12): pp. 1487–1502. 

Finkelstein, K., Coastal Resource Coordinator, NOAA Coastal Protection and Restoration 

Division. 2002. Personal communication. 

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Gunnarsson, J.S., M.E. Granberg, H.C. Nilsson, R. Rosenberg, and B. Hellman. 1999. 

Influence of sediment-organic matter quality on growth and polychlorobiphenyl 

bioavailability in Echinodermata (Amphiura filiformis). Environmental Toxicology and 

Chemistry 18(7): pp. 1534–1543. 

Hare, L., R. Carignan and M.A. Huerta-Diaz. 1994. A field study of metal toxicity and 

accumulation by benthic invertebrates; implications for the acid-volatile sulfide (AVS) 

model. Limnology & Oceanography 37:7. pp. 1653-1668. 

Lamberson, J.O., T.H. DeWitt, and R.C. Swartz. 2000. Assessment of sediment toxicity to 

marine benthos. In: Sediment Toxicity Assessment, G.A. Burton, Jr., ed. Chelsea, Mich.: Lewis 

Publishers, Inc. pp. 183–211. 

Lee, B-G., S.B. Griscom, J-S. Lee, H.J. Choi, C-H. Koh, S.N. Luoma, and N.S. Fisher. 2000a. 

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sediments. Science 287:5451, pp. 282-284. 

Lee, B-G., J-S. Lee, H.J. S.N. Luoma, Choi, and C-H. Koh. 2000b. Influence of acid volatile 

sulfides and metal concentrations on metal bioavailability to marine invertebrates in 

contaminated sediments. Environmental Science & Technology 34:21, pp. 4517-4523. 

Lee, J-S., B-G. Lee, S.N. Luoma, H.J. Choi, C-H. Koh, and C.L. Brown. 2000. Influence of 

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Lee, S.K., D. Freitag, C. Steinberg, A. Kettrup, and Y.H. Kim. 1993. Effects of dissolved 

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Mahony, D.J. 1995. Certain chemical aspects of heavy metal-sediment interactions: The role 

of organic carbon and the kinetics of metal sulfide oxidation. In: The Utility of AVS/EqP in 

Hazardous Waste Site Evaluations, D.A. MacDonald and S. M. Salazar, eds. NOAA Technical 

Memorandum NOS ORCA 87. Seattle, Washington. pp 23–26. 

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pentachlorophenol and salinity stress on phagocytic and chemotactic function in two 

species of abalone. Aquatic Toxicology 49(3): pp. 213–225. 

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elements into pelagic and benthic organisms. In: Sediment Toxicity Assessment, G.A. Burton, 

Jr., ed. Chelsea, Mich.: Lewis Publishers, Inc. pp. 127–149. 

Mudroch, A. and R.A. Bourbonniere. 1991. Sediment preservation, processing and storage. 

In: Handbook of Techniques for Aquatic Sediment Sampling, A. Mudroch and S. MacKnight, eds. 

Boca Raton, Fla.: CRC Press. pp. 131–170. 

Ortego, L.S. and W.H. Benson. 1992. Effects of dissolved humic material on acute toxicity of 

selected pyrethroid insecticides. Environmental Toxicology and Chemistry 11(2): pp. 261–265. 

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Reid, G.K. and R.D. Wood. 1976. Ecology of Inland Waters and Estuaries. New York: D. Van 

Nostrand Co. 485 pp. 

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salinity on NH4+ adsorption capacity, nitrification, and denitrification in Danish estuarine 

sediments. Estuaries 22(1): pp. 21–30. 

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silver, cadmium, and zinc to the estuarine amphipod Leptocheirus plumulosus through 

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281–293. 
HAZMAT 93-1-Appendix A 

APPENDIX A 

GLOSSARY 

Definitions

accuracy The closeness of a measured or computed value to its true value 

(e.g., sample mean to population mean). 

acute toxicity test A toxicity test in which the test organism is exposed to the 

contaminated material for only a small portion of its life cycle. 

acid volatile sulfides A class of operationally defined metal sulfides that react with aqueous 

acids at room temperature to liberate gaseous hydrogen sulfide and 

composed principally of amorphous ferrous and manganese sulfides. 

bioaccumulation The net result when the uptake of a chemical by a biological 

organism exceeds the depuration of the chemical from the 

organism. 

bioassay The determination of the presence or absence of a specific 

substance based on a biological response. 

bioassessment The assessment of environmental conditions by the use of 

biological organisms. 

bioavailability The availability of a substance to be taken up by biological 

organisms. 

bioconcentration The process by which a chemical is directly taken up (by 

absorption only) from water and is accumulated to levels greater 

than those found in the surrounding water. 

bioeffect A change in the condition or functioning of an organism, which 

reduces its potential viability, resulting from exposure to a toxic 

chemical(s). 

biomagnification The increase in tissue concentrations of a bioaccumulated chemical 

as the chemical passes up through two or more trophic levels. 

biomarker A biochemical, physiological, or histological indicator of either 

exposure to, or effects of, xenobiotic chemicals at the 

suborganismal or organismal level 

bulk sediment Whole or intact sediment, same as solid phase. 

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chronic toxicity test In general, a toxicity test in which the test organism is exposed to 

the contaminated material for at least the duration of one life cycle 

(the definition breaks down for long-lived organisms). 

correlation analysis Analysis to determine the relationship between two independent 

variables. 

endpoint (toxicity 

test) 

The response of the organism that is used as a measure of toxicity. 

exposure The contact with and uptake of an abiotic substance. 

flow-through A continuous flow of water through the test container; 

 (toxicity test) the water will be clean in sediment tests and will contain a fixed 

concentration of contaminants in a water test. 

in-situ toxicity test A toxicity test performed in the environment of concern as 

opposed to a laboratory toxicity test. 

liquid phase The sediment elutriate water from which all particles have been 

removed either by filtration or centrifugation. 

negative control A sample known to be nontoxic to the test organisms and in which 

they can function normally. 

nonparametric 

methods 

Methods not dependent on the characteristics of population 

parameters for their validity. 

null hypothesis A hypothesis formulated with the hope of rejection in favor of an

alternative hypothesis, with a known probability of being correct. 

parameter (ecological) A physical or chemical characteristic of an environment, e.g., 

sediment grain size or salinity. 

 

parameter (statistical) A characteristic of a statistical population, e.g., population mean. 

parametric methods Methods concerned with the characteristics of population 

parameters (e.g., population mean and variance). 

 

population 

(biological)

A group of individuals of the same species between which genetic 

material freely flows. 

population 

(statistical)

The totality of all possible observations of the variable of concern 

(takes into account method of observation). 

July 2003 A-2 
HAZMAT 93-1-Appendix A 

pore water The water present within the spaces between sediment particles; 

also called interstitial water. 

positive control One or a dilution series of water spiked with a toxic compound 

that produces a response (the endpoint) in the species used for the 

test. 

power (statistical) The probability of rejecting the null hypothesis and accepting the 

alternative hypothesis when the null hypothesis is in fact false. 

precision (statistical) A measure of the closeness of agreement of individual measures of 

the same quantity (closeness of replicate values).

pseudoreplication The taking and analysis of nonindependent observations as if they 

were independent observations (i.e., replicates). 

random sampling Taking of independent observations; e.g., with regards to spatial 

observations, the location of one observation has no influence on 

the location of any other observation. 

regression analysis Analysis to relate the behavior of a dependent variable to that of 

an independent variable. 

replicate One of a number of independent observations the total of which 

make up a sample. 

sample (statistical) A subset of a population. 

sampling (statistical) The process of taking observations of a population. 

sediment elutriate The water phase produced by mixing clean water with the test 

material and either allowing the material to settle or centrifuging it 

out. 

simultaneously 

extracted metals 

solid phase Whole or intact sediment, same as bulk sediment. 

static renewal Water is periodically replaced in test container either to prevent 

build up of waste products or to maintain the concentration of the 

contaminants in a water toxicity test. 

statistic A characteristic of a sample, e.g., sample mean, sample standard 

deviation. 

stratified random The division of the area to be sampled into various strata 

 A-3 July 2003 
HAZMAT 93-1-Appendix A 

sampling (subareas of similar characteristics) and then random sampling of 

each strata. 

suspended phase The sediment elutriate water still containing suspended particles. 

systematic sampling The first observation is selected randomly and each succeeding 

observation is taken at a fixed interval. 

toxicity test A test to determine the toxicity of an environmental sample 

utilizing the response of a biological organism. 

type I error Rejection of the null hypothesis when it is true. 

type II error Failure to reject null hypothesis when it is false. 

uptake the passing of a contaminant from the external environment 

surrounding an organism across a cell boundary layer and into the 

internal environment of the organism. 

variability The difference between replicate values (within sample variability) 

or between sample statistics (between sample variability). 

July 2003 A-4 
HAZMAT 93-1-Appendix A 

Abbreviations and Acronyms 

AET apparent effects threshold 

Ag silver 

AHH aryl hydrocarbon hydroxylase 

ALAD delta-aminolevulinic acid dehydratase 

ANOVA analysis of variance 

ASTM American Society for Testing and Materials 

AVS acid volatile sulfides 

BCF bioconcentration factor 

Cd cadmium 

CERCLA Comprehensive Environmental Response, Compensation, and 

Liability Act 

CI confidence intervals 

cm centimeter 

Cu copper 

DMSO dimethyl sulfoxide 

EC50 effective concentration 50 percent 

Eh oxidation-reduction potential 

EPA United States Environmental Protection Agency 

 A-5 July 2003 
HAZMAT 93-1-Appendix A 

EPT Ephemeroptera, Plecoptera, Trichoptera 

EROD ethoryresorufin-O-deethylase 

FCA foci of cellular alteration 

GHS glutathione 

GST glutathione transferases 

H2S hydrogen sulfide 

HBI Hilsenhoff Biotic Index 

Hg mercury 

KCI potassium chloride 

L liter 

LC50 lethal concentration 50 percent 

LOEC lowest observed effective concentration 

MESA Marine Ecosystem Analysis (NOAA Program) 

MFO mixed function oxidase 

ml milliliter 

ml/L milliliter per liter 

July 2003 A-6 
HAZMAT 93-1-Appendix A 

mm millimeter 

MN micronuclei 

MT metallothioneins 

NOAA National Oceanic and Atmospheric Administration 

NOEC no observed effective concentration 

NS&T National Status and Trends (NOAA Program) 

O&G oil and grease 

PAH polycyclic aromatic hydrocarbons 

Pb lead 

PCB polychlorinated biphenyls 

ppm parts per million 

ppt parts per thousand 

REMOTSTM remote ecological monitoring of the sea floor 

RI remedial investigation 

RTG rainbow trout gonad 

SAIC Scientific Applications International Corporation 

 A-7 July 2003 

SCE sister chromatid exchange 
HAZMAT 93-1-Appendix A 

July 2003 A-8 

SDN specific or degenerative necrotic 

SEM simultaneously extracted metals 

SFG scope for growth 

TIE toxicity identification evaluation 

TIC total inorganic carbon 

TOC total organic carbon 

TOM total organic matter 

TS total sulfides 

TVS total volatile solids 

Zn zinc 
HAZMAT 93-1–Appendix C

APPENDIX B 

PROTOCOLS 

The paper version of this appendix included copies of the noncopyrighted protocols, principally the Puget Sound Protocols. The 

complete Puget Sound Protocols, are available on the Internet at 

http://www.wa.gov/puget_sound/Publications/protocols/protocol.html,

while the copyrighted ASTM protocols can be purchased from the 

American Society for Testing and Materials via the Internet at 

http://www.astm.org/.

B-1 August 25, 1997 
HAZMAT 93-1–Appendix C

APPENDIX C 

Toxicity Test 

Sensitivity Comparison Table 

C-1 August 25, 1997
HAZMAT 93-1–Appendix C

Table C-1 Toxicity tests comparison summary. 

Test Treatments # Labs Relative Sensitivity 

Test Organisms Type Test Control Exp. Rep. # per Endpoint(s) Testing Rank Comments Ref.

Hyalella azteca Chironomus tentans Lumbriculus variegatus

S-R S-R S-R 

10d 10d 10d 

4545 50 

3/T 3/T 2/T 

10 10 10 

Survival Survival & growth Surv./reprod./ growth 

1-most mortality w/out food in 

sediment 2-<mortality w/out food in sediment; reduced growth 3-least sensitive-less mortality w/out food in sediment; # of worms increased and weight increased w/food addet to sediment 

•Test organisms-H. azteca 12-19d old; C. tentans 3rd instar; L. varigatus mixed adults

•Tests were conducted with & without feeding in sediment •The nutritional quality of the test sediments was a major factor influencing H. azteca test 

results •Conclusions:  •There is a potential for a significant percentage of false positive results in tests with clean sediments in which H. azteca & C. tentans is not fed 

 •Addition of food to sediments reduced the variability of assay results w/respect to survival  •Further attention should be given to the possible effects of sediment grain-size on growth of C. tentans •L. variegatus was the least sensitive organism

 1 

Cerastoderm edule Scrobicularia plana Nereis diversicolor Cirratulus cirratus Littorina litorea 

F-T F-T F-T F-T F-T 

17 wks 11 wks 17 wks 15 wks 12 wks 

1 1 1 1 1 

6 6 6 6 6 

3 3 3 3 3 

50 26 50 50 12 

Mortality % Mortality % Mortality % Mortality % Mortality % 

1 1 1 1 1 

1-100% death @ 2 wks high conc. w/inc. to 80% @ low conc. 17 wks2-100% death @ 10 wks high; 78% @ low 11 wks Not possible to rank 3-14% death @ 11 wks high; 0%@ low 4-8% death @ 12 wks high; 0% @ low 

•Data suggest: C. edule greater sensitivity TBT than S. plana; may be due to feeding regeme, 

tolerance, or ability to metabolize TBT •Few N. diversicolor were collected from any microcosms including the controls, and it is 

possible that some worms may have starved and others may have been ripe adults which spawned when temperature rose, and subsequently died •C. edule had a generally higher body burden of TBT than S. plana after 11 weeks, but S plana

seems to support a much greater body burden before death occurred in the high level TBT microcosm •Mean high level TBT = 1.786 ±0.047 •Mean low level TBT = 0.106 ±0.997 •Mean Control level TBT = 0.007 ±0.008 

 3 

Rhepoxynius abronius Neanthes (spp.) 

S 

S-R 

10d 20d 

1 pos. 1 

1 

5 5 

20 Survival

Growth (biomass) 

1 1 

2-> surv. than worms 1-< biomass, more sensitive 

•Both organisms were exposed to the same sediment contaminants & concentrations of metals, organics, phthalates, phenols. •One test measured PCBs •All three tests--storage of sediments can have a substantial effect on measurements of sediment toxicity; both for reference & contaminated sediments •Amphipod survival generally decreases w/storage time•Polychaete & Microtox mortality/bioluminescence varied unpredictably (responses either inc. or dec. w/storage time) •For both kinds of sediment, the test response declined dramatically after 12-14 weeks of storage 

 4 

Mytilus galloprovicialis Chlamys varia Crassostrea gigas Scrobicularia plana Dreissena polymorpha 

4wks4wks 4wks 2wks 

2

2 2 2 

30 30 30 30 

LT50 LT50 LT50 LT50 LT50 

1 1 1 1 1 

1--4.6 days @ 100 μg/l Ag 2--4.8 days @ 100 μg/l Ag 3--8.7 days @ 100 μg/l Ag 4--10.4 days @ 100 μg/l Ag 5--28 days @ 400 μg/l Ag 

•Rank based on 100 μg/l Ag--the 1st lowest conc. for all the species •No direct relationship exists between bioaccumulation & toxicity, may be due to the physicochemical forms of sequestration of Ag (e.g., Ag associated w/insol. forms which mainlyconsist of Ag-sulphide, a very stable compound whose tox. is therefore limited) 

•Sublethal effects: C. gigas--after 4wks, decrease of glycogen in intestinal tissue 

at 20 μg/l Ag M. galloprovincialis--dilation of ER in branchial cells, result death at 50-100 μg/l Ag C. varia--unable to attach, lost bysus & no thread produced at 20 μg/l Ag S. plana--after 2d exposure, pieces of siphons were lost at 50 μg/l AgD. polymorpha--lost byssus, lesions GI walls, tubes lysed, degeneration of ovaries 

 5 

August 25, 1997 C-2
HAZMAT 93-1–Appendix C

C-3 

Oncorhynchus mykiss Salvelinus fontinalis Ictalurus punctatus Lepomis macrochirus Pimephales promelas Fundulus heteroclitus 

96h96h 96h 96h 24h 96h 96h 

10 LC50 (mg/l) 

10 10 10 10 10 

LC50 (mg/l) LC50 (mg/l) LC50 (mg/l) LC50 (mg/l) LC50 (mg/l) LC50 (mg/l) 

1 1 1 1 1 1 1 

1--TNT @ 1.2 1--TNT/RDX @ 1.7 1--DNT +16 @ 7.2 1--Cd @ 00095 2--Zn @ 0.41 2--Pb @ 1.0 7--Cr @ 69.0 4--Malathion 0.17 3--Sevin @ 4.4 1--Chlordane @ 0.01 2--CH4Oxchlor0.0072 1--Treflan @ 0.011 4--DDT @ 0.007 2--Cu @ 0.017 5--Zn @ 0.96 6--Pb @ 4.0 6--Cr @ 50.0 2--TNT @ 2.4 3--TNT/RDX @ 5.3 2--DNT +16 @ 17.5 5--Malathion 9.0 5--2-4-D @ 500.0 8--Sevin @ 15.8 7--Chlordane @0.5 5--Treflan @ 0.42 6--DDT @ 0.016 3--TNT @ 3.0 2--TNT/RDX @ 4.2 1--DNT +16 @ 7.2 8--Cu @ 0.74 6--Zn @ 2.9 3--Malathion 0.103 4--2-4-D @ 375.0 4--Sevin @ 5.6 3--Chlordane @0.022 4--CH4Oxchlor0.058 5--DDT @ 0.008 3--Treflan @ 0.1 4--TNT @ 3.1 4--TNT/RDX @ 6.0 3--DNT +16 @ 22.0 6--Cu @ 0.55 4--Zn @ 0.87 7--Pb @ 5.0 6--Malathion 12.5 5--Sevin @ 6.7 5--Chlordane @0.052 7--DDT @ 0.19 4--Hg @ 2.0 

•L. variegatus appears much less sensitive to pesticides than the other invertebrates; in most 

cases, fish were also more sensitive than the worms •All of the compounds (except the nitroaromatics) were evaluated for sublethal effects on L. variegatus; in general, sublethal effects wee dose-related in terms of severity & frequency of occurrence &, except with Hg, Pb, & 2-4-D, occurred at levels below the LC50: •Fragmentation--affected worms broke up into large pieces (Cd, Hg, Pb, Sevin, chlordane, & malathion)  •Clumping--formation of tight spherical bunches or clumps (Cr, Cu, Zn, malathion, & chlordane)  •Localized swelling--small swellings or lumps along their bodies (Malathion, Sevin, & chlordane)  •Mucus production--(Pb, Cd)  •Overall swelling--during exposure, the diameter of the worms often doubled (Cd)  •Color changes--worms exposed to Sevin turned dark brown, and those exposed to Zn were gray 

 2 

August 25, 1997

Test Treatments # Labs Relative Sensitivity 

Test Organisms Type Test Control Exp. Rep. # per Endpoint(s) Testing Rank Comments Ref.
HAZ

MAT 93-1–Ap

pendix C

Test Treatments # Labs Relative Sensitivity 

Test Organisms Type Test Control Exp.

Rep. # per Endpoint(s) Testing

Ran

k

Com

ments Ref.

Lumbriculus variegatusHyalella azteca Daphnia ma

gna

Daphnia hyalina Daphnia pulex 

48h96h 48h 48h 64h 48h 48h 

2

2 

10 10 

5 5 5 

LC50 (mg/l) LC50 (mg/l) LC50 (mg/l) LC50 (mg/l) LC50 (mg/l) LC50 (mg/l) LC50 (mg/l) 

1 1 1 1 1 

5--TNT @ 4.9 5--TNT/RDX @ 8.8 5--Cu @ 0.23 6--DNT +16 @ 24.5 3--Cd @ 0.12 8--Zn @ 8.1 4--Pb @ 3.4 5--Cr @ 25.3 3--Hg @ 0.11 8--Malathion 30.9 3--2-4-D @ 122.2 7--Sevin @ 13.0 9--Chlordane @ 1.8 5--CH4Oxchlor 0.2 4--Treflan @ 0.3 9--DDT @ 0.13 4--Cu @ 0.15 2--Cd @ 0.074 7--Zn @ 6.3 3--Pb @ 1.8 3--Cr @ 13.3 2--Hg @ 0.1 7--Malathion 20.5 3--2-4-D @ 122.2 6--Sevin @ 8.2 8--Chlordane @1.4 5--CH4Oxchlor 0.2 4--Treflan @ 0.3 9--DDT @ 0.13 6--TNT @ 6.5 6--TNT/RDX @ 10.5 4--DNT +16 @ 22.7 7--TNT @ 11.9 7--TNT/RDX @ 11.7 5--DNT +16 @ 23.5" 1--Cu @ 0.0098 1--Zn @ 0.1 1--Cr @ 0.05 2--Malathion 0.009 2--2-4-D @ 100.0 1--DDT @ 0.001 1--Pb @ 0.6 1--Hg @ 0.006 1--Sevin @ 0.0064 4--Chlordane @0.029 3--CH4Oxchlor0.0078 3--Treflan @ 0.24 2--DDT @ 0.0036 

 2 

cont’d

August 25, 1997 

C-4
HAZMAT 93-1–Appendix C

Test Treatments # Labs Relative Sensitivity 

Test Organisms Type Test Control Exp. Rep. # per Endpoint(s) Testing Rank Comments Ref.

Tanytarsus dissimilus Simulium Ephemerella Damsel fly nymphAeroneuriaChironomus spp. Physa Gammarus lacustris Gammarus faciatus Asellus 

48h24h 48h 48h 

14d 

8h 

24h 96h 24h 48h 48h 

5 LC50 (mg/l)

LC50 (mg/l) LC50 (mg/l) LC50 (mg/l) LC50 (mg/l) LC50 (mg/l) LC50 (mg/l) LC50 (mg/l) LC50 (mg/l) LC50 (mg/l) LC50 (mg/l) 

1 8--TNT @ 24.8 

8--TNT/RDX @ 32.0 7--DNT +16 @ 47.0 9--Cu @ 3.9 4--Cd @ 2.0 5--Pb @ 3.5 2--Cr @ 3.5 4--Hg @ 2.0 10--Zn @ 69.5 8--Pb @ 19.5 4--Hg @ 2.0 2--Chlordane @0.015 8--DDT @ 0.04 3--Cu @ 0.05 3--Zn @ 0.8 4--Cr @ 17.3 7--Cu @ 0.58 1--Malathion 0.0018 2--Sevin @ 0.04 6--Chlordane @0.16 1--CH4Oxchlor0.0047 6--Treflan @ 8.8 3--DDT @ 0.0047 1--2-4-D @ 3.2 9--Zn @ 56.0 

 2 

cont’d

Chlorella spp. Scenedesmus spp. Selenastrum spp.

Agar 

plates 

Agar Agar 

96h 96h 96h 

4 conc.4 conc. 4 conc. 

See notes Growth inhibition 

Notes Notes 

Growth inhibition Grwth inhibit. 

1 1 1 

1--Cu @ 25 mg/l 2--Cr(VI) @ 100 mg/l no inhibition 2--Cu @ 100 mg/l 1--Cr(VI) @ 100 mg/l 2--Cu @ 100 mg/l 1--Cr(VI) @ 100 mg/l 

•Metal toxicity to algal cultures, growth of streaks of metal treated cultures on 2% BG11 - agar plates •Of the 4 metals investigated, Cu, Cr, Pb, and Cr2O7 the former 2 were more toxic than the 

latter two •4 concentrations--2.5, 1.0, 25, and 100 mg/l were the exposures •The algae were streaked out on 2% BG 11 - agar plates--(see number per column) •The variable factor here may be the tolerance of the species & the amount of toxic metal accumulated

 6 

C-5 August 25, 1997
HAZMAT 93-1–Appendix C

Test Treatments # Labs Relative Sensitivity 

Test Organisms Type Test Control Exp. Rep. # per Endpoint(s) Testing Rank Comments Ref.

Ceriodaphnia dubia Daphnia magnaHyalella azteca Selenastrum capricornutum 

48h48h 48h 48h 

1 (E&S); 1 

H2O 

1 (E&S); 1 

H2O 

1 (E&S); 1 

H2O 

1 (E&S); 1 

H2O 

3/T3/T 3/T 3/T 

20 20 10 

1x106/

ml

Survival (N) Survival (N) Survival (J) Growth 

1 1 1 1 

1-to elutriates 1-whole sediment 2-to elutriates 2-whole sediment 4-to elutriates 3-whole sediment 3-to elutriates 

•Determinations of assay sensitivity based on comparisons between the elutriate phase on one toxicity assay and the solid phase of another toxicity assay should not be made •Cladocerans spent significant time feeding on the sediment surface, increasing the potential for toxicant uptake •Relative sensitivity will frequently vary w/the site and/or toxicants being evaluated •The current study confirms the premise that multiple test assays are necessary to both detect sediment toxicity and differentiate degrees of toxicity •Microbial activity in elutriates-ß-galactosidase & ß-glucosidase were the most sensitive 

 7 

Daphnia magna 

Chironomus tentans Hyalella azteca Gammarus lacustris 

S-R S-R S-R S-R 

2d 

10d 10d 10d 

1 1 1 1 

5 5 5 5 

3 3 3 3 

20 15 15 10 

LC50 LC50 LC50 LC50 

1 1 1 1 

1--Cu @ 299 μg/l aqueous Cu 2--Cu @ 428 μg/l aq Cu 3--Cu @ 719 μg/l aq Cu 4--Cu @ 1152 μg/l aq Cu 

•Daphnia were added to the test beakers on day 8 of the 10 day exposure to Chironomus test 

beakers  Cu Dilutions

•Suvival data: Control 37.5 75.0 150 300 600

D. magna 15/20 14/20 7/20 0/20 0/20 0/20 C. tentans 11/15 13/15 8/15 1/15 0/15 0/15 

H. azteca 15/15 13/15 13/15 0.15 0/15 0/15 

G. lacustris 9/10 10/10 6/10 0/10 0/10 0/10 

 8 

August 25, 1997 C-6
HAZMAT 93-1–Appendix C

Test Treatments # Labs Relative Sensitivity 

Test Organisms Type Test Control Exp. Rep. # per Endpoint(s) Testing Rank Comments Ref.

Rhepoxynius abronius Rhepoxynius abronius Neanthes arenaceodentata Corophium vlutator Crassostrea gigas Crassostrea gigas Crassostrea gigas Crassostrea gigas Photobacterium phosphoreum Mya arenaria Bathyporeia sarsi 

S-R 

10d 10d 20d 10d 

2d 

24h 96h 24h 

1h 3h 

10d 

1 pos., 1 

neg. 

1 pos., 1 

neg. 

1 pos., 1 

neg. & 1 ref 

1 pos., 1 

neg. 

1 pos., 1 

neg. 

1 neg. 1 neg. 1 neg. 

1 clean, 1 

ref. sed. 

1 neg. 

16 sites 16 sites 16 sites 16 sites 16 sites 16 sites 16 sites 

2 

2 lab 

reps 

for 7 

sites 

3 at 2 

dilut 

20 

Few, 

not all 

20 20 

5 

10 

23 x103 20 x103

30 

53 x103

20 

Surv./reburial Surv./reburial/ avoidance Surv. & growth Survival/immobilization Surv./devel. Survival/development Survival & Metamorphosis Survival 

Bioluminescence 

Burrowing Surv./reburial 

1 USA 

1 Canada 

1 

1-UK 

1 Canada 

1-UK 

1-USA 

1-

Netherland

s 

1-UK 

1-USA 

1-

Netherland

s 

1-whole sediment -whole sediment -whole sediment 2-whole sediment 1-elutriate; whole sediment -elutriate -whole sediment; inconsistant results -elutriate -whole sediment; turbidity problem -whole sediment; inconclusive results 3-whole sediment 

•Labs: 

R. abronius (USA-Swartz) M. arenaria, C. gigas (USA-Phelps) 

R. abronius, N. arenaeodentata, C. gigas (Canada-Chapman) 

C. volutator, C. gigas, P. phosphoreum (UK-Roddie/Butler) 

C. gigas, B. sarsi (The Netherlands-van den Hurk) 

Limitations: •UK mean oyster larvae tests were below criteria for negative controls  •R. abronius exposed to drilling sites more sensitive than expected 

 •No control criteria for direct exposure in the Microtox assay  •M. arenaria specimens were stressed prior to testing; sediments held too long 

 •Differences between the German and drilling sites; sediment grain size, sediment sampling, sediment treatment Tests: •Originally for water column, adapted for sediment-oyster abnormality & Microtox  •Acute lethal tests specifically for sediments-all others  •Specific for sediment-sublethal tests (growth, reproduction, full life-cycle tests; e.g.,Neanthes) 

 9 

Rhepoxynius abronius Mytilus edulis Tigriopus californicus 

10d

48h 

4wks 

1 1 1 

9 9 9 

5 8 

20 

1 

Survival & avoidance Survival & abnormal developmt.Reprod.-# young/adult 

1 1 1 

2-survival 2/9 signif. diff. from control 2-avoidance 1/9 signif. diff. from control 1-survival 8/9 signif. diff. from control 1-abn. dev. 5/9 signif. diff. from control 1-# young 5/9 signif. diff. from control 

•The responsiveness of the organisms-the number of statistical differences from reference; lethal (survival) endpoints were compared and sublethal endpoints were compared to rank the relative sensitivity •Exposure similarities: Few differences in concentration observed 

 among stations for major metals (Al, Si, Fe, Mn, Mg, Ca, Na, Ti) 13 trace elements that were always detected (As, Cr, Cu, Ni, Zn,  Pb, Hg, Sn, Ag) Organic compounds-station ranks--LPAHs, HPAHs & other  OC--IS (Islais Waterway)>OA(Oakland)>SP(San Pablo Bay) 

Detected at all stations--DDT, DDE, DDD •Aggregate index--mean of the combined values for 5 chemical groups to achieve a single composite value that will identify contaminated sites; IS = 6.29, OA = 1.97, & SP = 1.00 

10 

C-7 August 25, 1997
HAZMAT 93-1–Appendix C

Test Treatments # Labs Relative Sensitivity 

Test Organisms Type Test Control Exp. Rep. # per Endpoint(s) Testing Rank Comments Ref.

Photobacterium phosphoreum Pimephales promelas 

F-T 

5 min 

96h 

68 chemicals Few- 10% Bioluminescence (EC50) 

Mortality (LC50) 

1 1 

1-LC50 prediction from EC50for alcohols (r2=0.96), ketones (r2=0.81), ethanes (r2=0.72) 

2-general groupings1-general groups & not a semihomolo-gous series 

•The reliability of the bacterial assay for predicting fish LC50 is greatest when the data are derived from a semiholologous series of chemicals rather than from general groups (e.g., inorganic vs organic) •Prediction of LC50 values from bacteria EC50 values not sufficient to justify except for order 

of magnitude estimates•Semihomologous series-a plot of the endpoint against the number of carbon atoms for each chemical; it was proposed that the that if the EC50 had the same dependence on the carbon number as the fish LC50, comparable mechanisms of toxicity wer operative & the two endpoints would be correlated 

11 

Lubriculus variegatusDiporeia hoyi 

S-R S-R 

2 wks 

1 wk 

1 site 1 site 

6 sites 6 sites 

5 5 

10 

5 

Survival Survival 

1 1 

1--5/6 stations signif. diff. from controls 2--2/6 stations signif. diff. from controls 

•Worm survival in the more toxic sediments from the harbor's center (sites 1, 2, 3) was greater for Lumbriculus than for the deep-water amphipod Pontoporeia Diporeia hoyi•The sites contained high levels of PCBs (2300 μg/g), PAHs, and metals associated w/the industrial area 

13 

Leptocheirus plumulosus Eohaustorius estuarius Lepidactylus dytiscus Hyalella azteca Streblospio bendicti Crassostrea gigas Mya arenaria 

10d28d 10d 10d 10d 10d 

96h 43h 

1 ref. 1 ref. 1 ref. 1 ref. 1 ref. 1 ref. 1 ref. 1 ref. 

5 dilut.5 dilut.5 dilut.5 dilut.5 dilut.5 dilut.5 dilut.5 dilut.

Mortality

Mort.gro.fert Mort.rebury Mort.rebury Mortality Mortality Mort.meta-morphosisReburial 

4 3 2 1 1 1 1 1 

3-10d mortality 2-10d/1-28d 1-Cd sensitivity 1-most conc./discrim.4-10d mortality 2-10d mortality 2-least conc./discrim.5-10d mortality 1-Cd sensitivity 2-least conc./discrim.1-10d mortality 2-least conc./discrim.1-Cd sensitivity 3-difficult to compare w/other species 

•Protocols--DeWitt et al. '92; Schlekat et al. '92; Hall et al. '91; ASTM '90 E1367-90 Vol 11.04; McGee & Schlekat '93; Phelps & Warner '90; Phelps '90 •22 organic contaminants in sediment from Curtis Cr.; 5 metals (e.g., Cd, Cu, Ni, Pb, Zn); total SEM/AVS <1.0 •Conclusions--  •Mortality in most short-term (10d) tests were similar in their sensitivity to contaminated Chesapeake Bay sediment  •Short-term sublethal responses were not more contaminant-sensitive than acute mortalityfor any species  •Chronic L. plumulosus were more sensitive than the acute toxicity test responses by only2-3x  •Moderate among-lab variability in 10d L. plumulosus mortality, large between lab differences in 28d mortality, growth, and fertility due to methodologies used by each research group 

14 

August 25, 1997 C-8
HAZMAT 93-1–Appendix C

Test Treatments # Labs Relative Sensitivity 

Test Organisms Type Test Control Exp. Rep. # per Endpoint(s) Testing Rank Comments Ref.

Photobacterium phosphoreum Culex pipiens Oryzias latipes Poecilia reticulata 

Uronema parduczi 

Aedes aegypti Hydra oligactis Xenopus laevis Pseudomonas putidaChlorella pyrenoidosa Selenastrum capriconutum Lymnea stagnalis Chilomonas paramecium Ambystoma mexicanumScendedesmus pannonicus Entosyphon sulcatum Pimephales promelas Oncorhynchus mykiss Daphnia magnaDaphnia pulex Microcystis aeruginosa 

5 min 1 4 2 1 20--least sensitive 

1-6.93 > MAS 2--3.48 > MAS 3--2.54 > MAS 4--2.04 > MAS 5--1.99 > MAS 6--1.8 > MAS 7--1.74 > MAS 8--1.29 > MAS 9--1.19 > MAS 10--1.17 > MAS 11--1.09 > MAS 12--1.03 > MAS 13--0.96 > MAS 14--0.93 > MAS 15--0.75 > MAS 15--0.75 > MAS 16--0.7 > MAS 17--0.66 > MAS 18--0.65 > MAS 19--0.53 > MAS 

•In comparing the sensitivity of the Microtox Assay System (MAS) w/that of a different test on aquatic toxicity, the geometric mean of the ratio for all of the tested chemicals of Microtox 5-min. EC10 & the concentration causing minimal effect in the other test expresses the number of times the specific test is more susceptible than the MAS •Test compounds: mercuric chloride, Cd nitrate, n-Propanol, n-Heptanol, ethylacetate, 

ethylpropionate, acetone, trichloroethylene, allylamine, aniline, benzene, pyridine, o-Cresol,Salicylaldehyde, pentachlorphenate •Chemicals tested: bacteria-14, green algae--12-14, protozoa-14, crustaceans-15, insects-15, coelenterates-15, molluscs-15, fishes-15, and amphibians-15 

12 

Daphnia magna Thymallus thymallus S-R 

72h 72h 

168h 1 

6 9 2 

70 70 

300 

MortalityMortalityMortality

1 1 1 

2--7% @ pH6 1--22% @ pH8 3--2.6% @ pH8.1 

•Exposure concentration of TBTCl at 6.0 μg/l for both species •pH affects the toxicity of TBT; higher at higher pH values •TBT must be in soluable form, and not bound to organic materials or coloidal substances to be available to organisms •TBT is lipophilic 

17 

C-9 August 25, 1997
HAZMAT 93-1–Appendix C

Test Treatments # Labs Relative Sensitivity 

Test Organisms Type Test Control Exp. Rep. # per Endpoint(s) Testing Rank Comments Ref.

Paracorophium excavatum Proharpinia hurleyi 

10d10d 

4 4 

6 conc. 6 conc. 

2 2 

10 10 

Survival Survival 

1 1 

1-based on LC50 value of 18.3 mg/kg 2-based on LC50 value of 23 mg/kg 

•P. hurleyi sensitive to sand grain size-best survival in fine sand, and not in very fine, medium,or coarse sand; best in 16% salinity & not 8%; best in enriched sediment w/organic content 7% Protocol: Swartz et al. 1985 •Exposure was to Cd in sediment-range 2.8-70 mg/kg 

18 

Crab zoea Sand dollar adult Cabezon (sculpin) Sea urchin emb. Sea urchin adult Sea urchin sperm Oyster embryo Sea urchin emb. Oyster spermSand dollar sperm Crab zoea Oyster embryo Sea urchin sperm Cabezon (sculpin) Mussel embryo Salmon sperm Sea urchin emb. Oyster embryo Sea urchin emb. Sand dollar sperm Sea urchin sperm Cabezon (sculpin) Oyster sperm Sea urchin sperm Crab zoea Salmon sperm Sea urchin sperm Oyster embryo Crab zoea Mussel embryo Shiner perch Salmon smolts Sand shrimpCrab zoea Sand shrimpShiner perch Salmon smolts Crab zoea

S S S 

F-T 

S S S S S S S S S S S S S S S S S 

F-T F-T F-T 

S 

F-T F-T F-T 

S 

96h 72h 

120h 

96 

1h 

48 

120h 

96h 48h 

1h 

72h 

120h 

48h 

120h 

1h 1h 

96h 

1h 

48h 96h 72h 96h 96h 96h 96h 96h 96h 96h 96h 

LC50 LC50 LC50 LC50 LC50 LC50 LC50 LC50 LC50 LC50 LC50 LC50 LC50 LC50 LC50 LC50 LC50 LC50 LC50 LC50 LC50 LC50 LC50 LC50 LC50 LC50 LC50 LC50 LC50 LC50 LC50 LC50 LC50 LC50 LC50 LC50 LC50 LC50 

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

1--0.25 mg/l Cd 2--0.4 mg/l Cd3--Juv. 0.5 mg/l Cd 3--0.5 mg/l Cd4--0.7 mg/l Cd1--2 μg/l Cu 2--6 μg/l Cu 2--6 μg/l Cu 3--12 μg/l Cu 4--26 μg/l Cu 1--0.6 mg/l Pb2--0.7 mg/l Pb3--1.0 mg/l Pb4--juv. 1.5 mg/l Pb 1--4.4 μg/l Ag2--11 μg/l Ag 3--15 μg/l Ag 4--19 μg/l Ag 1--23 μg/l Zn 2--28 μg/l Zn 3--148 μg/l Zn 4--juv. 191 μg/l Zn 1--0.4 μg/l DDT 2--1.0 μg/l DDT 3--1.1 μg/l DDT 4--2.4 μg/l DDT 1--1.4 μg/l Dieldrin 2-- 23 μg/l Dieldrin 3--24 μg/l Dieldrin 4--48 μg/l Dieldrin 1-Endosulfan1.1μg/l 2-Endosulfan1.7μg/l 3-Endosulfan2.3μg/l 4-Endosulfan 15μg/l 1--Endrin 0.45 μg/l 2--Endrin 0.5 μg/l 3--Endrin 1.2 μg/l 4--Endrin 2.0 μg/l 

•This work primarily investigated the use, refinement & comparative sensitivity of a sea urchin sperm cell bioassay for marine pollution monitoring. This test exposed sea urchin or sand dollar sperm cells to test solutions for 60 min. prior to fertilization of the eggs. Elevation of the fertilization membrane was used as the test endpoint. An oyster sperm and a salmon sperm test (for brackish waters) were also investigated •The "validation" portion of this work compared the sensitivity of sperm cell tests to other common test organisms available in the Pacific Northwest 

15 

August 25, 1997 C-10
HAZMAT 93-1–Appendix C

Test Treatments # Labs Relative Sensitivity 

Test Organisms Type Test Control Exp. Rep. # per Endpoint(s) Testing Rank Comments Ref.

Hyalella azteca Chironomus tentans 

F-T F-T 

10d 10d 

1 1 

5 conc. 

spiked sed.

5 conc. 

spiked sed.

4/T 4/T 

10 

5 

Survival-LC50 values SurvivalLC50 values; growth (wt) 

1 1 

2-Dieldrin LC50 7.6 μg/l (water only) 1-Dieldrin LC50 1.1 μg/l (water only) 

•Examination of the dieldrin 10d water only LC50 values for both species indicates that these organisms are two of the most sensitive freshwater invertebrate species tested with dieldrin •Growth was diminished (relative to control) in spiked sediment exposure •Some interaction may occur between sediment/dieldrin/H. azteca, limiting dieldrin exposure to this species 

19 

Photobacterium phosphoreum Spirillum volutans Pseudomonas fluorescens Aeromonas hydrophila 

S S S S 

15min 

18h 18h 18h 

1 pos. & 1 

neg. 

1 pos. & 1 

neg. 

1 pos. & 1 

neg. 

log 4 conc.log 4 conc.log 4 conc.

2 2 2 2 

25ml 25ml 25ml 

Bioluminescense Opticle densityOpticle densityOpticle density

1 1 1 1 

1-ave. rate = 1.53 for 5 metals & 8 compounds 2-ave. rate = 2.38 for 5 metals & 8 compounds 3-ave. rate = 2.61 for 5 metals & 8 compounds 4-ave. rate = 3.76 for 5 metals & 8 compounds 

•Protocols: Microtox-Beckman Instruments Interim Manual No. 11067B-9-80; S. volutansBowdre & Krieg 1974; P. fluorescens-ATCC-13525; A. hydrophila-ATCC-23213 

•Metals tested: Hg, Zn, Cu, Pb, Ni•Other compounds tested: ∝Napthol, NaLauryl SO4, NaCyanide, 3,3 dichlorobenzidine,

Phenol, N-Nitrosodiethylamine, dichloromethane, Nitrotriacetic Acid 

16 

Hyalella azteca Chironomus riparius 

S 

F-T F-T 

29d 29d 29d 

1 1 1 

3 3 3 

4 4 4 

20 20 50 

Mortality & growth 

(lgth) 

Emergence

1 1 

1-Waukegan sediments 2-Homer sediments 2-Waukegan sediments 1-Homer sediments 

•Survival of amphipods reduced in Waukegan treatments (10d & 29d), but not in control treatments; body length not affected; delay in sexual development observed in Waukegan-2 sediments after 29d F-T tests •Confounding factors--Sensitivity of C. riparius-1st instar 1-27x more sensitive than 4th instar 

to Cu and 127x more sensitive than 2nd instar to Cd exposure; indigenous animals may have affected emergence 

21 

Neanthes arenaceodentata Rhepoxynius abronius Eohaustorius estuarius Photobacterium phosphoreum 

20d 20d 20d 

1 1 1 

12w/3 

dilution 

12w/3 

dilution 

12w/3 

dilution 

5 5 5 

Mortality & biomass MortalityMortality & reburial Bioluminescence 

1 2-mortality (25%) 

2-biomass (41.6%) 1-mortality (50%) 1-mortality (50%) 3-nonreburial (8.3%) 1-red. bioluminescence (100%) 

•Statistical sensitivity-the number of statistical differences from the reference (control)--reported here as a percentage in the rank column •The authors classify N. arenaceodentata as moderately sensitive 

•Sediment contaminantes included 9 metals (Commencement Bay), 7 low molecular weight PAHs, 9 high molecular weight PAHs (Eagle Harbor), and polychlorinated biphenyls (Harbor Island) 

22 

Photobacterium fischeri Oncorhynchus mykiss Pimephales promelas 

F-T 

S 

F-T F-T 

S 

5 min 

5min 

96h 24h 

1 blank 1 blank 

7 9 7 7 9 

10 μl10 μl 

Bioluminescence Mortality (LC50) Mortality (LC50) 

1 1 1 1 

2-mean = 0.75% 1-mean = 1.20% 1-mean = 0.44% 2-mean = 0.75% 

•Processed water from shale retorting & coal gasification used as toxicant substances •Rainbow trout were more sensitive to processed waters than luminescent bacteria (EC50 & LC50 correlation coeff. = 0.82, F-T) •Fathead minnow & bacteria were not significantly correlated (EC50 & LC50 correlation 

coefficient = 0.41, F-T) •Bacterial system more sensitive than fathead minnow in static tests (EC50 & LC50 correlation 

= 0.97) •Fish more sensitive to phenolic compounds than bacteria 

23 

C-11 August 25, 1997
HAZMAT 93-1–Appendix C

Test Treatments # Labs Relative Sensitivity 

Test Organisms Type Test Control Exp. Rep. # per Endpoint(s) Testing Rank Comments Ref.

Photobacterium phosphoreum Photobacterium phosphoreum & 

Dunaliella tertiolecta 

Fish species

5 min

1h & 24h 

1 1 

35 compounds 35 compounds 

3-5 3-5 11.25 

ml each

spp. 

Bioluminescence Bioluminescence LD50 

1 1 

Many (lit.

search) 

2-Microtox generally more sensitive than the Tchan proceedure 3-least sensitive in general than Microtox or fish survey; exception-some herbicides were more sensitive 1-generally more sensitive than either microbial bio-assays 

•Compounds included metals (e.g., Hg, Na, Zn, Cd, Be, K, Ni), DMSO, hydrocarbons, herbicides Protocols: proceedure of Tchan utilizing a bacterium & an alga, & Microtox by Beckman Instruments, Inc.•Most of the test compounds were detected at lower concentrations by fish bioassays and implies that the microbial bioassays are less sensitive 

25 

Rhepoxynius abronius Ampelisca abdita Mytilus edulis Strongylocentrotus purpuratus Dinophilus gyrociliatus 

S 

F-T 

S S S 

10d 10d 

48h 

20min 

7d 

2 2 2 4 5 

15 samples 

15 smpls 15 smpls 15 smpls 15 smpls 

5 5 5 5 5 

20 20 

100-900 

20 20 

Survival, avoidance Mortality, avoidance Survival, norm development Cell changes, fert., abnorm developmt Survival, eggs/female 

1 1 1 1 1 

1-survival most sensitive;reburial & avoidance insensitive 2-surv. insensitive (< R. abronius); avoid. not sensitive 1-embryo test high; abn. develpmt most sensitive 3-< M. edulis for developmentinter-mediate sensitivity 

4-rel. resistant to some chem.;eggs sensitive to complex effluents 

•M. edulis-high concordance w/R. abronius tests; highest precision & discriminatory power •R. abronius-relatively high discriminatory power & intermediate precision •A. abdita-high analytical precision; not correlated w/sedimentological variables & relatively high correlation w/several toxicants •S. purpuratus-responsive to benzo(a)pyrene exposure resulting in cellular effects;

intermediate sensitivity, precision and discriminatory power •D. gyrociliatus-resistant to endosulfan & pentachlorophenol; survival insensitive; intermediat 

sensitivity, precision & discriminatory power w/relatively high correlation w/PAHs 

24 

Leptocheirus plumulosus Hyalella azteca 

10-20d 10-40d 10-20d 

1 1 1 

CC % 

100,50, 25,12.2 

12.5,6.3,3.1,

1.5 

100,50, 25,12.2 

3 20 

Survival Growth (Lgth,Wt) Survival 

1 1 

1 (J) 

2 (A) 

3 (J) 

•Temperature variations (2°C recommended) & feeding regime affect the growth and reproduction of L. plumulosus

•Increase the number of tests/concentration, & number of replicates •Significant positive relationship between length & dry weight in L. plumulosus 

•Juveniles more sensitive to use for sublethal endpoints 

26 

Rhepoxynius abronius Eohaustorius washingtonianus 

4d

10d 

4d 

10d 

Mortality LC50 Mortality LC50 

1 1 

2-30 to 100x more tolerant to TBT 1-non-tolerant to TBT 

•R. abronius 4d LC50 = 138 ppm, 10d LC50 = 29 ppm •E. washingtonianus LC50 = 4.7 ppm, 10d LC50 = 0.3 ppm 

•The decrease from 4 to 10 day was substantial, indicating that tributyltin is a slow-acting toxicant •Survivors exhibited differences in reburial behavior related to their tolerance of the toxicant •BCF for R. abronius (2000-3000) was an order of magnitude lower than for E. washingtonianus

27 

August 25, 1997 C-12
HAZMAT 93-1–Appendix C

Test Treatments # Labs Relative Sensitivity 

Test Organisms Type Test Control Exp. Rep. # per Endpoint(s) Testing Rank Comments Ref.

Hyalella azteca Chironomus tentans Lumbriculus variegatus

F-T F-T F-T 

10d 10d 10d 

1 1 1 

5 conc. 5 conc. 5 conc. 

10

10 or 5 

10 

LC50 LC50 LC50 

1 1 1 

1-Cu, Zn, Cd, Ni, Pb, DDT, DDD, DDE 2- Dieldrin, Chlorpyrifos 3/4-Cd w/other spp. in WQCD & AQUIRE 3-Cu 2-Zn, DDT, DDD, DDE 1-Dieldrin, Chlorpyrifos 3/4-Zn, 5/7-Cu w/other spp. in WQCD & AQUIRE data base 1-Cu 3-Zn 2-Cd, Ni, Pb, DDE3/4-Ni, 4/4-Zn, 4/5-Pb, 4/7-Cu w/other spp. in WQCD & AQUIRE 

•Water only tests in soft water (hardness 40 mg/l as CaCO3) •Toxicity of the three species was compared to the genus mean acute & chronic data found in US EPA WQC documents & AQUIRE data base-they are reasonalbly represent the range of sensitivities of other aquatic species, & occasionally are amone the most sensitive species •No one species was consistently most sensitive to the 10 toxicants •It appears that H. azteca is more sensitive to broad spectum toxicants than the other two 

species •L. variegatus was never the most sensitive species tested; but more sensitive than midges for Cu & Pb •Sensitivity is a function of the test species &test regime: Static or flow-through, nominal or measured chemical concentrations? •Ranks are reported for metals from Water Quality Criteria Documents (WQCD) & AQUIRE data bases 

30 

C-13 August 25, 1997
HAZMAT 93-1–Appendix C

Test Treatments # Labs Relative Sensitivity 

Test Organisms Type Test Control Exp. Rep. # per Endpoint(s) Testing Rank Comments Ref.

Photobacterium phosphoreum Daphnia Oncorhynchus mykiss Pimephales promelas Amphipod Oyster

Bioluminescence Acute lethal Acute lethal Acute lethal 

1-(15/27) organics2-inorganic (pure) 1&2-oil refinery wastewater 1-fossil fuel water 4-oil sands tailing 1-sewage plant efflu. 1-industrial efflu. (4/5) 2-industrial efflu. (1/5) 1-sedimt. contam. (2/8) 2-sedimt. contam. (4/8) 3-sedimt. contam. (3/8) 3-sedimt. extracts 1-(15/27) organics 1-inorganic (pure) 1-oil refinery water 2-oil sands tailing 1-industrial efflu. (1/5) 3-industrial efflu (4/5) 1-sedimt. contam. (5/6) 2-sedimt. contam. (1/6) 1-(13/27) organics 4-inorganic (pure) 1-oil sands tailing 2-fossil fuel process 1-industrial efflu. (1/4) 2-industrial efflu. (3/4) 4-sedimt. contam. (1/1) 1-(13/27) organics 3-inorganic (pure) 2-oil refinery wastewater 3-oil sands tailing, refinery,fossil fuel wastewater 2-industrial efflu. (1/1) 1-sedimt. contam. (1/2) 2-sedimt. contam. (1/2) 2-sedimt. extracts 1-sedimt. extracts 

•The evaluations were made on chemical groupings. •Microtox was more sensitive than or as sensitive as the acute lethality tests for pure organics, but less sensitive to most inorganics.•Microtox not as sensitive to effluents or leachates w/high components of insecticides, herbicides, inorganics, pharmaceuticals or textiles, or highly lipophilic contaminants.•As complexity and toxicity of industrial effluents increased, the sensitivity of Microtox increased.•Limitations of the available data: variation of sensitivity with the compounds and organisms tested; lack of standardization in approach and failure to provide details; published reviews commonly failed to identify fish species and test details for cited data.•Data and studies available for comparison were separated by chemical groupings into organic chemicals, inorganic chemicals, municipal wastes, oil refinery wastewaters, industrial effluents/complex wastes, and sediment extracts or landfill leachates.•Micotox has a lower CV than many other bioassays with decreasing variablility with increased toxicity.•Possible that lyophilized bacteria show an increase in sensitivity due to toxicants 

(multichlorinated organics) crossing cell membranes.•Variability of Microtox is higher w/metals than organics. •Microtox good for field screeningindustrialized effluents, greater sensitivity to effluents than pure compounds, more sensitive to commercial formulations of herbicides and pesticides than pure chemical forms. •Increased sensitivity to effluents may be due to pH, NH3, and phenols. •Conclusions: Organics--as sensitive as acute lethal tests to most pure substances, more sensitive to complex

compounds.Inorganics--not as sensitive as Daphnia and less sensitive than rainbow trout and fathead minnow, except Hg, As, and Co. Municipal wastes--favorable results w/Microtox. 

Petroleum wastes--useful test. 

Industrial wastes--good for screening for relative toxicity. Other wastes--less sensitive to insecticides, herbicides, pharmaceutical effluents, textile effluents, and highly lipophilic contaminants.

28 

August 25, 1997 C-14
HAZMAT 93-1–Appendix C

Test Treatments # Labs Relative Sensitivity 

Test Organisms Type Test Control Exp. Rep. # per Endpoint(s) Testing Rank Comments Ref.

Rhepoxynius abronius Dendraster excentricus Neanthes spp.Crassostrea gigas 

10d 

72h 

20d 

48h 

1 2 1 1 

7/site; 3 

stations 

7/site; 3 

stations 

7/site; 3 

stations 

7/site; 3 

stations 

5 5 

10 

5 

20 

2x104

2x104

Mortality % Mortality % Abnorm. %Mortality % Mortality % Abnorm. %

1 1 1 1 

1--mortality (high) 2--mortality (med.) 1--abnorm. (slightly) 3--mortality (least) 2--abnorm. 

•Bivalve test--estimated number of initial larvae not representa-tive of true number of initial larvae; high mortality due to unmeasured aspect; unusual sensitivity due to unevaluated factor •Amphipod test--bioassay appears to be the most sensitive •Echinoderm test--only positive numbers used to calculate the mean mortality (i.e., 3/7 stations) 

31 

Photobacterium phosphoreum Arbacia punctulata Pimephales promelas Daphnia magnaMenidia menidia Mysidopsis bahia Arbacia punctulata S. purpuratus S. droebachiensis C. gigas 

FT 

S S S S S S S 

15min 

4h 1h 

96h 48h 96h 96h 48h 48h 48h 48h 

1 NaCl 4 conc. 

6-10 conc. 

Bioluminesces Growth (E) Mortality (S) MortalityMortalityMortalityMortalityEC50 LC50 LC50 LC50 

1-trichloroethaniol(ave. rank for organics (ARO)= 3.25) (ARO = 3.75) 1-pentanedione, methylpropanol, methyl pentanedione, ethocxyethoxyethanol (ARO = 2.50) 1-hexachloroethane (ARO = 3.12) 1-pentachlorophenol (ARO = 2.00) 2-silver (ave. rank for metals (ARM)= 4.0) 1-lead (ARM = 3.6) Ave. metals = 3.2 (E) Ave. metals = 3.2 (S) Ave. metals = 1.2 (E) Ave. metals = 2.6 (S) Ave. metals = 2.6 (E) Ave. metals = 3.2 (S) Ave. metals = 1.8 (E) Ave. metals = 1.8 (S) 

•Protocols: Beckman Instruments, Inc. 1982. Microtox System Operating Manual. Fullerton CA. p. 8-11; sea urchin embryo-Jackim & Nacci (1984); Nacci & Jackim (1985) ASTM STP 891, pp. 382-394; sea urchin sperm-Dinnel, P., et al. (1983). •Conclusions: 

Some chemicals measures similarly by both rapid and acute tests Only parent compounds measured--not metabolic by-products Sperm cell tests overestimated the general toxicity of compounds In metal exposures, rapid tests measured toxicity similar to standard embryo tests, but maynot have predictable relationships for acute toxicity in certain marine fish and macroinvertebrates Some rapid tests predicted general toxicity 

29 

C-15 August 25, 1997
HAZMAT 93-1–Appendix C

Test Treatments # Labs Relative Sensitivity 

Test Organisms Type Test Control Exp. Rep. # per Endpoint(s) Testing Rank Comments Ref.

Rhepoxynius abronius Eohaustorius washingtonianus 

F-T F-T 

7d 7d 

66 

300300 

Higher conc. of toxic intermediate compound & covalent binding of xenobiotic to macromolecules same as above 

1 1 

1 2 

•Higher levels of covalent binding of BaP intermediates in R. abronius than in E.washingtonianus suggest a higher potentialfor disruption of cellular processes and subsequent impairment of the overall health of R. abronius in PAH-contaminated sediments •R. abronius was shown to be more susceptible than E. washingtonianus to contaminated 

sediments •There were notable differences in relative proportions of individual metabolites produced by R. abronius & E. wasthintonianus (e.g., higher amounts of BaP 7-8-diol to BaP-9-10-diol was > for 

R. abronius; BaP-7-8-diol is more cytotoxic, mutagenic and carcinogentic) •The concentration of [3H]BaP in the sediment did not change significantly during the 7d 

exposure; average value was 5.1 pmol BaP/g sediment dry wt 

33 

Photobacterium phosphoreum 

Oncorhynchus mykiss Daphnia magnaSpirillum volutans 

S S 

5 min 

96h 48h 

5 min 

1 

1/series 

1 neg. & 1 

pos. 

4 dilution 

log seriesdilution log seriesdilution log seriesdilution 

2 1 2 

2 & 1 

0.5 ml 

5 

0.1 

ml/l

Bioluminescence EC50Mortality LC50 Mortality LC50 Reduced reversal swimming

1 1 1 1 

2.75-metal average 4/4-NH4, KCN 2/4-Phenol, styrene, chloroform 1/4-dichloroethane 1.5-ave. complex effluents 2.25-metal average 1.6-NH4, KCN 1-Phenol, styrene, chloroform 2-dichloroethane 2.3-ave. complex effluents 1.25-metal average 2.3-NH4, KCN 3-Phenol, styrene, chloroform 3-dichloroethane 1.7-complex effluents 3.5-metal average 2-NH4, KCN 4-Phenol, styrene, chloroform 4-dichloroethane 

•Ranking shown in decending order from greatest to least sensitive •The Microtox test generally demonstrated the greatest sensitivity to industrial effluents •In general, the rainbow trout was found to be the most sensitive for several toxicants •The daphnid assay was more sensitive than Microtox when the rainbow trout bioassay was not the most sensitive •Micotox may be a poor indicator of ammonia toxicity •Rainbow trout & Microtox were the most sensitive tests for the organic compounds •Daphnid & spirillum bioassays were generally the least sensitive tests for organic compounds •Rainbow trout were 22 & 30x more sensitive to Zn & Cu than the Microtox test; for these metals, the daphnid was the most sensitive with spirillum the least sensitive, especially for As & Hg •Regarding potassium cyanide toxicity, the rainbow trout was 11, 40, and 87x more sensitive than the spirillum, daphnid, & Microtox assays respectively •Limitations: Microtox proceedure requires the addition of 2% NaCl, & ideally the 

utilization of freshwater luminescent bacteria would be more desirable The pH of the diluent is distictly different from that of the aquatic environmentsl samples & may affect test relevance The revised Microtox proceedure was not available when this study was conducted Color of the effluents & turbidity may affect the instrument readings for bioluminescence in the Microtox tests 

32 

Armandia brevis Dendraster excentricus Rhepoxinius abronius 

F-T 

20d 28d 10d 

1 ref. 

1 posi-tive 

1 ref. 

1 pos. 

1 ref. 

1 pos. 

17 sites, 4 

expo-sure 

17 sites, 2 

exp. 

9 sites, 1 

exp. 5 

10 20 10 

Growth (weight) Growth (lngth/wt) MortalityReburial 

1 1 1 

1-red. growth 2-red. growth 1-mortality 

•Reduced growth of polychaetes was significantly correlated with increasing proportions of contaminants in sediments, 14/17 •Growth of sand dollars was significantly correlated w/conc. of various contaminants in sediments; as contam. conc. of LPAHs, HPAHs, PCBs, & some individual selected elements increased, sand dollar growth decreased, 14/17 •Signif. amphipod mortality occurred in 2/9 sediments; signif. reduced reburial was not observed; neither mort. nor nonreburial correlated signif. w/sed. contam., org. carbon content,or grain size 

34 

August 25, 1997 C-16
HAZMAT 93-1–Appendix C

Test Treatments # Labs Relative Sensitivity 

Test Organisms Type Test Control Exp. Rep. # per Endpoint(s) Testing Rank Comments Ref.

Ampelisca abdita Eohaustorius estuarius Leptocheirus plumulosus 

S S S 

10d 10d 10d 

1 1 1 

BRH % 

7,20,33 9,25,42 

10,28,47 

5 5 5 

20 20 20 

MortalityMortalityMortality

6 8 7 

1 2 3 

•Interlaboratory comparison study; protocol from EPA600/R-94/025 •Black Rock Harbor % contaminated sediment tested in experimental groups •All species identified moderate to highly contaminated sediments •More variability of data for low contam. sediments between labs •All species appropriate for measuring toxicity of sediments 

35 

Photobacterium phosphoreus Pimephales promelas Daphnia magna

5 min

24h 24h 

Bioluminescense LC50 LC50 

1 1 1 

1-Most sensitive test 2-81% agreement w/P. promelas& Micotox 3-62% agreement w/Microtox 

•An effluent study comparing Microtox with fathead minnow and daphnia •No test was able to predict 100% presence of toxicant to another species 

37 as 

cited in

16

Photobacterium phosphoreum Rhepoxynius abronius Corophium volutator Neanthes sp.Macoma balthica Pseudopleuronectes americanus 

F-T 

5 & 15 

min 

10d 

96h 

10d 

96h 

20d 30d 

2wks 

KCr2 ref 

Sea H2O 

2 sites 

CdCl2

2 sites 

CdCl2

2 sites & 

CdCl2

1 ref site 1 

cont.

9 

6 sites 5 sites 4 sites 1 sites 

3 

5 1 5 1 5 2 

20 10 20 10 

5 

60 

Bioluminescence EC50Survival LC50 Survival LC50 Biomass Bioaccumulation Histopathology

1 1 1 1 1 1 

1-solvent extract 2-solid phase 4-pore water 3- 5- 6- 

•Microtox tests: pore water, org. solvent extract, solid phase •Only the 3 Microtox and the R. abronius useful in assessing the degree of contamination at the regulatory levels of contaminants •Microtox test was the most sensitive bioassay for sediment toxicity assessment •R. abronius was relatively sensitive to the Halifax sediments; most suporting study for the Microtox bioassay •Macoma exposure too short for significant uptake of contaminants •Sample size too small in winter flounder test, but lesions found •Contaminants in Halifax Harbor: Metals (e.g., As, Cd, Cu, Hg, Pb, Zn); PAHs•Protocols: Microtox--supplied by MicrobicsTM 

Neanthes sp.--Johns et al. 1990 

R. abronius & C. volutator--Sed. Tox. Test. using 

 Infaunal Amphipod R. abronius Environ. Canada, 1989 

Macoma--method developed by Environ. Canada 

 Atlantic Regional Laboratory 

P. americanus--Luna, 1968 

36 

Hyalella azteca Chironomus tentans 

DDT, DDE, DDD 

DDT, DDE, DDD 

38 cited

in 20 

Hyalella azteca Chironomus tentans Lumbriculus variegatus

S-R 

F-T F-T 

10d 10d 10d 

1 ref. 1 ref. 1 ref. 

11 11 11 

2 2 3 

10 10 10 

Survival Survival Surv. & Reprod. 

1 1 

1-Cu-LC50 @ 31 μg/l 3-Cu-LC50 @ 54 μg/l 2-Cu-LC50 @ 35 μg/l 

•LC50 values were for water only exposure to copper sulfate •Reduced survival from sediment exposure (Keweenaw Waterway sites:  •H. azteca--8/11 were signif. toxic from control  •C. tentans--7/11 were signif. toxic form control  •L. variegatus--0/11 were signif. toxic from control; reproduction decreased 6/11 signif. diff.

from control 

39 

Rhepoxynius abronius Crassostrea gigasPhotobacterium phosphoreum 

S 10d

48h 

15min 

12 

4 ref. 

5 H2O 

4 ref. 

1@0 

2ref.tox. 

4 ref. 

46 46 

46@100,50,

25,12.4 ref. 

5 20 Survival 

Survival Abnormal Bioluminescence 

1 1 1 

2-39% signif. mortality over ref. 

3-35% signif. abnormal over ref. 1-63% signif. decreases over ref. 

•Microtox utilizing the saline extract procedure. •Agreement among the three bioassays was evaluated using correlation analysis.•Protocols--oyster-Chapman & Morgan (1983); amphipod-Swartz et al. (1985); MicrotoxSchiewe et al. (1985). •Statistical comparisons showed that the results of the Microtox, oyster embryo and amphipod bioassays were significantly correlated and concurred in determination of the presence or absence of toxic effects in 41% of the sediment samples. 

40 

5 sites 

1 

C-17 August 25, 1997
HAZMAT 93-1–Appendix C

Codes:Test type S = static Sensitivity Rank 1 = highly sensitive Life Stage J = juvenile  F-T = flow-through 2 =moderately A = adult 

S-R = static-renewal 3 = less than moderately sensitive E = embryo 

Duration d = days 4 = least sensitive S = sperm h = hours Reps i.e., 3/T = three per treatment N = neonates August 25, 1997 C-18
HAZMAT 93-1–Appendix C

Table C-1 References:

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August 25, 1997 C-22
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C-23 August 25, 1997