PA/600/8-87-044
x>EPA
United States
Environmental Protection
Agency
Environmental
Research Laboratory
Corvallis OR 97333
EPA/600/8-87/044
August 1 987
Research and Development
Role of Acute Toxicity
Bioassays in the
Remedial Action
Process at Hazardous
Waste Sites
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EPA/600/8-87/044
August 1987
ROLE OF ACUTE TOXICITY BIOASSAYS
IN THE REMEDIAL ACTION PROCESS
AT HAZARDOUS WASTE SITES
by
L. A. Athey
J. M. Thomas
J. R. Skalski
Pacific Northwest Laboratory
Rich!and, Washington 99352
W. E. Miller
Corvallis Environmental Research Laboratory
Corvallis, Oregon 97333
Project Manager
D. A. Neitzel
Earth and Environmental Sciences Center
Pacific Northwest Laboratory
Richland, Washington 99352
Pacific Northwest Laboratory
Richland, Washington 99352
U.S. Environmental Protection Agency
P i,-:i 5, Library (5PL-16)
£-o"s. Deartorn Street, Room 1670
Cnicago, IL 60604
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NOTICE
This document has been reviewed in accordance with
U.S. Environmental Protection Agency policy and
approved for publication. Mention of trade names
or commercial products does not constitute endorse-
ment or recommendation for use.
11
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SUMMARY
This document is written to aid in making decisions regarding the use of
standardized aquatic and terrestrial acute toxicity bioassays at hazardous
chemical waste sites. Other types of bioassays, including those for use with
receiving waters and in situ testing, are not discussed. The use of acute
bioassays at hazardous chemical sites is explained and illustrated for use in
the remedial action process. Step-by-step guidelines are presented whereby
decisions can be made concerning the design of site-specific bioassay
studies.
The use of acute laboratory bioassays as a method of assessing toxicity
of samples from hazardous chemical sites is a relatively recent development.
Therefore, we have included currently available bioassay results from three
actual hazardous chemical waste site studies to illustrate the ability of
standardized bioassays to define toxicity at hazardous waste sites:
Soils from the Rocky Mountain Arsenal were bioassayed and the results
used to prepare a cleanup map based on phytotoxicity.
Bioassay results from sediment samples taken from a wood treatment site
were compared to chemical analysis results to determine whether
bioassays can be used effectively to guide remedial actions at
creosote-contaminanted sites.
Bioassay results from soil, sediment, and surface-water samples at two
hazardous chemical waste sites were used to evaluate site toxicity,
determine sources of chemicals, and illustrate the use of staged
sampling and compositing.
References to major U.S. Environmental Protection Agency documents and
other papers that explain standard methods for sampling soil, sediment,
surface water, and ground water are included.
With the development of a suitable rationale for regulatory action based
on bioassay results, bioassays offer great promise as a standard
site-specific method of environmental risk assessment at hazardous chemical
waste sites.
iii
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ACKNOWLEDGMENTS
The technical help of Dr. Dave Thome, Dr. Bill Trautman, and Mr. Brian
Anderson of the Rocky Mountain Arsenal (Colorado) as well as the cooperation
of Dr. Royal Nadeau at EPA-Edison, New Jersey were essential 1n accomplishing
the work in Sections 8.1, 8.2, and 8.3. In addition, Dr. David Charters and
members of EPA's Emergency Response Team, Edison, New Jersey, collected the
samples used in Section 8.2. At the Pacific Northwest Laboratory, Stacy
Freeman typed the original manuscript, Linda Parker finished the final
report, and Kathy Borgeson, Steve Weiss, and Susan Kreml supplied editorial
expertise.
The information in this document has been funded wholly or in part by
the U.S. Environmental Protection Agency, Environmental Research Laboratory
in Corvallis, Oregon, under Related Services Agreement TD 1598 with the U.S.
Department of Energy, Contract DE-AC06-76RLO 1830, to Pacific Northwest
Laboratory. It has been subject to the Agency's peer and administrative
review, and it has been approved for publication as an EPA document.
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CONTENTS
SUMMARY in
ACKNOWLEDGMENTS v'
FIGURES x
TABLES xii
1.0 INTRODUCTION 1
1.1 OBJECTIVES OF THIS DOCUMENT 1
1.2 AUDIENCE FOR WHICH THIS DOCUMENT IS INTENDED .... 1
1.3 WHAT IS A BIOASSAY? 1
1.4 ORGANIZATION OF THIS DOCUMENT 2
1.5 MEASUREMENT EQUIVALENTS 3
2.0 CONCLUSIONS 4
3.0 DESCRIPTION AND USE OF STANDARDIZED ACUTE BIOASSAY STUDIES
AT WASTE SITES 5
3.1 WHERE BIOASSAYS FIT IN THE REMEDIAL ACTION PROCESS . 5
3.2 QUESTIONS BIOASSAYS CAN (OR CANNOT) ANSWER .... 5
3.3 ADVANTAGES AND LIMITATIONS OF BIOASSAYS COMPARED
TO CHEMICAL ANALYSES 8
3.4 DESIGN DECISIONS FOR ACUTE BIOASSAY STUDIES AT CHEMICAL
WASTE SITES 9
3.4.1 Assemble Information Relevant to the Problem . . 11
3.4.2 Prepare a Statement of the Study Objectives . . 13
3.4.3 Define the Evaluation Criteria and Reliability
Requirements for the Results 13
3.4.4 Determine What Is to Be Sampled in the Field . . 16
3.4.5 Choose the Test Organisms for the Bioassays . . 16
3.4.6 Define the Data Analysis Techniques .... 17
3.4.7 Design the Field Sampling and Laboratory Studies . 17
3.4.8 Determine the Sample Collection Methods ... 18
3.4.9 Define the Operational Procedures .... 18
3.4.10 Review the Design 19
3.4.11 Periodically Evaluate Progress in the Field
and Laboratory 19
3.4.12 Analyze and Evaluate the Results 20
vii
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4.0 BACKGROUND FOR ACUTE BIOASSAY USE AT CHEMICAL WASTE SITES . . 21
4.1 INTRODUCTION 21
4.2 DEVELOPMENT OF BIOASSAYS FOR WASTE SITE STUDIES . . .22.
4.3 SENSITIVITY, APPLICATION, AND INTERPRETATION OF WASTE SITE
BIOASSAY RESULTS 23
4.3.1 Bioassays Using Pure Chemicals 23
4.3.2 Bioassays Using Waste Site Samples with Known
Chemistry 24
4.3.3 Bioassays Using Waste Site Samples with Inferred
Chemical Constituents 28
5.0 STATISTICAL TECHNIQUES FOR WASTE SITE BIOASSAY STUDIES ... 32
5.1 THE ROLE OF PRIOR WASTE SITE INFORMATION IN BIOASSAY STUDY
DESIGN 32
5.2 ANALYSIS TECHNIQUES FOR DETECTING CONTAMINATION AND
ESTIMATING ACUTE TOXICITY 32
5.3 -TECHNIQUES FOR ESTIMATING SPATIAL DISTRIBUTION ... 36
6.0 AN OVERVIEW OF WASTE SITE BIOASSAY STUDY DESIGN .... 38
6.1 COMPONENTS OF VARIABILITY AND THEIR IMPACTS ON STUDY
DESIGN 38
6.2 ONE-STAGE VERSUS MULTISTAGE STUDY DESIGNS .... 41
6.3 SAMPLING STRATEGIES FOR COLLECTING FIELD SAMPLES ... 43
7.0 METHODS OF SAMPLING MEDIA FOR WASTE SITE BIOASSAYS ... 46
7.1 SOIL ............ 46
7.2 SEDIMENT . 47
7.3 GROUND WATER 47
7.4 SURFACE WATER 48
8.0 CASE STUDIES: ILLUSTRATIONS OF BIOASSAY METHODS AND DESIGN
DECISIONS 50
8.1 SOIL SAMPLING AT THE ROCKY MOUNTAIN ARSENAL .... 50
8.1.1 Assemble Information Relevant to the Problem . . 50
8.1.2 Prepare a Statement of Study Objectives ... 55
8.1.3 Design the Field Sampling and Laboratory Studies . 55
8.1.4 Analyze and Evaluate the Results 57
viii
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8.2 SEDIMENT SAMPLING AT A WOOD TREATMENT SITE
71
8.2.1 Assemble Information Relevant to the Problem 71
8.2.2 Prepare a Statement of Study Objectives . 71
8.2.3 Define the Data Evaluation Criteria and
Reliability Requirements ... 73
8.2.4 Determine What Is to Be Sampled . . 73
8.2.5 Choose Test Organisms for the Bioassay. 73
8.2.6 Define the Data Analysis Technique . 73
8.2.7 Design the Field Sampling and Laboratory Studies 73
8.2.8 Determine the Sample Collection Methods 75
8.2.9 Define the Operational Procedures. . 75
8.2.10 Review the Design 76
8.2.11 Periodically Evaluate Progress . . 76
8.2.12 Analyze and Evaluate the Results . . 76
8.3 SOIL, SEDIMENT, AND SURFACE-WATER SAMPLING AT TWO HAZARDOUS
WASTE SITES 81
8.3.1 Assemble Information Relevant to the Problem . . 81
8.3.2 Prepare a Statement of Study Objectives . . 86
8.3.3 Define the Evaluation Criteria and
Reliability Requirements for the Results . . 86
£.3.4 Determine What Is to Be Sampled in the Field . 88
8.3.5 Choose Test Organisms for the Bioassays . . 88
8.3.6 Define the Data Analysis Techniques ... 89
8.3.7 Design the Field Sampling and Laboratory Studies 89
8.3.8 Analyze and Evaluate the Results .... 90
9.0 NEEDED ENHANCEMENTS OR ADDITIONS TO BIOASSAY TECHNOLOGY FOR USE
AT HAZARDOUS WASTE SITES 101
9.1 BIOASSAY STUDIES USING ADDITIONAL PURE CHEMICALS, MIXTURES,
AND CHEMICALLY CHARACTERIZED WASTE SITE SAMPLES . . .101
9.2 ADDITIONAL WASTE SITE STUDIES 102
9.3 DECISION RULES TO RATE WASTE SITE SAMPLE TOXICITY . . .102
9.4 SCREENING BIOASSAYS 102
9.5 DEVELOPMENT AND ENHANCEMENT OF LABORATORY COMPOSITING
STRATEGIES AND CONCOMITANT FIELD DESIGNS FOR DETECTING
MOVEMENT AND EXTENT OF CONTAMINATION 103
REFERENCES 104
ix
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FIGURES
3.1 Bioassay Data Implementation in the Remedial Action Process . 6
8.1 Principal Physical Features of the Rocky Mountain Arsenal,
Commerce City, Colorado 52
8.2 Location of the Study Site in Basin A at the Rocky Mountain
Arsenal 53
8.3 Location of Logarithmic Sampling Points in Basin A
at the Rocky Mountain Arsenal 56
8.4 Observed Mean Lettuce Seed Mortality at Each Basin A Plot,
0- to 15-cm Soil Fraction 58
8.5 Observed Mean Lettuce Seed Mortality at Each Basin A Plot,
15- to 30-cm Soil Fraction . . 59
8.6 Comparison of Earthworm and Lettuce Seed Mortality Using
Basin A Soils from the Rocky Mountain Arsenal .... 63
8.7 Comparison of Lettuce Root Elongation and Lettuce Seed
Mortality Using Basin A Soils from the Rocky Mountain
Arsenal 64
8.8 Estimated Lettuce Seed Mortality for the 0- to 15-cm Soil
Fraction from the Rocky Mountain Arsenal 66
8.9 Estimated Lettuce Seed Mortality for the 15- to 30-cm Soil
Fraction from the Rocky Mountain Arsenal 67
8.10 Comparison of Lettuce Seed Mortality Predicted from Kriging
to Actual Lettuce Seed Mortality 0- to 15-cm Soil
Fraction 69
8.11 Comparison of Lettuce Seed Mortality Predicted from Kriging
to Actual Seed Mortality, 15- to 30-cm Soil Fraction . 70
8.12 Wood Treatment Site in Mississippi 72
8.13 Location of Samples Collected from the Wood Treatment Site
in Mississippi 74
8.14 Bioassay Results from Sediment Elutriates at the Wood
Treatment Site in Mississippi 78
8.15 Bioassay Results from Sediments at the Wood Treatment
Site in Mississippi 80
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8.16 Seven Sampling Locations at or near Hazardous Waste Site 1 . 82
8.17 Sampling Locations at Landfill Site 2 85
8.18 Grid Sampling at Location F-3 of Site 1 91.
8.19 Transect Sampling at Location F-4 of Site 1 . . . .91
8.20 Opportunistic Sampling at Location C-11 of Site 2 . . .92
xi
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TABLES
1.1 Measurement Conversion.Chart
3.1 Questions That Need to Be Addressed to Support Remedial
Action Decisions at Hazardous Chemical Waste Sites ... 7
4.1 EC50 Response in Percent Soil for Earthworms and Percent Elutriate
for Other Organisms Exposed to Chemical Contaminants in Hazardous
Waste (Ada Samples Only), Waste Site Soil, Soil Elutriate,
and Ground-Water Samples 25
4.2 EC50 Response or Percent Inhibition Caused by Chemical
Contaminants in Rocky Mountain Arsenal Soil, Soil
Elutriate, Waste Water, and Ground-Water Samples .... 29
8.1 Examples of Some Chemicals Found in Soils, Air, Water, Animals,
and Plants at the Rocky Mountain Arsenal 54
8.2 Intercomparison of Lettuce Seed Mortality, Lettuce Root
Elongation, Earthworm Mortality, and Algal Inhibition
for Two Fractions of Basin A Soils Obtained from
the Rocky Mountain Arsenal 61
8.3 Bioassay Results from Phase 1 Samples Collected from the
Wood Treatment Plant in Mississippi 77
8.4 Description of Composite Components Used in Screening
Bioassays of Samples Taken from the Locations Shown in
Figures 8.18 to 8.20 93
8,5 Bioassay Results from Three Stages of Sample Analysis at
Sitel . . . . . . « « .94
8.6 Bioassay Results from Three Stages of Sample Analysis at
Site 2 98
8.7 Description of Samples Collected at Additional Site 2
Locations Where Positive Bioassay Results Were Obtained . . 99
xii
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1.0 INTRODUCTION
1.1 OBJECTIVES OF THIS DOCUMENT
This document provides guidance for deciding how bioassays may be
incorporated into the remedial action process at a hazardous chemical waste
site. It also provides aids for designing, executing, and interpreting the
results of bioassay studies. Alternative bioassay study designs are
discussed, and illustrations showing their use are presented. Because of the
focus on hazardous chemical waste sites, information on bioassessment for
other purposes is generally excluded (e.g., in situ bioassay, bioassay of
receiving waters). The intent of this document 1s not to prescribe the way
to use bioassays to help assess the toxicity of hazardous waste sites.
Because specific site problems vary to such a great extent, application at
any particular location will require a site-specific design depending on the
waste(s), their location, and the intended use of the bioassay information.
Thus, the purpose of this document is to present a description of acute
bioassay studies, the background for their use at chemical waste sites, and
field designs and sampling methods as well as case studies.
1.2 AUDIENCE FOR WHICH THIS DOCUMENT IS INTENDED
This document is intended for site managers, state agency personnel, and
other individuals responsible for making decisions about remedial action at
hazardous chemical waste sites. It can be used by readers with little
experience in performing bioassay studies. The document presents an overview
of the decisions that must be made to determine how bioassay studies should
be included in the remedial action process and information for choosing among
alternative field study designs. References are provided to guide readers to
sources of additional technical detail.
1.3 WHAT IS A BIOASSAY?
A bioassay is a technique by which organisms (i.e., whole plants or
animals), biological systems (e.g., tissues), or biological processes (e.g.,
enzymatic activity) are used to measure the biological effects of a
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substance. In the context of hazardous chemical waste site management,
bioassays may be defined as the exposure of biological indicators to
field-collected environmental samples in order to detect the presence of
toxicity and/or to identify potential for toxic effects on resident species.
Typically, a hazardous waste site bioassay involves laboratory testing of
soil, soil leachates, water, or sediment samples using a standard array of
test organisms under controlled laboratory conditions.
1.4 ORGANIZATION OF THIS DOCUMENT
Section 2.0 summarizes the conclusions of this document.
Sections 3.1 through 3.3 present information to be used in deciding how
! bioassays may be used for a particular chemical waste site problem. Section
3.1 presents an overview of where and how bioassays can be used in the
remedial action process. Questions that bioassays can or cannot answer are
presented in Section 3.2, followed by a discussion in Section 3.3 of the
advantages and disadvantages of using bioassays instead of chemical analysis
to define site problems.
Section 3.4 contains a list of steps to follow in planning, executing,
and interpreting the results of a bioassay study. This section also includes
a discussion of the decisions to be made in designing and executing the field
sampling, laboratory analyses, and data evaluation.
Section 4.0 contains a detailed discussion of the development and use
of bioassays. This section is followed by explanations in Sections 5.0, 6.0,
and 7.0 of the basis for choosing among alternative study designs.
Section 8.0 contains examples of bioassay studies that illustrate the
information presented in the previous sections, and Section 9.0 presents a
discussion of enhancements that could improve bioassay technology and
directions for future research.
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1.5 MEASUREMENT EQUIVALENTS
Metric units are used 1n this report. A conversion chart for
appropriate English measurements is provided as Table 1.1.
TABLE 1.1. Measurement Conversion Chart
Measurement Used
Square mile
Acres
Meters
Centimeters
Feet
Inches
Kilograms
Grams
Liters
Mill inters
Multiply By
2.60
0.40
3.28
0.39
0.305
2.54
2.21
0.035
1.06
0.03
For
Square kilometers
Hectares
Feet
Inches
Meters
Centimeters
Pounds
Ounces
Quarts
Fluid ounces
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2.0 CONCLUSIONS
Based on our review of specific bioassessment procedures and their use
at four different sites, we concluded that acute toxicity bioassays are a
valuable and cost-effective method for use in the remedial action process at
hazardous chemical waste sites. They have two important advantages over
chemical analyses: 1) they can directly measure the effects of a known or
suspected contaminant on plants and animals, and 2) they are, in general,
Inexpensive compared to complete chemical analyses.
With the development of a suitable rationale for regulatory action based
on bioassay results, bioassays offer great promise as a standard
site-specific method of environmental risk assessment at hazardous chemical
waste facilities.
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3.0 DESCRIPTION AND USE OF STANDARDIZED ACUTE BIOASSAY
STUDIES AT WASTE SITES
3.1 WHERE BIOASSAYS FIT IN THE REMEDIAL ACTION PROCESS
Bioassay studies are appropriate before, during, and after remedial
action as a cost-effective way to detect the presence of toxic wastes and/or
to determine their biological availability at known or suspected hazardous
chemical waste sites. The diagram of the remedial action process shown in
Figure 3.1 indicates the many ways in which bioassay data are useful. A
detailed explanation of this process is found in Ford and Turina (1985).
Bioassay studies may be used before remedial action to detect the
presence of hazardous materials and to determine if immediate remedial action
is needed. Because bioassays directly evaluate the effect of chemical wastes
on biota, they are powerful and efficient tools for ranking sites requiring
remedial attention. Bioassay data are also useful for gauging the areal
extent of needed remedial action, evaluating remedial action alternatives,
and assessing and characterizing waste sites.
During remedial action, bioassay studies may be used both to monitor the
cleanup process and to evaluate cleanup impacts on the site. Bioassays are
also useful after remedial action has been taken to evaluate the efficacy of
the cleanup activities.
3.2 QUESTIONS BIOASSAYS CAN (OR CANNOT) ANSWER
The questions that often need to be addressed in support of remedial
action decisions may be grouped into four major areas: 1) Where are the
contaminants? 2) Are the contaminants toxic? 3) What quantities of the
contaminants are present? and 4) What are the contaminants? Specific
questions under each of the four categories are listed in Table 3.1.
Bioassay studies are a cost-effective method for evaluating "where"
questions because they can detect contamination, determine contaminant
distributions, and define contaminant migration beyond site boundaries.
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Planned
Response
Discovery
I
Stabilization
Preliminary
Survey
Decision for
Further
'Response
Site
Characterization
Assessment
±
Identification
of Alternatives
I
Analysis of
Alternatives
Selection of
Remedial
Action
1
Design of
Remedial
Action
BEFORE
BIOASSAY
Study Data
Implementation
of Remedial
Action
Monitoring
of Remedial
Action
DURING
Post
Closure
Evaluation
AFTER
FIGURE 3.1. Bioassay Data Implementation 1n the Remedial Action Process
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TABLE 3.1. Questions That Need to Be Addressed to Support Remedial
Action Decisions at Hazardous Chemical Waste Sites
Category Specific Questions
WHERE?
TOXICIITY?
WHAT QUANTITY?
Is the site contaminated?
Are specific areas contaminated?
How much of the total site is contaminated?
What is the distribution of the contaminants?
Have the contaminants migrated off site?
How toxic are the contaminants separately or
together?
What quantities of the contaminants are present on
the site (inventory)?
Is the contaminant concentration above a prescribed
limit [in parts per million (ppm) or a related
measure]?
What is the chemical composition of the
contamination?
Is a specific chemical present on the site?
Is a particular class of toxic chemicals (e.g.,
organics, metals) present on the site?
While bioassays currently are the only reliable means of evaluating the
integrated bioactivity of complex chemical wastes, they usually cannot
identify specific chemical contaminants present. However, they provide a
quantitative indication (EC50 or LC50) of total toxicity relative to the
percentage of sample water, soil or leachate that is required to produce the
EC50 or LD50. In addition, they may give some indication of the class of
chemicals to which the contaminants belong (e.g., organics, metals). Some
examples in which the identification of specific chemicals have been
attempted are given in Sections 8.1 and 8.3.
WHAT?
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3.3 ADVANTAGES AND LIMITATIONS OF BIOASSAYS COMPARED TO CHEMICAL
ANALYSES
Bioassays have two important advantages over chemical analyses. They
can directly measure the effects of a known or suspected contaminant on
biota, and they are, in general, inexpensive compared to complete chemical
analyses.
Complete chemical analysis of a waste site does not provide information
as to whether the waste will cause harm to biological organisms. For
example, heavy metals may be present at a particular site in high
concentrations, but may be chelated by organic compounds in soils or
sediments and thus be almost completely unavailable to the biota (Thomas et
tal. 1984a). Conversely, presumably harmless chemicals may have toxic
effects. Results of studies by Miller et al. (1985) have shown that "inert"
compounds in a herbicide formulation were, in fact, active toxicants or
synergistic components. Also, the toxic effects of chemical mixtures are not
readily predictable from knowledge of the toxic effects of the individual
chemicals (Miller et al. 1985). Chemical waste sites almost always contain a
mixture of chemicals. Therefore, bioassays are valuable tools for measuring
the integrated toxicity potential of waste mixtures (Roop and Hunsaker, 1985)
whether chemical concentrations have been measured or in cases where
chemistry of the mixture is uncharacterized. Thus, bioassays are capable of
providing guidance on toxicity limitations, even in situations where federal
or state environmental chemical concentration limits do not exist.
Acute toxicity bioassays are generally less expensive than conventional
priority pollutant chemical analyses. Schaeffer et al. (1982) reported
average analytical costs as high as $2000-$5000 per single soil sample for
the U.S. Environmental Protection Agency (EPA) priority pollutants. Costs
have declined to $1500-$2000 per sample/3' which are closer to the average
$1000-$1500 cost of conducting acute toxicity tests with algae and Daphnia.
However, cost alone should not be the determinant in choosing between the use
of priority pollutant chemical analyses and bioassays to define the hazard at
Personal communication from Dr. G. B. Weirsma of Idaho National
Engineering Laboratory, August 15, 1986.
8
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waste sites. All available resources must be allocated to attain the best
balance of both chemical and biological data. The concept and use of
bioassays to support and/or direct chemical analyses of environmental samples
is discussed by Miller et al/a' They indicate this integrated approach, in
addition to being cost effective, is the most feasible current way to define
the effects of environmental variables such as solubility, pH, antagonism,
synergism, and time of exposure upon the toxicity of complex chemical
mixtures.
Acute toxicity bioassays are inferior to chemical analysis for
evaluating the danger presented by a hazardous chemical waste when 1) no
known bioassay exists for a suspected or known chemical toxicant, 2) there
is a need to identify a specific pollutant (e.g., an EPA Priority Pollutant),
3) there is a need to conform to an established regulatory standard for a
particular pollutant in the environment (i.e., pollutant concentration or an
amount), or 4) there is a need to identify sublethal or chronic effects for a
specific chemical waste.
3.4 DESIGN DECISIONS FOR ACUTE BIOASSAY STUDIES AT CHEMICAL WASTE
SITES
Design of bioassay studies includes planning the collection of field
samples and the laboratory analyses of those samples. It is imperative that
the entire project, from objectives to expected results, be thoroughly
planned before the actual fieldwork is started. Without proper planning, the
study will waste both time and resources. Further, all individuals who will
contribute to the project should be involved as early as possible in project
planning. Personnel who should be involved at the initial stagel of the
project include the project manager, a statistician, a field biologist, a
chemist, the scientist who will oversee the laboratory work, possibly a
hydrologist, meteorologist, or modeler when appropriate, and risk assessment
and Quality Assurance/Quality Control experts.
* ' Miller, W. E., J. C. Greene, and S. A. Peterson. 1987. Protocol For
Bioassessment of Hazardous Waste Sites. 2nd Edition. Corvallis
Environmental Research Laboratory, Corvallis, Oregon. Final draft.
9
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The steps to be used in designing and executing a bioassay study are
listed below. Each step is explained further in the following sections.
1. Assemble infonnation relevant to the problem.
2. Prepare a statement of the study objectives.
3. Define the evaluation criteria and reliability requirements for the
results.
4. Determine what is to be sampled in the field.
5. Choose test organisms for the bioassays.
6. Define the data analysis techniques.
7. Design the field sampling and laboratory studies.
8. Determine the sample collection methods.
9. Define the operational procedures.
10. Review the design.
11. Periodically evaluate progress in the field and laboratory.
12. Analyze and evaluate the results.
These steps are listed in the order in which they should be applied by
the planning team. However, study design is an iterative process, and
decisions made at later steps in the process may require the review and
revision of decisions made at previous steps.
These 12 steps should be considered whether the study is an initial site
investigation, a feasibility study, or a full remedial action. The amount of
effort and degree of technical sophistication required for each "step will
vary depending on the objectives and cost of the study and the reliability
required of the results. For example, in a full and potentially expensive
remedial action investigation, the entire team should be involved from the
beginning. During preliminary investigations, the objectives, uses of the
results, and methods should be carefully planned before sampling; however,
the advice of team members from each area of expertise may not be necessary.
Depending on the quality of information gathered during the preliminary
investigations, the results of the initial studies may be used to design the
remedial action program.
10
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3.4.1 Assemble Information Relevant to the Problem
The first step in the design of a bioassay study is to assemble and
review all available information relative to the hazardous waste site. This
includes the history of waste disposal to the site, regulations regarding the
waste, requirements for conducting a site investigation in support of
remedial action, and resources available for conducting the bioassay studies.
Information that should be obtained at each hazardous waste site
includes:
1. the type of waste likely to be found on the site
2. the likely chemical and physical properties of the waste
3. the environmental media in which the waste is most likely to be found
(e.g., does the waste adhere to soil particles or is it more likely to
be found in the interstitial soil water?)
4. the location on the site where the waste is most likely to be found
5. the known toxicity of the waste to particular organisms or groups of
organisms. Sources of the needed information Include records of waste
disposal, manufacturer's records, expert chemists, the chemical
literature, environmental impact statements (both for the site and the
chemicals), and local experts.
Each hazardous chemical waste site is unique in some respect; therefore,
each site should be evaluated as a separate problem. Several site
characteristics will influence the design, execution, and interpretation of
the results of the bioassay study. A more thorough site study may be
required if the hazardous waste poses an increased risk because of:
1. human activity on the site (e.g., farming)
2. proximity of the site to human communities
3. the possibility of contaminant transport outside and/or inside the site
boundary (e.g., streams, ground water, or wind)
4. the presence of threatened or endangered species, or
5. the presence of commercially or recreationally valuable species on the
site.
11
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In addition, the presence of human activity or endangered or valuable species
on the site may limit the areas from which samples may be collected. Other
site characteristics (e.g., topographical) may also limit the type, number,
or location of samples that can be taken during the study.
The study planning should not proceed to the field study design (Step 7)
without an initial site visit. Preliminary studies may also be necessary to
gather some of the information.
Documentation of the history of waste disposal to the site may provide
valuable information about the types of contaminants that will be found and
their probable location and distribution. Possible sources of information
regarding the site disposal history include waste site records,
manufacturer's records, and local experts.
It is also necessary to review federal and state regulations regarding
the known or suspected waste materials. The texts of the Toxic Substances
Control Act (TSCA), the Comprehensive Environmental Response, Compensation
and Liability Act (CERCLA), and the Resource Conservation and Recovery Act
(RCRA) should be consulted for guidance to the federal regulations regarding
individual hazardous substances. The EPA hotline (1-800-424-9346), a good
source for the most recent information concerning federally regulated waste
materials, is operated during normal business hours (Eastern Standard Time).
Some, although not all, states have their own regulations regarding waste
substances in the environment. The EPA hotline, state environmental
agencies, and regional EPA offices may provide information about or access to
documents containing state regulations.
While this document provides guidance for deciding whetherbioassays are
appropriate for a waste site problem and for planning and carrying out
appropriate field and laboratory studies, it does not cover all aspects of
conducting a site study in support of remedial action. The reader should
consult Ford and Turina (1985) for this information.
Finally, a realistic appraisal must be made of available study resources
in order to preclude designing a study that is too expensive to complete
within the available budget or manpower. An elaborate study poorly or
incompletely done will yield less reliable information than a completed but
12
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smaller scale study. Further, most of the statistical analyses that are used
to describe bioassay results will either be questionable or invalid if
significant amounts of data are missing. The relationship between
anticipated costs and the available resources should be considered in every
stage of design planning.
3.4.2 Prepare a Statement of the Study Objectives
It is critical that an unambiguous statement of the study objectives be
prepared and understood by all of the project staff before the study begins.
This will minimize confusion and wasted effort during the design and
execution of the field and laboratory work.
Each objective must be phrased as a specific question. The advantage of
specific questions is that they can generally be studied as testable,
statistical hypotheses. A question such as "Does the waste site pose a
threat to the adjacent community?" 1s not acceptable because there are many
different ways of evaluating the impact of the hazardous waste on the
community. Examples of questions that can be answered or tested are "What is
the spatial distribution of the contaminant in the soil?" or "Is the
concentration of the contaminant in the ground water measured in onsite
wells significantly different from that measured in offsite wells?" The
reader is referred to Table 3.1 for additional examples of specific questions
that can be answered or easily modified into testable hypotheses (depending
on site problems).
3.4.3 Define the Evaluation Criteria and Reliability Requirements for
the Results
Bioassay results are expressed as the percentage of test organisms
affected at various dilutions of soil, water, or sediments sampled at the
site. An EC50 or LC50 is often calculated from a series of such dilutions.
This parameter represents the concentration of field-collected material at
which 50% of the organisms are affected. Either the EC50 (percent affected;
e.g., percent inhibition of lettuce roots) or, alternatively, LC50 (percent
mortality) resulting from testing the dilutions of the site samples may be
used as a measure of toxicity of a substance. Depending on the study
objectives, either measure of toxicity may be used for: 1) comparison to a
13
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critical value which, if exceeded, indicates the need for further action;
2) comparison to the toxicity of samples from a "clean" site; or 3) mapping
the contaminant distribution and/or bounding the areas of the site at which
the toxicity of the samples is greater than some critical value, indicating
that further action may be necessary. At this point in the study planning,
the project manager must define the critical value, or determine the
magnitude of the difference in toxicity between "clean" and possibly
contaminated sites, that will indicate further analysis or remedial action is
necessary. Porcella (1983) states that an EC50 caused by a 1:5 dilution
(i.e., 20%) of the bioassayed sample would generally be cause for concern.
However, his choice of this value was arbitrary and may be too high in some
situations (see Section 9.3). The critical value should be evaluated
'separately for each problem, based on site circumstances. Thomas et al.
(.1984a) found results from tests of undiluted material useful for preparing
site "toxicity maps."
The project manager must also decide on the reliability requirements for
the results. For this purpose, consultation with the project statistician is
strongly recommended. The statistician can aid in defining the required
precision and accuracy. A major factor in determining needed precision and
accuracy is the use that will be made of the study results. At one extreme,
the reliability (precision) and accuracy of data must be well known if it is
to be used in litigation. In contrast, the reliability requirements for
results of preliminary or pilot studies are not as stringent. The field and
laboratory work must be designed to meet the necessary requirements.
The results from bioassays contain a certain amount of "inherent"
variability, or "noise." The "noise" is a result of the natural variability
of similar field samples, variability in field conditions and sampling
techniques when the samples are taken, and variability in sample handling and
analysis in the laboratory. These sources of variation are discussed further
in Section 6.1. Qualified laboratories routinely calibrate their analytical
instruments, to achieve precise and accurate results. Field and laboratory
handling procedures can be standardized. Therefore, most of the "noise" is
introduced by the natural variation between field samples. Thus,
improvements in the field sampling design have the largest impact on
increasing the precision and accuracy of the results.
14
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Unless the entire site is sampled, there is some probability that the
results based on the samples collected will lead to an under- or
overestimation of the extent of the chemical contamination. To determine the
desired level of accuracy, the project manager must 1) evaluate the risk
posed by underestimating the amount or distribution of the contaminant and
thus failing to take appropriate remedial action, and on the basis of this,
decide on an acceptable possibility of missing a "hot spot"; and 2) evaluate
the cost of performing unnecessary remedial action, and on that basis,
decide on an acceptable probability of failing to detect "clean areas."
Finally, the project manager must determine the desired level of
precision for the bioassay results. Precision is a function of the "noise"
in the data and the number of samples analyzed, and is usually expressed as a
standard deviation or confidence interval around the EC50 or LC50. When the
objective of the study is to compare the site toxicity to a critical value or
samples from a "clean area," the level of precision should be such that
uncertainty about the toxicity measure does not impair the ability of the
test results to clearly establish the relationship between the site toxicity
and the critical value or toxicity of "clean" soil.
When mapping contaminant distributions, the precision of concentration
contours estimated by kriging (described in Section 5.3) should be such that
the contours are located unambiguously with respect to critical site areas
(such as the waste site boundaries or the habitats of endangered or valuable
species), or do not significantly under- or overestimate the size of areas
that may require further attention. Precision can usually be increased by
collecting and/or analyzing a larger number of samples. If there is a
preliminary estimate of expected variability in the data, a qualified
statistician can calculate the number of samples required to achieve the
desired level of precision and power for discriminating between clean and
contaminated areas. Precision may also be increased by reducing the "noise"
in the data by exerting more careful control over the field and laboratory
handling procedures.
15
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3.4.4 Determine What Is to Be Sampled in the Field
The choice of which medium or media to sample in the field (i.e., soil,
soil water, surface waters, ground water, or air) depends on the objectives
of the study. The medium might be chosen to maximize the capability of
detecting contamination on the site or because it is the medium that poses
the greatest threat to humans or other organisms. The medium or media chosen
for sampling should be either: 1) the ones to which the contaminants were
known or suspected to have been disposed, 2) the one(s) to which the
contaminant(s) are likely to migrate or adhere to as a result of their
chemical or physical properties, 3) the one(s) that are most likely to be in
contact with humans or rare, endangered, or commercially or recreationally
valuable species, or 4) the one(s) that are most likely to transport the
contaminant(s) within or off the site. Thus, the decision should be made
only after consultation with the project biologist, chemist, hydrologist
and/or meteorologist, and risk assessment expert.
3.4.5 Choose the Test Organisms for the Bioassays
Several species should be used to test each sample because of the
differences in tolerance to certain substances between the different
organisms (Thomas et al. 1984a). Porcella (1983) outlined a set of standard
procedures for conducting bioassay tests on terrestrial and aquatic samples.
He recommended using a freshwater algae (Selenastrum capricornutum), a
daphnid (Daphnia magna), and a freshwater fish (the fathead minnow,
Pimephales promelas) to assess toxicity in soil elutriates and water samples.
Soil and sediment samples are tested using soil microorganisms, common plant
seeds, and earthworms. Kenaga (1978) compared the acute toxicity test
responses of a variety of terrestrial and aquatic organisms to 75
insecticides and herbicides, and recommended that one species of fish
(including the fathead minnow), one species of aquatic arthropod (including
Daphnia magna), and laboratory rats (Rattus norvegicus) be tested to give the
range of responses representative of a variety of aquatic and terrestrial
species, including mammals.
Bioassay tests should be conducted using a suite of organisms. The
organisms listed in Porcella (1983) and Kenaga (1978) are generally easy to
16
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obtain and culture, represent important levels in the ecological food web,
and are widely used in testing, so that the test results may be comparable
to those obtained from tests using other substances and these same organisms.
In addition, these organisms are common and thus the tests are likely to be
less expensive.
Nonstandard organisms may be appropriate for bioassays 1f 1) standard
organisms have previously shown no response to the known or suspected
contaminants, 2) the response of a particular organism, not included in the
standard list, will be more specific for the suspected contaminants, or
3) the response of a specific organism, not included in the standard list, is
needed. However, tests using nonstandard organisms are likely to be more
expensive because of difficulties in obtaining, culturing, and standardizing
the new bioassay (including quality assurance procedures). In addition,
extensive preliminary testing using the nonstandard organism may be required
to establish reliability. Some of the problems and additional costs can be
identified in consultation with the field biologist and the scientist
supervising laboratory bioassay tests.
3.4.6 Define the Data Analysis Techniques
The choice of data analysis techniques depends on the study objectives
and will determine the number and location of the field samples collected for
laboratory analysis. This decision should be made in consultation with the
project statistician. Methods of data analysis applicable to chemical waste
site problems are discussed in Section 5.0.
3.4.7 Design the Field Sampling and Laboratory Studies
The approach to the bioassay study and the design of the field sampling
and laboratory analysis depend on the study objectives and reliability
requirements. These choices may also influence the data analysis technique.
Decisions regarding the study design should be made in concert with the
project statistician, the field biologist, and the scientist supervising the
laboratory work. Section 6.0 provides some guidance for developing an
overall strategy for allocating available resources to the field and
laboratory work to maximize the reliability of the results and minimize the
study costs. Alternative field sampling designs are discussed in Sections
17
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5.0 and 6.0. Procedures for conducting bioassays are in Porcella (1983), and
are not repeated here. Finney (1978) presented alternatives to the
statistical estimation of EC50s and LC50s included in Porcella (1983). These
alternative methods may be needed if a standard deviation or confidence
interval is required for the EC50/LC50.
3.4.8 Determine the Sample Collection Methods
Methods for collecting soil, groundwater, surface water, and air samples
are presented and discussed in Ford et al. (1984) and are not repeated here.
Some modification to the sampling devices and procedures described in Ford et
al. (1984) may be necessary because of particular characteristics of the
waste site. In addition, it should be noted that all sample collection
methods are in some way selective and operate with varying efficiencies under
different conditions. Therefore, the collection devices should be thoroughly
tested, before the fieldwork is begun, under conditions that approximate
those at the site.
The amount of sample collected should be at least threefold (where
possible and not cost prohibitive) that required for the laboratory analysis
to allow for repeated analysis of samples with unusual results or the loss or
damage of the samples. In addition, it may be prudent to archive a portion
of the sample to answer additional questions that may be raised after study
completion. Finally, extra samples allow additional compositing decisions to
be made long after field samples are collected and screening assays have been
interpreted (see Section 8.3).
3.4.9 Define the Operational Procedures
Several operational considerations must be taken into account when
designing and executing the field and laboratory work for a bioassay study.
Ford and Turina (1985) provide guidance for establishing operating procedures
for hazardous waste site investigations. Appropriate quality assurance and
quality control procedures need to be established for field sampling and
laboratory work before the study begins. Chain-of-custody procedures for
samples need to be established. In addition, worker safety must be ensured
both on the field site and in the laboratory.
18
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Finally, the project manager must consider the possible impacts of the
field sampling procedures to the hazardous site and surrounding areas.
Extensive field sampling may interfere with physical or biotic features and
thereby the functioning of the site, or may mobilize certain toxins.
Consultations with chemists, biologists, waste site management personnel, and
public officials in surrounding communities are recommended.
3.4.10 Review the Design
Before field sampling is started, the study design should be reviewed
again and the following questions should be addressed:
Is there sufficient information about the problem to design a cost-
effective study?
Is a preliminary study needed to obtain sufficient information?
Will the final results meet study objectives?
What constraints do the statistical, sampling, and laboratory methods
impose on the interpretation of the results?
Are there sufficient resources available for the study to obtain the
desired precision?
Depending on the answers to these questions, portions of the study may need
to be redesigned. This process should continue until a satisfactory design
is achieved.
3.4.11 Periodically Evaluate Progress in the Field and Laboratory
No matter how carefully the study was planned or how thoroughly the
methods were tested in advance, it 1s likely that adjustments will be
necessary 1n both field and laboratory procedures during the course of the
study. Some procedures may turn out to be impractical or otherwise
unsatisfactory, while additional sampling or bioassay tests may be suggested
by preliminary results. Therefore, it is important to continually monitor
and evaluate current results from each phase of the study. However, a
statistician should be consulted before changes are made in either the field
and/or laboratory procedures to be sure that the results from the entire
study can still be interpreted as planned.
19
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3.4.12 Analyze and Evaluate the Results
The quality and completeness of the information obtained from the study
should be examined. The following questions should be answered on the basis
of final study results:
Do the data meet the precision requirements established at the
beginning of the study?
Do the results satisfy the objectives of the study?
Are there indications that further experimental work is needed? In
addition, the information obtained during the study should be assessed
to determine its usefulness for design of additional studies at the same
site and at other sites.
Finally, the data from the study will need to be interpreted in the
context of risk assessment and waste management decisions. To accomplish
this, consultation with experts in the area of risk assessment is
recommended. A discussion of risk assessment is beyond the scope of this
document, but is discussed in Douglas (1985). If done correctly, the study
should provide valuable input into decisions about possible remedial
measures.
20
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4.0 BACKGROUND FOR ACUTE BIOASSAY USE AT CHEMICAL WASTE SITES
4.1 INTRODUCTION
Historically, bioassays have been used to establish chemical criteria
values for specific purposes (i.e., to determine freshwater, drinking water,
and air quality standards). These criteria, for the most part, were
developed with single pure chemical dose-response bioassays conducted in a
well-defined medium under controlled laboratory conditions. A major
criticism of these tests is that generally organism response to
environmentally derived samples that contain complex chemical mixtures cannot
be predicted from individual bioassays using the specific chemical
components. However, the problems are more severe when the toxicity
potential of a waste is estimated from Priority Pollutant content instead of
bioassay results. Reliance on laboratory-derived chemical criteria to
predict toxicity of field samples introduces the following potential problems
and concerns:
The data bases for most chemicals are not complete, so reliable
criteria are unavailable.
Most chemicals for which reliable criteria have been developed are not
commonly found at chemical waste sites.
The application of laboratory-derived criteria to complex field samples
usually results in conservative and, therefore, overly restrictive
toxicity estimates and sometimes misinterpretation because of the lack
of knowledge about cause-and-effect relationships.
The Water Quality Criteria Documents (Federal Register, 1980) cautioned
against additive use of a single chemical criterion by stating "It is
impossible in these documents to quantify the combined effects of these
pollutants and persons using criteria should be aware that site-specific
analyses of actual combinations of pollutants may be necessary to give
more precise indications of the actual environmental impacts of a
discharge."
There are no criteria for contaminated soils and sediments on which to
base decisions about environmental hazards.
21
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Even though about 19,000 uncontrolled waste sites have been
inventoried, it is unrealistic to assume that each of these sites can be
thoroughly investigated. Costs to identify specific individual priority
pollutant chemicals and associated sampling efforts become prohibitive.
Bioassays, on the other hand, can be used to screen for potential hazard
within and between waste sites. Early research established that standard
test organisms could define the toxicity potential of complex chemical
mixtures in waste site samples. The response of a living organism to a
complex chemical mixture integrates the effects of environmental variables
such as solubility, pH, antagonism, synergism, and time of exposure, all of
which affect test-organism toxicity. In effect, the bioassay produces a
direct estimate of the environmental toxicity of a sample regardless of the
causal factors. Bioassays can also be performed on composited samples in
order to reduce per sample costs.
4.2 DEVELOPMENT OF BIOASSAYS FOR WASTE SITE STUDIES
Concern about the pitfalls of chemically derived criteria led to the
development of a biological assessment protocol for evaluating the
environmental hazard potential of waste site contaminants. In October 1981 a
workshop was conducted to discuss the conceptual basis, ecological factors,
and regulatory requirements that would influence the development of a
hazardous waste biological assessment protocol. The attendees also
considered the National Contingency Plan prioritization, cleanup, field
application, and evaluation procedures. The resulting protocol (Porcella
1983) contains standardized aquatic and terrestrial bioassays that have been
used to define the toxicological properties of inorganic and organic
chemicals. The bioassays included in Porcella (1983) are an alga
(Selenastrum capricornutum), a macroinvertebrate (Daphnia magna), seed
germination/root elongation (in lettuce, Lactuca sativa), an earthworm
(Eisem'a foetida), and fathead minnow larvae (Pimephales promelas). In
addition, the Microtox (Photobacterium phosphoreum) microbial (Beckman 1982)
and Neubauer seed germination (Thomas and Cline 1985) bioassays are often
used. Ongoing research studies have been designed to: 1) ascertain the
ability of bioassays to define the areal extent of contamination at waste
22
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sites (Thomas et al. 1984b); 2) determine the predictive capability of bioassays in idc
waste site ecological impact zones (Thomas et al. 1984c); and 3) define the
ability of sensitive laboratory test organisms in measuring and/or monitoring
the effectiveness of cleanup operations (research in progress).
4.3 SENSITIVITY, APPLICATION, AND INTERPRETATION OF WASTE SITE BIOASSAY
RESULTS
4.3.1 Bioassays Using Pure Chemicals
Available research using pure chemicals suggests that the algal
(Selenastrum) and Daphnia bioassays are the most sensitive to heavy metals,
followed in order of decreasing sensitivity, by Microtox, lettuce root, and
iearthworm bioassays (Miller et al. 1985; Thomas et al. 1986). Root
elongation (lettuce root bioassay) is the most affected by the herbicide
2,4-D, followed in increasing order of tolerance by algae, Microtox, Daphnia,
and earthworms. Herbicides other than 2,4-D were not tested. Algae and
Daphnia are the only organisms sensitive to the insecticides aldrin,
dieldrin, chlordane, and heptachlor. These results indicate that algae might
be the most broadly sensitive test organism for assays using site soil or
sediment elutriates and surface-water or ground-water samples, since they
were inhibited by water solutions of all the major chemical subgroups that
have been studied. A larger group of chemicals must be tested to ensure the
generality of this finding.
Thomas et al. (1986) found that a commercial formulation of the
herbicide 2,4-D was more toxic than the pure 2,4-D acid. This differential
response shows that bioassays can identify additional (or perhaps
synergistic) toxicity in chemical mixtures. In addition, these results
indicate that a potential problem may exist if toxic effects for pure
chemicals are used to estimate the toxicity of commercial formulations
containing "other inert ingredients." Based on its 2,4-D content, the
commercial product should have had minimal effects on aquatic organisms.
However, laboratory bioassays of this commercial chemical formulation (as it
would be used in the field) indicate the material was more toxic than its
active ingredient. Decreased algal and Daphnia EC50s obtained with
commercial aldrin and endrin also suggested the presence of a toxic
23
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constituent not present or bloactive 1n the chemically pure reference
standards (Miller et al. 1985). These results demonstrate the ability of
algae and Daphnia to define differences 1n chemical toxiclty caused by
formulation differences and/or other Impurities. This supports speculation
that the "inert components" might themselves be toxic or synergistic. Based
on the foregoing studies using pure chemicals, 1t appears that a multimedia
bioassessment protocol can distinguish among a variety of relatively subtle
differences in the chemical composition of complex mixtures. Extension of
this observation to environmentally derived samples permits the use of the
bioassay procedure to identify the presence of a toxicant, regardless of the
specific chemical content of the sample.
4.3.2 Bioassays Using Waste Site Samples with Known Chemistry
The responses of selected test organisms to hazardous waste site soils,
soil elutriates, and ground water in which major contaminants have been
chemically identified are shown in Table 4.1.
An evaluation of the EC50 values In this table Indicates how each of the
waste sites can be ranked 1n order of their relative toxiclty. However,
caution should be exercised when using these values as primary decision
guidelines. For example, it is apparent that the oil slop, drilling fluid,
and wood preservative wastes (waste samples from Ada, Oklahoma; see Table
4.1) are highly toxic to all the organisms tested and, because they are toxic
at very low levels, may constitute an environmental hazard. Both the United
Chrome ground-water and 01 in soil elutriate samples are also obviously
hazardous. When potential aquatic impacts are of primary concern, the algae,
Daphnia, and Microtox tests are the most applicable bioassays because these
tests use aqueous soil elutriates or water. The earthworm bioassay (and also
the Neubauer bioassay; Thomas et al. 1984c) can be used to define the
environmental hazard of non-water-soluble contaminants. For Instance, both
the Thiokol and LaSalle earthworm soil contact results, in concert with the
other bioassays, show a potential for environmental damage but little
apparent potential for transport via water.
Differential responses to the same major chemical components in various
waste site samples are also shown in Table 4.1. For example, the response of
24
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TABLE 4.1. EC50 Response 1n Percent Soil for Earthworms and Percent Elutriate for Other Organisms Exposed to
Chemical Contaminants In Hazardous Waste (Ada Samples Only), Waste Site Soil, Soil Elutriate,
and Ground-Water Samples
Bioassay Response (EC50)
Waste Site
Major Contaminants
Algae
Daphnia
Hi crotox RE
(a)
Earthworm
Western Processing
No. 1
No. 11
No. 17
£ No. 22
Hollywood
Holder Chemical
Big John Houldt
Sapp Battery
Thtokol
Sharon Steel
Rocky Mountain
Arsenal No. 92
Eddystone Arsenal
Time Oil Well 12A
Heavy metals
Heavy metals, solvents,
phthalates
Heavy metals, phenols,
solvents, pesticides
Heavy metals, phthalates,
solvents
Pesticides
Pesticides, herbicides
(d)
PAH , other organics
Heavy metals
Diphenylamine
Heavy metals, tar, PAH
Heavy metals, insecticides,
organosulfur compounds
j
Heavy metals
Trichloroethane solvents
28
1.8
0.2
22
69
10
5.6
80
NE
29
(b)
2.2
11
NE
41
37
NE
NE
77
(c)
55
NE
24
2.1
5.4
41
NE
0.6
6.4
12.8
20
22
3.6
87
70
NE
30
25
58
85
90
18
28
NE
NE
99
3
NE
91
NE
3.6
NE
NE
NE
NE
61
NE
NE
25
70
10
NE
35
75
<5
NE
NP
(e)
-------
TABLE 4.1. (contd)
Bioassay Response (EC50)
ro
en
Waste Site
Ada (waste samples)
Oil Slop
Drilling Fluid
Wood Preservative
Olin
Nease Chemical
United Chrome Shallow
Well No. 1
Hogtown Creek
LaSalle
Number of Tests Where
EC50 Was Obtained
Total Number of Tests
% of Total Tests Where
Major Contaminants
2,4-dlmethyl phenol,
phthalates
Heavy metals, phthalates
Phenol , creosol , PCP
Pesticides
Pesticides
Chromium
Phenol, 2,4-dimethyl phenol
PCS
an
an
Algae
0.03
0.07
0.0*
0.10
99
0.03
11
NE
19
21
90
Daphnla
0.02
0.51
0.22
14
NE
0.05
24
NE
18
21
86
HI crotox
0.13
0.46
0.05
15
13
8.5
11
NE
16
21
76
RE("
4.3
2.5
0.59
4.5
NE
0.6
-<47,
NE
8
21
38
Earthworm
NP
NP
NP
10.2
NE
NP
24
5-10
11
16
69
EC50 Was Obtained
(a) RE lettuce seed root elongation test.
(b) NE no observable toxic effect at ,100% concentrations of soil, ground water, wastes, or soil elutriates.
(c) Earthworm 14-day soil test EC50 values.
(d) PAH « polynuclear aromatic hydrocarbons.
(e) NP « test not performed on this sample.
(f) -( ) * * Inhibition in 100% test sample when an EC50 value was not obtained.
-------
algae to the heavy metals in the Western Processing No. 1 sample is about
midway between those of the Sapp Battery and Eddystone Arsenal samples (other
bioassay results contributed little additional information). All of these
samples were predominantly contaminated with metals. However, differences, in
solubility, pH, ionic strength, organic content, etc. may have affected
sample toxicity to different degrees, in addition to actual differences in
metal concentrations. Based on algal toxicity, the rank order of these
metal-contaminated sites is Eddystone > Western Processing No. 1, which is >
Sapp Battery (when the statistical error of the ECBOs is ignored). The
bioassays of Western Processing No. 11 and No. 22 soils indicate that
earthworms may not be greatly affected by phthalates or the metals and
solvents present. However, as stated previously, earthworms were the only
test organism to exhibit a toxic response to diphenylamine and PCBs in the
Thiokol and LaSalle soil samples. Thus, bioassay response can be used to
define toxicity potential even when specific chemical information is not
available. The results from the pesticide-contaminated Hollywood, Olin, and
Nease chemical waste sites reinforce this observation.
The results shown in Table 4.1 indicate that a suite of test organisms
can be used to define the toxicity of complex waste mixtures so that sites
containing chemicals that are most toxic can be easily ranked for remedial
action investigations. Moreover, differences in bioassay response among
test organisms could also aid in the prioritization of additional tests to
provide the most information at the least cost. A planned sequence of
preliminary bioassay analyses at single or multiple sites could be used to
define the most probable impact areas and identify those waste sites that
require extensive chemical and biological investigation.
Algal toxicity caused by water-soluble contaminants was observed in 90%
of the waste site samples (see Table 4.1). The lettuce root elongation test
only exhibited a toxic response in 38% or 8 of 21 samples assayed, whereas
Daphnia, Microtox, and the earthworm soil contact tests showed toxicity in
86%, 70%, and 69% of the waste site samples, respectively. In addition to
detecting a large fraction of toxic samples, algal bioassays were usually
also the most sensitive (i.e., their respective ECSOs were generally lower)
Thus, this assay may be the most useful to screen sites. Based on the sar
27
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criteria, Daphm'a and Microtox could also be good candidates to screen for
environmental hazards.
4.3.3 Bioassays Using Haste Site Samples with Inferred Chemical
Constituents
A field study was conducted by Thomas et al. (1984a) at the Rocky
Mountain Arsenal near Denver, Colorado. The site had been used for the
manufacture of antipersonnel gases, herbicides, and insecticides and as an
ordnance testing area. Over the years, myriad organic and inorganic
compounds were carried through ditches to a series of interconnecting holding
basins for disposal.
Grab samples of soils and water from the arsenal were used to ascertain
whether bioassays (including the Neubauer lettuce seed germination test;
Thomas and Cline 1985) would respond to the unknown chemical mixtures
contained in these samples. The Neubauer test was added to the bioassay
series because the earthworm soil contact test was not very sensitive to pure
chemicals (see Section 4.3.1). Addition of this assay allowed a better
comparison of the toxic properties of soil elutriates and the soils
themselves. Because of the known high salt content in the water at one site
basin, preliminary wheat and lettuce seed germination bioassays were
conducted (Thomas and Cline 1985). These assays showed that copper, sodium,
nickel, or arsenic, singly or in combination, were not toxic at levels found
in the basin water.
Bioassays of soil elutriates from site 085 indicated that the algal
assay was approximately 10 times more sensitive than the Daphnia assay
(Table 4.2). No response was observed for Microtox and lettuce root
elongation, while the earthworm soil test for sample 085 had an EC50 value
>252>. Such results are typical of those for low levels of heavy metals (see
Section 4.3.1). In contrast, soil sample 092 (from a lime pit) showed a
different response pattern of increased toxic sensitivity to Daphnia,
Microtox, lettuce root elongation, and earthworms, suggesting a stronger
influence from the organic components in this sample. Both the basin waste
water (a holding basin for toxic wastes) and a sample of well water (located
1n another part of the same area) v/ere toxic to most organisms tested. Basin
28
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TABLE 4.2. EC50 Response or Percent Inhibition Caused by Chemical Contaminants in Rocky
Mountain Arsenal Soil, Soil Elutriate, Waste Water, and Ground-Water Samples
Rocky Mountain Arsenal
IS}
to
Sample Number
085
092
Basin Water
Basin Well Water
1-5
6
7
8
9
Algae**'
8.3
6.4
0.002
27
s(f)
S
NE
S
S
DaphniaV9; Modified Microtox'
86
25
0.003
21
72
94
NE
NE
NE
NElc)
3.0
0.006
NE
NE
NE
NE
NE
NE
l°; REvu; Neubauer Earthworm
NE
61
1
12
72(g)
32
19
26
>25
>5
0.5*e'
91(g) 62
100(g) 55
100(g) <25
92(9) 58
13(g) NE
(a) EC50, % elutriate or % water.
(b) RE lettuce root elongation test, EC50 % elutriate or % water.
(c) NE = no biologically significant toxldty was observed.
(d) Earthworm 14-day soil test EC50 values, % soil.
(e) LC50 value In % basin F water.
(f) S growth stimulation.
(g) 72/100 « 72% inhibition of lettuce root elongation 1n 100% soil elutriate or seed germination In 100% soil
(Neubauer test). Neubauer re'sults are the mean of three replicates of 40 seeds each.
-------
water was toxic to all organisms tested, with EC50 values of <1.0%, while
basin well water was much less toxic to algae, Daphnia, and lettuce root
elongation, and was not toxic at all in the Microtox assay. The assay
results for soil elutriates from samples 1 to 9 are unique in that, except
for site 7, these soil elutriates stimulated algal growth rather than
depressing it. This lack of an algal toxic response was partially
corroborated by low toxicity obtained with Daphnia assays, which showed an
EC50 of 72% elutriate for samples 1 through 5 (subsamples of a single large
sample collected adjacent to 085, but 1 year later) and site 6 (EC50 of 94%).
In contrast, lettuce root elongation results based on 100% soil elutriates
(samples 1 through 8) were highly toxic as assessed by percent inhibition
(footnote g, Table 4.2).
The earthworm and modified Neubauer soil contact bioassays for the same
samples confirm some of the lettuce root elongation results and, unlike the
other soil elutriate tests, show the presence of toxicity at sites 1-8 and
low toxicity at site 9. Earthworm ECBOs ranged from <25% to 62% soil for the
same samples. The sample from site 7 was the most toxic (Table 4.2).
A comparison of the soil elutriate and earthworm and lettuce seed soil
contact bioassay results based on the Rocky Mountain Arsenal samples suggests
the following: 1) These soils contain very low levels of water-soluble heavy
metal and pesticide contaminants; 2) if the heavy metals are soluble, they
bind to organic compounds so that they are not available and thus are not
toxic to algae, Daphnia, and Microtox; and 3) the water- soluble toxic
components in these soils are leached as a function of time or are strongly
adsorbed by the clay and organic fractions, but are available to earthworms
and lettuce seeds.
Thus, in bioassays of the Rocky Mountain Arsenal samples of which the
contaminant history was unknown, soil contact bioassays showed that
earthworms and lettuce seeds were the most negatively affected, while
elutriates of the same soil stimulated algal growth. The lettuce root
elongation test, using undiluted soil elutriate, also indicated the presence
of toxic components, but was less sensitive than the lettuce seed solid-phase
soil contact test. These results suggest (in the absence of chemical
30
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analyses) the presence of low levels of water-soluble metals and possibly
pesticides.
31
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5.0 STATISTICAL TECHNIQUES FOR WASTE SITE BIOASSAY STUDIES
This section contains descriptions and discussions of statistical
techniques useful for estimating the location, distribution, and toxicity of
known or suspected contaminants on hazardous chemical waste sites. These
techniques address the "Where?" and "Toxicity?" questions for which bioassays
are well suited (Table 3.1). Section 5.1 contains a short discussion of the
role of prior information in the design and analysis of bioassay studies,
followed in Section 5.2 by a description of techniques for detecting
contamination and methods for comparing site toxicity to a regulatory
"critical" value or, alternatively, to that of a "clean" area. Finally, a
discussion of techniques for mapping the distribution of toxic materials, on
or off the waste site, is presented in Section 5.3.
5.1 THE ROLE OF PRIOR WASTE SITE INFORMATION IN BIOASSAY STUDY DESIGN
Any prior information on the source or location of contamination can
greatly improve the efficiency of the field sampling strategy and thereby
increase the precision and accuracy of the results. When no reliable
estimates can be made of the waste source(s) or boundaries, generalized field
sampling schemes must be employed. Although a well-designed general sampling
plan should be capable of detecting and estimating the distribution of toxic
substances, it may not yield sufficiently precise estimates for some
important sites. Also, sample analyses may show that a large sampling effort
was unnecessarily directed to areas of little or no interest. With some
previous knowledge about the expected contaminant distribution, the
allocation of sampling effort can be optimized to focus on those areas in
which precise estimates of toxicity are needed.
5.2 ANALYSIS TECHNIQUES FOR DETECTING CONTAMINATION AND ESTIMATING ACUTE
TOXICITY
Bioassay studies designed to answer "Where?" and "Toxicity?" questions
usually involve determining if toxicity in specific hazardous waste site
areas exceeds prescribed limits. For this purpose, samples may be collected
according to the most appropriate sampling design discussed In Section 6.3,
32
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based on the study objectives and the amount of prior information available
about the source or distribution of the contaminant. Confidence interval
techniques and compositing are two different approaches that may be used to
statistically evaluate the results.
In order to use confidence interval techniques, individual samples must
be analyzed and an estimate of the mean toxicity, usually an EC50 or LC50 or
the average mortality resulting from diluted material, must be calculated for
the whole site or separately for subareas within the site. The variance and
95% confidence intervals are then calculated for these means. Methods for
calculating the variance and confidence intervals around several mortality
estimates are described in most standard statistical texts (e.g., Snedecor
and Cochran 1967). The estimated mean and variance may be used to compare
the site toxicity either to a regulatory "critical" value or to the estimated
mean toxicity of samples taken from a "clean" site.
If, when comparing the mean site or subsite toxicity to a critical
value, the 95% confidence interval around this mean toxicity does not include
the critical value, the site or subsite toxicity is clearly distinguished
from the critical value and further analyses may be unnecessary. On the
other hand, when the critical value is contained within the confidence
interval, more samples are needed to obtain a narrower interval or the site
and critical values cannot be differentiated. This is equivalent to
performing a statistical t-test to decide whether the site or subsite mean 1s
significantly different compared to the critical value. For example, a mean
EC50 of 40.99, with 95% confidence intervals extending from 19.65 to 63.40,
is clearly distinguished from a critical value of 80. However, an EC50 of
59.99, with 95% confidence intervals extending from 38.95 to 82.40, cannot be
declared significantly lower than a critical value of 80. Thus, further
sampling and or analysis will be necessary to more clearly define the
relationship between the measured and critical values.
The variance estimates are required for the statistical comparison of
the mean sample toxicity between "clean" sites and those sites suspected of
contamination. For example, it may be appropriate to take remedial action if
an EC50 from a suspected contaminated site is significantly lower than an
EC50 from a clean site. Moreover, the same data can be used to estimate the
33
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power of the statistical test to discriminate between clean and contaminated
sites. Additional sample analyses might be required to increase the power of
the test if the results do not meet the established reliability criteria for
the study.
Analysis of variance techniques can be used to statistically compare the
means from two or more sites. Formulas for calculating the power of the
F-tests embedded in the analysis of variance can be found in most standard
statistical texts (e.g., Snedecor and Cochran 1967). In some cases, the
statistical significance of differences in toxicity between samples from
"clean" sites and possibly contaminated sites is not as important as
obtaining estimates of the magnitude of the difference between the sites.
For example, it may be appropriate to take remedial action if an EC50 from
the suspected contaminated site is one-quarter or one-half that from a
control site or some other such site-specific relationship. The variances
for the two calculated mean values may be used to construct confidence
intervals around the differences between the two means as a way to gauge the
estimated precision of the toxicity estimates.
Compositing involves combining portions of several field samples into a
single or composite sample. Samples from the entire site may be composited,
or separate composites can be made from samples from different areas of the
site. The component samples of any composite whose toxicity exceeds the
critical value (generally relaxed to account for dilution of toxicity of
single-composite components) may be analyzed separately, while the
constituents of the composites not exceeding the critical value will be
ignored in further analyses. In this way, whole areas without appreciable
toxicity can be identified with a minimum number of analyses.
In order to use composited samples, several assumptions are needed.
Among these are 1) complete mixing of components occurs; 2) there 1s a linear
bioassay response to increasing waste concentration; and 3) no chemical or
physical interaction occurs during the mixing of the composite components.
Adequacy of mixing procedures can be evaluated by using replicate bioassays
on the same composite, while linearity can be tested using several components
that contain known, but graded, amounts of toxicants. Unfortunately, known
34
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compounds may not be the ones of greatest interest because the chemical
composition of waste site samples is often unknown. However, for those
samples with a few known contaminants (based on information obtained as
suggested in Section 3.4.1), the likelihood of interactions can be estimated
using experimental results from the aquatic toxicology literature.
Compositing dilutes sample contaminates, and this can cause failure to detect
one or more toxic samples mixed with "clean" samples. The following
"rule-of-thumb" for determining the maximum number of samples that should be
combined into a composite is adapted from that proposed by Ska! ski and Thomas
(1984) for chemical analyses. The maximum number of samples that should be
grouped into a single composite (n) is
n<
MDL
where MDL is the Minimum Detection Limit for the bioassay tests, usually
estimated from the effect rate measure among control samples, and MAL is the
Maximum Acceptable Limit for the contaminant, or the percentage of bioassay
effect that indicates further or remedial action may be necessary.
Ordinarily, composite samples are subsampled for laboratory analysis.
The toxicity value obtained from the composite subsamples is a good estimate
of the mean toxicity of the component samples, so long as the composite
sample was thoroughly mixed before the subsamples were removed. However, the
analysis of the composite sample supplies no information about the
variability among component samples, some of which may have extreme
concentration values. Therefore, variances and confidence intervals cannot
be calculated for the average toxicity value estimated from results based on
a single composite sample. The individual samples must be analyzed if these
variance estimates are needed.
In normal analytical chemistry work, detection limits present problems,
especially for cancer-causing chemicals or materials that must be completely
removed from the environment (e.g., no lower limit). Thus, statistical
questions can and do arise about very low environmental concentrations (e.g.,
1.05 ppm when the detection limit for the instrument or procedure 1s 1 ppm).
Frequently, a particular chemical procedure calls for a control blank (all
35
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chemicals and procedures used as if an actual sample were present) to be
subtracted from actual sample results. The result of the subtraction can be
a negative number [2 ppm (sample) - 2.5 ppm (control) » -0.5 ppm] or below
the detection limit [below 1 ppm (sample) - 1.5 ppm (control) » -0.5 ppm or
more] in an uninterpretable way. Gilbert and Kinnison (1981) addressed these
problems for low levels of radioactivity.
At this point in the development of bioassays for assessing hazardous
chemical waste sites, detection limits per se have not been a problem for two
reasons. First, the criteria for toxicity as developed by Porcella (1983)
are based on LC50 or EC50 values and are not point estimates from a single
sample. Second, even if point estimates of mortality (or reduction compared
to control values) were used for a 100% sample, mortalities very near 10%
would be unlikely to elicit any toxicological interest (see Section 8.1.4).
Clearly, the lack of toxicological interest is because observations near 10%
mortality (or reduction) are close to the likely upper limit for samples
containing no toxic chemicals. Even though the below-detection-limit problem
does not directly affect bioassays as currently conducted, the distribution
of control-sample results markedly influences compositing schemes (see
Sections 8.2 and 8.3), and has some implications for decision rules for
action, such as Porcella (1983) proposed. Both these topics are addressed in
Section 9.0.
5.3 TECHNIQUES FOR ESTIMATING SPATIAL DISTRIBUTION
One way to express the spatial distribution of a contaminant at a
hazardous chemical waste site is to construct a contour map of toxicant
concentrations. Such maps can be used to estimate the boundaries of zones
requiring further attention or remedial action, or to predict toxicity at
specific site areas. In either case, a set of samples is collected from
various locations on the waste site and analyzed. Toxicity at all other
locations is then predicted by interpolation between toxicity values obtained
from these sample sites. Several data interpolation methods may be employed
to accomplish this, including trend surfaces, spatial splines, and kriging.
Kriging is a potentially powerful and cost-saving method for estimating
the spatial distribution of a toxicant. It is a weighted moving-average
36
-------
technique (i.e., predicted values are weighted averages of the data in
surrounding locations) in which the derivation of the weights takes into
consideration the distance between the predicted point and the sampling
location, the variance structure of the data, and any systematic trend or
drift in the observations. Detailed explanations of kriging can be found in
Clark (1979), Journal and Huijbregts (1978), and Mason (1983). Kriging has
one important advantage over other interpolation techniques for the purpose
of defining the areal distribution of contaminants for remedial action
decisions: the procedure produces variance estimates for the predicted
toxicity values. This variance estimate may be used to 1) evaluate the
precision of the toxicity estimates between sampling locations for
comparison to the study reliability requirements, and 2) indicate areas in
which further sampling and/or sample analyses will Improve the toxicity
estimate as well as areas in which additional sampling or analytical effort
is unnecessary, thereby minimizing sampling and analytic costs. Further,
the benefit of additional or more intensive sampling, in terms of the
reduction of the kriging variance or error about interpolated values and
resulting increase in the contour map precision, can be predicted from the
analysis of the original small data set.
The kriging technique is most efficient when field samples are collected
according to a systematic sampling design. The results of kriging analysis
are less reliable with small data sets or when sampling locations are very
unevenly spaced. Examples illustrating the use of kriging to analyze
bioassay data may be found in Section 8.0. Other interpolation and
contouring techniques do not yield a variance estimate. Trend surface
analysis involves the use of toxicity values at sampling points to define a
(typically) polynomial "toxicity surface." Toxicity at any point on the
site can then be predicted from the mathematical equation describing the
surface. Splining is a technique for fitting curves smoothly through the
data points and is widely used for curve smoothing in computer software
packages. At this time, there are no compelling reasons to choose either
trend surfaces or spatial splines to estimate the areal distribution of a
contaminant. For purposes of remedial action decisions, the lack of
variance estimates for predicted values make these techniques second choices
to kriging.
37
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6.0 AN OVERVIEW OF WASTE SITE BIOASSAY STUDY DESIGN
This portion of the document describes the factors to consider when
allocating effort to field and laboratory work so that the most cost-
effective bioassay study design is achieved. Section 6.1 describes possible
sources of variation in bioassay results that may decrease the reliability of
data-based conclusions, and discusses the impact of variability on the study
design. This is followed in Section 6.2 with a discussion of the use of
multistage designs that may maximize the information gained from the study
while minimizing the study costs. Finally, Section 6.3 contains descriptions
of the different overall field sampling strategies useful for bioassay
studies and provides guidance for choosing among them.
6.1 COMPONENTS OF VARIABILITY AND THEIR IMPACTS ON STUDY DESIGN
The results from bioassay studies contain a certain amount of
variability, or "noise." One of the objectives in study design is to
quantify and minimize this "noise" so the results will meet the reliability
requirements established for the study (Section 3.4.3). The variability in
the data comes from several sources in both the field and laboratory: 1) the
natural variability among similar field samples, 2) variability in field
conditions when the samples are taken, 3) variability in the sampling
techniques, 4) variability in sample handling in the laboratory, and
5) variability in the bioassays (e.g., variability among test organisms, and
in the test conditions). Variability from sources 2 through 5 can often be
minimized by careful field and laboratory procedures. The natural variance
between samples is beyond investigator control, but some fraction may be
anticipated and accounted for in the field sampling design.
Contaminant concentrations in the field usually vary naturally both
spatially and temporally. Spatial variation tends to be much higher in soils
and sediments because mixing is very slow compared to the much faster mixing
in air and water. Conversely, temporal variation in air and water may be
much higher than in soils and sediments because of the more rapid transport
of material in those media. Temporal variation in soils and sediments is not
negligible, however. Concentrations of contaminants in soils and sediments
38
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may change seasonally or after rains or other climatic events. Finally, the
variation in contaminant concentrations may be a combination of both spatial
and temporal components; that is, the relative concentrations in two
different locations may not remain constant over time.
In general, the greater the natural spatial and temporal variation in
similar field samples, the greater the sampling effort required to
characterize the site. However, both spatial and temporal variation can be
anticipated and incorporated into the design in order to increase the
efficiency of the field sampling strategy. With respect to spatial
variation, stratified sampling designs may be used to obtain separate
toxicity estimates from areas on the site that are likely to differ in
contaminant concentrations (e.g., near the source of the contamination versus
farther from it, or different media in which contaminant concentrations are
likely to vary because of the chemical or physical properties of the waste).
Stratified sampling designs are discussed more thoroughly in Section 6.3.
The sampling effort may be allocated unequally over the site with more
emphasis on areas where the reliability of toxicity estimates is more
important (e.g., where contaminant concentrations are likely to approach a
critical value, or at the site boundaries).
Sampling strategies that account for temporal variation must contain a
series of samples obtained at several distinct times. In the usual field
study, unless rates of contaminant transport are being measured, sampling
usually occurs only at a single time. Therefore, the timing of sampling
should be carefully considered in the study design. Depending on the
objectives of the study, samples might be taken at times when 1) human,
animal, or plant exposure to the waste is likely to be greatest (e.g., during
the growing season or during periodic events when humans use the site), or 2)
transport of hazardous material is likely to occur (e.g., when ephemeral
streams are flowing on the site).
Designs that account for both spatial and temporal variation are only
now being addressed by the statistical research community. Additional
comments on progress in this area may be found in Section 9.0.
39
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Sample collection, handling, and analysis should be standardized to
reduce variation. Sample collection techniques should be the same for all
samples that will be compared among one another. All samples should
represent the same weight or volume of the medium being sampled. Field
technicians should receive the same instructions on what should and should
not be included in the sample and actions to be taken when sampling problems
are encountered. For example, a decision must be made whether to include
surface vegetation and leaf litter in soil samples, and additional
instruction is needed to deal with events that prevent a complete sample from
being taken (e.g., encountering a rock). Sampling procedures should be kept
as simple as possible because complicated protocols are difficult to repeat
consistently in the field. Even with a uniform sampling method, results can
be influenced by individual technicians; therefore, 1f the study 1s
sufficiently small, all of the samples should be collected by one person. If
more than one technician is needed to collect samples, personnel should be
assigned so that one person does not collect all of the samples from a
"clean" site while another collects all the samples from contaminated areas
(i.e., confounding "clean" and "contaminated" with differences In sampling
techniques used by different technicians).
The performance of sampling devices may vary dramatically depending on
the field conditions under which the samples are taken. For example, the
quality and reliability of soil samples taken with coring devices may be
very different at different levels of soil moisture. All samples should be
collected on the same day or on consecutive days when field conditions are
relatively uniform. If field conditions change during sampling, resampling
of some areas may be useful for calibrating results based on samples taken
under the different circumstances.
Samples should be transported and stored as uniformly as possible. A
random arrangement of samples in storage containers is often advisable for
more volatile contaminants. The random arrangement prevents confounding the
differences between samples with systematic variations in storage
conditions.
In the laboratory, whole samples are usually mixed and subsampled for
analysis. Incomplete mixing of the sample will result in a larger variance
40
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for subsamples. When soils and sediments are being analyzed, preliminary
tests should be conducted to determine whether the sample mixing process is
sufficiently thorough.
Variation among the test organisms will also add to the "noise" in the
results. The response to chemicals is often dependent on the taxonomic
position, age, size, physiological condition, and genetic strain of an
organism. If the objective of the bioassay study Involves the comparison of
toxicity in different areas or sites, then uniformity among test organisms
is desired, and these factors should be controlled as much as possible.
However, if the objective of the test 1s to characterize the response of a
i particular organism to the suspected toxic material, then test organisms of
varying ages, sizes, etc. may be desirable since they more closely represent
the population of that organism as a whole. Individual organisms should be
assigned randomly to test containers and kept under the same conditions
during the test. Growth chambers, in which temperature, light, and moisture
are controlled, are often used for this purpose. Test containers should be
randomly arranged 1n the growth chamber so the sample differences are not
confounded with systematic differences in various areas of the growth
chamber.
6.2 ONE-STAGE VERSUS MULTISTAGE STUDY DESIGNS
In a one-stage study, field samples are collected according to some
sampling strategy and the resulting samples are subjected to bioassays at the
same time. Using this design, all of the field sampling and laboratory
analysis would be completed prior to finding that either the number of
samples collected and analyzed far exceeded that needed to satisfy
reliability requirements, or that the number of samples collected was
insufficient to satisfy those reliability requirements and the results
consequently are not useful for remedial action decisions without further
work. Either mistake can be costly In terms of both time and resources.
In a multistage study, a smaller amount of work is performed at each
stage and the information gained Is used to optimize the work performed at
subsequent stages. The steps in a multistage design might be as follows:
41
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1- Preliminary investigation. The purpose of a preliminary investigation
is to test the field sampling methods and obtain an estimate of the
expected variability in the bioassay test data. Analysis of the
preliminary samples also provides the opportunity to discover and
correct difficulties in laboratory techniques.
2. Collection of field samples. The variability estimates based on the
preliminary study can be used to design a field sampling strategy with
the appropriate number of samples for the required precision and
accuracy. All or a portion of the needed samples might be collected at
this stage.
3. Initial laboratory analyses. A subset of the field samples or the
composited samples (see Section 5.2) may be analyzed to obtain initial
estimates of the location, distribution, or toxicity of known or
suspected hazardous materials, depending on the study objectives. If
the initial estimates satisfy the reliability requirements for the
results, then the study may be terminated. If a shorter confidence
interval is needed for estimates at a particular location on the site,
or the results from a composite sample indicate that the toxicity of
one or more of the component samples may exceed the critical value,
then sample analysis should continue.
4. Subsequent laboratory analyses. Either another subset of samples from
areas shown to be of interest in the initial analyses or the components
of a composite sample exceeding the level of concern may be analyzed.
The initial estimates of location, distribution, or toxicity may be
revised and tested against the established reliability requirements.
Analyses will continue until sufficiently precise and accurate results
are achieved.
The allocation of effort to the various design stages depends on the
scale, cost, and reliability requirements of the study. For small, less
expensive projects, or for projects in which the reliability requirements are
lower, an extensive preliminary investigation or use of small sample subsets
may not be warranted. For large or expensive studies, extra effort in the
preliminary stages may result in considerable savings in cost and effort
during subsequent sampling and analyses.
42
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Either sample collection, analysis, or both may be done in phases during
a multistage study. When sampling costs are small compared to analytical
costs, all of the samples that could possibly be needed for all study stages
should be collected at one time. Collecting all samples at a single time
minimizes the variation caused by. sampling techniques and eliminates the need
to account for temporal variation (i.e., between sampling periods). However,
samples that are collected in phases are appropriate when the cost of
collecting samples is very high (e.g., when wells must be drilled).
Multistage bioassay study designs can lead to appreciable cost
reductions for large, expensive projects when 1) little is known about the
location, distribution, or toxicity prior to the study; 2) fairly precise
and accurate information is needed; and 3) sufficient time exists to evaluate
the results from each stage before proceeding to the next. Single-stage
studies may be more appropriate if 1) the approximate distribution and field
variability of the contaminants are known, 2) the field conditions allow
proven sampling techniques to be used, 3) the reliability requirements for
the data are low, or 4) there is an emergency response.
6.3 SAMPLING STRATEGIES FOR COLLECTING FIELD SAMPLES
The three basic design strategies for collecting field samples are
simple or stratified random, systematic, and judgment sampling. These
strategies are described in Ford and Turina (1985) and are discussed here
only as they apply to bioassay studies at hazardous chemical waste sites.
All remedial action decisions for the hazardous waste site will be based
on the results obtained from samples collected on the site. Therefore, it is
important that the samples obtained accurately represent the conditions on
the site. The traditional approach to collecting a representative sample is
to randomly select the sampling locations over the entire site with the aid
of a random number table or similar device. This procedure is called simple
random sampling and is an appropriate strategy when no prior information is
available on the likely location or distribution of the contaminant.
43
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With stratified random sampling, sampling sites are randomly chosen
within several defined site areas or strata. Appropriate strata for a
hazardous chemical waste site might be any division of areas in which it is
anticipated that toxicity will differ; for example, areas at Increasing
distance from the known or suspected source of chemical contamination.
Stratified random sampling is often a useful technique even if there is
insufficient information available to identify distinct strata a priori, and
is likely to produce a more widespread distribution of sampling locations on
the site than will a simple random sampling strategy. The construction of
arbitrary strata can allow the variability in toxicity between different
areas of the site to be estimated and may help identify actual strata. For
these reasons, stratified random sampling is a particularly useful approach
for preliminary studies.
Systematic sampling involves the collection of samples at regular
intervals over the site. This method is sometimes preferred to random
sampling strategies because it ensures even coverage of the site. However,
as Eberhardt and Thomas (1986) warn, a systematically distributed contaminant
may not be detected when using this strategy. Systematic sampling (often 1n
grids) is often the preferred method to provide input data for kriging and
other mapping techniques.
Judgment sampling relies on the sampler's judgment of what constitutes
a representative site sample. The purpose of these samples might be to
assess the presence or absence of contaminants in obvious places (e.g., a
streambed if transport is a concern), or for use in special studies of a
preliminary nature (e.g., are toxic chemicals nearer the spill source?).
However, judgment sampling is biased and such results should not be used in a
statistical analysis. In addition, judgment samples can be collected along
with samples from the designed study in the event that unusual or interesting
circumstances arise or are discovered during sampling. A statistical
evaluation of the data from the judgment samples can be used to suggest
additional sampling or to make statements without accompanying error
statements or probablistic assertions. In cases where prior Information
about a possible spill location becomes available during sampling, that
information should be used to advantage in the survey design. In fact, design
44
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modifications can be made onsite (e.g., an extra grid, transect, or stratum).
Locating field sampling sites in order to implement any of the sampling
designs discussed is not a trivial matter. At least one-third of the field
effort should be devoted to locating and marking the sampling sites. Each
sample location should be accurately recorded to aid in the Interpretation
of the bioassay results, to accurately define areas in which remedial action
may be necessary, and to permit return to any sampling site to collect
necessary additional material.
45
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7.0 METHODS OF SAMPLING MEDIA FOR WASTE SITE BIOASSAYS
Bioassays may be performed on soil, sediment, ground water, or surface
water. These different media present different problems in field sampling
and in the performance of laboratory tests that need to be taken into
consideration in the planning and execution of bioassay studies. The special
considerations for sampling and analyzing samples from each medium are
discussed in Sections 7.1 through 7.4. Sampling methods have been presented
in several other documents, and that information will not be repeated here.
However, references to helpful documents are included in each section.
7.1 SOIL
Soil is usually collected with grab or core samplers. Discussions of
sampling techniques and other considerations in soil sampling may be found in
Mason (1983), Barth and Mason (1984), DeVera et al. (1980), Ford et al.
(1984), and Sisk (1981).
Mixing in soils occurs very slowly so that the concentrations of toxic
chemicals will vary substantially from location to location. In fact, as
much as half the variation between similar soil samples often occurs within a
distance of 1 m (Mausbach et al. 1980). This variation must be taken into
account in the field sampling design, and a larger number of samples may need
to be collected to adequately characterize the soils on the waste site. The
extreme heterogeneity of soils also requires that extra effort be made to
ensure that soil samples are thoroughly mixed before being subsampled for
compositing (see Section 5.2) or other purposes. Tests should be conducted
in the laboratory to ensure that the mixing procedure is adequate.
During some bioassay tests, organisms are exposed to the whole soil
sample (e.g., the earthworm bioassay). More commonly, however, the soils are
eluted and organisms are exposed to the extracts. Soil samples containing
sand or silt are more readily extracted than those containing clay. Special
techniques and equipment may be needed to elute clay samples (Miller et al.
1984).
46
-------
Soil samples are frequently collected with coring devices to obtain a
depth profile of the chemical waste. Because contamination of the lower
layers may occur during the insertion of the coring tube into the soil,
outer edges of the core should be removed, if possible, before the sample is
analyzed. With any type of sampling device, a decision must be made whether
to include, in the samples, surface vegetation, plant roots, rocks, small
organisms, or other material that might be collected along with the soil.
Sampling equipment should always be cleaned between samples to avoid
cross-contamination. Samples should be collected and stored in containers
made of inert materials that will neither absorb materials from the sediments
nor add toxicants. In addition, soil samples should be stored uniformly
under conditions that will prevent or retard chemical degradation and
microbial metabolism.
7.2 SEDIMENT
Sediment is usually collected with devices similar to those for
collecting soils. References that discuss sediment sampling techniques and
problems include DeVera et al. (1980), Ford et al. (1984), American Public
Health Association (1985), and Sisk (1981).
The major problems encountered when sampling sediment are similar to
those for soil sampling. However, sediment samples usually have a higher
moisture content compared to soil samples and therefore less integrity.
Clean core samples are more difficult to obtain in sediments. Depending on
the depth and size of the overlying water body, sediment sampling may
present additional problems with sample site location and the use of
sampling equipment.
7.3 GROUND WATER
Ground-water samples are usually pumped from wells or collected from
seeps. Discussions of ground-water sampling techniques and concerns may be
found in Ford et al. (1984), U.S. Environmental Protection Agency (1977),
Sisk (1981), Gibb et al. (1980), and Dunlap et al. (1977).
47
-------
Representative groundwater samples for bioassays are particularly
difficult to obtain. Changes in pressure, oxidizing/reducing conditions, and
other factors may alter the chemical composition of samples as they are
brought to the surface. Certain toxic compounds in seeps may precipitate
from ground water when the chemicals encounter the strong oxidizing
environments of receiving streams.
Samples pumped from wells may be contaminated with surface soils or
drilling fluids inadvertently added to the ground water during the boring of
the wells. Well casings and pumps may absorb or add toxic materials to the
samples. Gibb et al. (1980) discovered that pumping depth, type of pump
used, and sample filter sizes had an effect on the measured chemical
composition of ground-water samples, the magnitude of which depended on the
yield, depth, diameter and water level of the well, the pumping rate, and
the general character of the water being pumped, as well as the specific
toxicants. Because of these problems, sampling conditions and techniques
should be standardized when collecting ground-water samples. Well casings,
pumps, and sampling equipment should be made of inert materials. The type of
pump used, the water level of the well, and other measurements should be made
at the time of sampling and recorded on data sheets. Since ground water
contains many trace constituents, extra care should be taken to avoid
contamination of the samples, and storage should be under uniform conditions
(4°C) to prevent or slow chemical conversions in the storage containers.
Several characteristics of water, other than the concentration of toxic
substances, may affect the outcome of bioassay tests. The pH or hardness of
the water sample may render it toxic to the assay organisms. Most of these
factors are accounted for in the bioassay procedures. '
7.4 SURFACE WATER
Surface water is usually collected by dipping it into sample bottles.
Remotely operated devices may be used to collect samples at various depths.
Miller, W. E., J. C. Greene, and S. A. Peterson. 1987. Protocol for
Bioassessment of Hazardous Waste Sites. 2nd edition. CorvaTTFs
Environmental Research Laboratory, Corvallis, Oregon. Final Draft.
48
-------
Water from very shallow levels is often collected with pumps. References
that discuss surface-water sampling methods include Ford et al. (1984),
DeVera et al. (1980), Gibb et al. (1980), and American Public Health
Association (1985).
Representative surface water samples are relatively easy to collect.
Mixing occurs quickly in water so these samples do not have the extreme
heterogeneity of soil samples; although stratification and stagnant layers in
surface waters do occur, thorough sample mixing 1n the laboratory is
relatively simple to achieve.
Several characteristics of the water, other than the concentration of
toxic substances, may affect the outcome of the bioassay tests. The pH or
hardness of the water sample alone may render 1t toxic to the bioassay
organisms. Bioassay procedures include methods to account for these
factors.(a^
In.vivo reactions proceed readily in surface water samples, so samples
should be analyzed quickly and/or stored (4°C). The length of time between
collection and analysis should be noted for each sample.
* ' Miller, W. E., J. C. Greene, and S. A. Peterson. 1987. Protocol for
Bioassessment of Hazardous Waste Sites. 2nd Edition. Corval1 is
Environmental Research Laboratory, Corvallis, Oregon. Final Draft.
49
-------
8.0 CASE STUDIES: ILLUSTRATIONS OF BIOASSAY
METHODS AND DESIGN DECISIONS
The field studies in this chapter were selected to illustrate the steps
in executing a bioassay study (Section 3.4), statistical principles (Sections
5.0 and 6.0), and sampling of various media (Section 7.0). The site bioassay
studies evaluated site toxicity and defined areal extent, if toxicity was
detected. Because the sites were large and resources were limited, many
extra samples were collected and some composited, which illustrates the
principles presented in Section 5.2.
Rocky Mountain Arsenal.soil sample bioassay results (Section 8.1) were
used to prepare a map that could be used for cleanup decisions. In Section
8.2, bioassay results are compared to results of chemical analyses on
sediment samples collected from a stream on a wood treatment plant site in
Mississippi. Other objectives of the Canton study were to determine whether
standard bioassay organisms can be used to detect creosote contamination and
to map contaminant distribution. Section 8.3 includes soil, sediment, and
surface-water sampling at a waste site presumed to be clean (Friedman) and at
a second site thought to be toxic (Combe). Both waste sites are in New
Jersey.
8.1 SOIL SAMPLING AT THE ROCKY MOUNTAIN ARSENAL
The Rocky Mountain Arsenal was used principally by the U.S. Army and the
Shell Chemical Company to manufacture, test, and dispose of toxic chemicals.
2
Certain areas of the 26-square-mile (67.6-km ) arsenal have been contaminated
by various spills during waste disposal operations. The site is surrounded
by homes, farms, and businesses (e.g., Stapleton Airport, Commerce City, and
Denver). In 1974, diisopropylmethylphosphonate (DIMP) and dicyclopentadiene
(DCPD) were detected in the surface water draining from a manmade bog at the
northern boundary of the arsenal.
8.1.1 Assemble Information Relevant to the Problem
Decontamination wastes and process waste streams containing toxic
materials consisting of salts, heavy metals, and pesticides were
50
-------
deposited in defined areas on the Rocky Mountain Arsenal (Figure 8.1).
Basins A and F were the two major sites of waste material storage.
Initially, all waste materials were placed in Basin A (Figure 8.2, Section
36). Three basins (C,D,E,) in Section 26 were constructed from 1952 through
1955 to contain overflow from Basin A. In 1955, Basin F was constructed to
contain overflow from Basins C, D, and E. Beginning in 1957, all liquid
waste from the Rocky Mountain Arsenal and Shell operations was pumped into
Basin F. Basin C was also used as a temporary holding pond for Basin F.
Certain portions of the arsenal were leased to private industry for
chemical manufacturing. Shell Chemical Company, the major lessee, has leased
a considerable portion of the manufacturing facilities at the Rocky Mountain
Arsenal since 1952. Shell has made major alterations and additions to the
facilities for the manufacture of various pesticides. The Rocky Mountain
Arsenal has been used for the manufacture and disposal of waste residuals of
GB, a chemical warfare agent, TX, a biological anticrop agent, and cyanogen
chloride and phosgene. Since 1970, several major chemical demilitarization
actions have been conducted. These actions include the incineration of both
the anticrop agent TX and a mustard agent.
In May 1974, DIMP and DCPD were detected in surface water draining from
the manmade bog on the northern boundary of the arsenal. DIMP Is a
persistent compound produced in small quantities (<3%) during the manufacture
of GB. DCPD is a chemical used in the production of pesticides. Detection
of these two compounds resulted in the construction of a dike north of the
manmade bog to eliminate surface drainage from the arsenal.
In December 1974, the Colorado Department of Health detected small
concentrations of DIMP [0.57 parts per billion (ppb)] in a well near the city
of Brighton. This discovery indicated that DIMP was also present in the
ground water traveling in a northerly direction from the Rocky Mountain
Arsenal boundary. A 2-year study by the U.S. Geological Survey confirmed the
direction of ground-water flow.
Because of the foregoing manufacturing and disposal actions, various
analyses of chemicals in air, water, soil, and certain organisms have been
conducted over the years. Table 8.1 contains a partial list of some of the
chemicals identified.
51
-------
Sewage
Treatment
Plant
Administrative AreaQ
Warehouse Area
Lake Mary
Stapleton
International
Airport
FIGURE 8.1. Principal Physical Features of the Rocky Mountain Arsenal,
Commerce City, Colorado
52
-------
STAPLETON
INTERNATIONAL
AIRPORT
FIGURE 8.2. Location of the Study Site in Basin A (which includes
most of Section 36) at the Rocky Mountain Arsenal. The
areas in Section 26 labeled C, D, E, and F are or were
waste ponds.
53
-------
TABLE 8.1. Examples of Some Chemicals Found In Soils,
Air, Water, Animals, and Plants at the Rocky
Mountain Arsenal
Aldrin
Arsenic compounds
Benzene
Chlordane
Chloroform
Dieldrin
Endrin
Lewisite
Lewisite oxide
Mercury salts
Mustard gas (HD)
Thiodiglycol
Methylphosphonic acid
Isopropyl methylphosphonate
Diisopropyl methylphosphonate
Dicyclopentadiene
p-Chlorophenyl methyl sulfone
Hexachloronorboradiene
Tetrachloroethylene
1,4,-Thioxane
Methylene chloride
Toluene
Xylene
54
-------
8.1.2 Prepare a Statement of Study Objectives
Objective 1 was to assess the toxicity of a trench site in Basin A. If
one or more bioassays identified toxic components, a field study would be .
designed to supply data needed for kriging to devise a contour map useful for
cleanup decisions (objective 2). Finally, an assessment of contaminant
mobility was needed (objective 3). To meet these objectives, a toxic site at
the Rocky Mountain Arsenal had to be located and the most sensitive bioassay
selected.
8.1.3 Design the Field Sampling and Laboratory Studies
Previous results (see Section 4.3.3; Table 4.2) indicated that an area
in Basin A was toxic (objective 1) and would be useful for addressing the
second study objective. Soils from the Basin A location caused a major
reduction in lettuce seed germination (insoluble compounds were likely
causative) and had a variable effect on other bioassays (little likelihood of
soluble compounds; objective 3). Because of these results and, in part,
because it appeared that plant growth diminished with distance from the
trench, a sampling location was established near the trench in Basin A (to
meet objective 2).
The results also suggested a possible gradient of contamination on the
west side of Section 36, extending north-south from a trench that drains
Basin A and runs to the west. This possibility of a toxicity gradient
offered good prospects for the kriging method, which generally requires a
physical mechanism of toxicant dispersal (Journal 1984) for valid error
predictions. Thus, four parallel transects were established on the west side
of Basin A, each beginning on the north bank of the trench and running south
for 90 m (Figure 8.3). A logarithmic scale was used beyond the south trench
edge because it was assumed that contamination might have been moved by some
physical means (e.g., wind or water). The transects were 15 m apart and
labeled L, M, N, and P. The first three sample points of each transect fell
within the trench and the fourth was on top of the south bank. Sample
numbers 5 through 9 were 15, 20, 30, 50, and 90 m, respectively, south of the
north trench edge (Figure 8.3). Each of the 36 sampling points was marked
with a stake.
55
-------
9S
CD
00
CO
o> r
<-» o
o
O
S13
n o
C 01
V) (/>
n 01
O9
01
Vt
(/I
n
o
o>
Distance (m) from North Bank
-------
A drilling company was hired to do most of the soil sampling. At each
sampling point a split spoon was used to take two 7.6-cm-diameter soil cores.
One core was taken from a 0- to 15-cm depth, and the second was taken from a
15- to 30-cm depth. Together, these cores weighed approximately 4 kg.
Between sampling points the split spoon and drill bit were decontaminated by
washing with methanol and rinsing with distilled water. All samples were put
in plastic bags, sealed, and labeled. The area being sampled and any
problems encountered (e.g., mud, accessibility) dictated exactly how the
cores were taken and the variations on the basic sampling scheme (see below).
In Basin A, the first two points in each transect were in the trench,
which was very wet and soft. The samples from these points were difficult to
'obtain. It was impossible to sample by depth, so a hand trowel was used to
take two surface samples (to approximately 15 cm deep) from these points.
The split spoon was used to take most of the other samples in Basin A (points
4 through 9 in transects L, M, N, and P). Sample points L, N, and P-3 were
just over the south bank of the trench and could not be reached from the
drill rig. At these points, the split spoon was hammered into the ground and
extracted by hand. Only a 0- to 15-cm sample was obtained from each of these
points; the soil from 15 to 30 cm deep was too wet to stay in the split
spoon. A surface sample of undefined depth was taken from the M-3 transect
because the entire profile was very wet.
8.1.4 Analyze and Evaluate the Results
Bioassays of Field Study Soils
Lettuce seeds were used in the bioassays of the Rocky Mountain Arsenal
field study soils, because they are more sensitive than other seeds (Thomas
et al. 1986) and previous work has shown these soils to be phytotoxic (Table
4.2). The mortalities (mean from three subsamples from each core fraction;
Figures 8.4 and 8.5) indicate that four samples in transect M and three in
transect P showed large differences as a function of depth, suggesting that
the contaminants had either migrated below 15 cm or were purposely placed
there. We found no records to support the latter argument and concluded that
the toxic material had migrated.
57
-------
DISTANCE (m) FROM NORTH DITCH BANK IN BASIN A
00
^
t9
CT
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70
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-------
4 f\
10
Z
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220
z
Z 30
CO
I
I 40
ff
i 50
5
O
c
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9^ ilOPJ | 83J **
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-
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ut
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9
TRANSECT LETTER
FIGURE 8.5. Observed Mean Lettuce Seed Mortality at Each Basin A
Plot, 15- to 30-cm Soil Fraction. Means enclosed with a
box are >75%, an enclosing circle indicates means from
30% to 75%, and numbers with no symbol are means <30%.
59
-------
The results for all other bioassays (Porcella 1983) conducted using
Basin A field study soils are shown in Table 8.2. Sample unavailability
either precluded analyses or curtailed the number of assays that could be
conducted. Daphnia or Microtox bioassay results are not included because no
mortality was observed.
Results from the algal bioassay revealed that small quantities of
elutriate from all but four samples were stimulatory. The four elutriate
samples that inhibited algae were obtained on or very near the waste trench
on transects L and N. According to the criteria outlined in Porcella (1983),
these sites would be classified as moderately toxic. Interestingly, only a
1% to 14% elutriate from transect M samples was needed to stimulate algal
growth. Because there was little effect on the algal bioassay, it appears
that the toxic components detected using lettuce seeds (see Figures 8.4 and
8.5) were not water-soluble heavy metals, herbicides, or insecticides (except
perhaps at sites L-2, L-4, and N-2), since results using pure chemicals
(Section 4.3) showed depressed algal growth in the presence of these
contaminants. This evidence suggests the presence of water-insoluble
contaminants that are not likely to migrate (objective 3).
Results of the earthworm bioassay are presented as a function of lettuce
seed mortality in Figure 8.6. Except for plot P-3, earthworms were only
affected by Basin A soil when lettuce seed mortalities were greater than 70%.
In contrast, the five soil samples that caused 20% to 70% lettuce seed
mortality resulted in no earthworm deaths. Thus, for these Basin A samples,
it appears that lettuce seeds are more sensitive to lower levels of an
insoluble toxic component than are earthworms (objective 3).
Results from the lettuce root elongation (based on elutriates) and the
lettuce seed mortality (based on intact soil) bioassays are compared in
Figure 8.7. There was a slight correspondence, based on samples from
transect L. It appears that the phytotoxic component that impairs lettuce
seed germination may not be water soluble (objective 3) or does not affect
root elongation.
Cleanup Decisions Based on Bioassays and Kriging
One way to depict the lettuce seed mortality patterns observed at each
depth (see Figures 8.4 and 8.5) is to prepare a contour map based on the
60
-------
TABLE 8.2. Intercomparison of Lettuce Seed Mortality (MM), Lettuce Root Elongation (RE),
Earthworm Mortality (tw), and Algal inhibition (b) tor Iwo factions (0-lt
and 15-30 cm depth) of Basin A Soils Obtained from the Rocky Mountain Arse
Transect
L (15-30 cm)
PLOT
1
2
3
4
5
6
7
8
9
MN(8)
100
100
86
22
12
100
36
20
15
RE(b) EW(C)
53
56 100
42
0
21*
*
9 0
18
14
33 0*
s(d,
11
39"
50~
49~
25
27
23
23
40
M (15-30 cm)
MN
100
100
78
83
16
100
50
29
65
RE EW
58 100
100
14
45* 0
10 100
21 0
33 0
20 0
S
4
9
14
1
13
7
10
N (15-30 cm)
MN
83
100
97
100
9
100
95
10
52
RE EW
20 100
31 100
44
4
» *
35 0
8
30 100
17
15 0
S
17
69~
12
26
25
30
5
21
39
-------
TABLE 8.2. (cont'd)
91
ro
Transect
P (15-30 cm) L (0-15 cm)
PLOT MN RE EW S MN RE EW
1
2
3
4
5
6
7
8
9
100
100
91
96
66
97
100
100
13
25
2
10+
23
15
8
0
1
100
100
0
50
0
100
100
100
0
15
24
4
17
14
8
19
30
100
100
86
10
14
100
11
14
12
53
56
42
21
29
40
27
11
20
11
100 39
50
10
9
25
20
35
27
(a) Percent lettuce seed mortality. Each value is the mean mortality of three replicate plates of 40 seeds grown in each soil.
(b) Percent of control lettuce root length (based on soil elutriate). Values accompanied by a superscripted plus (+) were greater than
controls.
(c) Earthworm percent mortality using 100% soil (one replicate of 10 earthworms). Values with an asterisk (*) are questionable
since soils had been previously used to prepare elutriates.
(d) EC50 values in % elutriates are denoted by a superscripted minus (-), while values with no superscript were stimulatory.
-------
100
90
80
-I 70
§g
I O
t- tr
OC LU
< n-
LU ~"
60
50
30
20
10
0 -
TRANSECT
L
O M
D N
A P
AO
OD
J
D
10
1
20
30
40
50
60
70
80
90 100
LETTUCE SEED MORTALITY
(PERCENT IN 100% SOIL)
FIGURE 8.6. Comparison of Earthworm and Lettuce Seed Mortality Using Basin A
Soils from the Rocky Mountain Arsenal
63
-------
UJ uj
li
^3
1
-» LU
2g
*- s
2 0
O C.
"X
U. f-
O 0
£5
a ->
U H-
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uj o
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+40
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-50
-60
O TRANSECT
L
D O M
DM
A P
9
A
A * A g~
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_
-------
observations (objective 2). We estimated contours for a map of lettuce seed
mortality using a relatively new statistical technique called kriging, which
was developed for use in the mining industry and is used principally in
Europe and South Africa (see Section 5.3). Kriging provides a variance
estimate that can be used to construct a confidence interval for the true
value. Results based on block kriging are presented in Figures 8.8 and 8.9.
The results clearly show the lettuce seed mortality differences at the two
depths. Estimated contamination is greater from 15 to 30 cm deep than from 0
to 15 cm deep. This contamination difference was also indicated by the
qualitative analyses of results (see Figures 8.4 and 8.5).
Contour maps are useful for making site cleanup decisions. As a
scenario, we selected 30% lettuce mortality as a criterion for cleanup of the
Basin A site (see Figures 8.8 and 8.9). In the absence of any other
guidance, the cleanup criterion was selected as two standard deviations above
the mean control mortality (i.e., 16.7 ± 14.0, n « 6). The shaded areas
below the heavy solid black lines would be targeted for cleanup. The cleanup
decision would be different for the 0- to 15-cm-deep (Figure 8.8) and the 15-
to 30-cm-deep fractions (Figure 8.9). While this difference complicates
decision making, the available data and the kriging maps show that the field
situation is complex, and cleanup decisions based solely on either soil
fraction would not result in a "clean" site. Cleanup using the 30% mortality
contour of the 15- to 30-cm samples would remove all known contamination, but
additional samples taken below this depth would be needed to assure that the
site meets the 30% mortality cleanup criterion. The need for additional
samples below 30 cm was not evident from preliminary profile data in Thomas
et al. (1984c).
It appears that bioassays of field samples and subsequent kriging
analyses (objective 2) offer a practical method to aid in cleanup decisions
based on environmental toxicity, especially when accompanied by error
estimates for the mortality isopleths. We did not present confidence limits
here because of some questions about the possibility that the observed
toxicity was caused by pollutants that were "dumped" rather than spread from
the trench by wind or water. Journal (1984) argues that the confidence
limits may be invalid unless movement is caused by physical forces. The
65
-------
90
80
<
1 70
CD
Z
60
u
5 50
5
o
c
40
i»
U
i 20
10
P
0 -10
CZ]10+.20 £
20+ -30
30+ -50
50+ -75
75+-100
oc
O
5
*
15 30
DISTANCE (m) FROM NORTHEAST CORNER
45
FIGURE 8.8. Estimated Lettuce Seed Mortality (based on kriging)
for the 0- to 15-cm Soil Fraction from the Rocky
Mountain Arsenal
66
-------
cr>
73
m
00
10
DISTANCE (m) FROM NORTH DITCH BANK IN BASIN A
S
O O «/i
c -j c+
3 -
H- rf 3
Oi 3- 01
- fl> r+
3 CD
»- O.
-i i r-
m re
n> rt- r*
3 O rt-
Oi C
«co o
ore
S !<
3 re
re
to ex
o
-*3
»o
-nr«-
O -fc
r*rt-
o re
3 o.
<-» o
3" 3
CD
O -
o to
?r- -fc
«< 3
U3
-J Ol W ls> -
a, o o o o o
% MORTALITY
-------
limits in our study averaged between ±10% and 25%, and depended on data
density, whether block or point kriging was used, and the contour of concern.
An arbitrary crosshatching has been superimposed on Figures 8.10 and
8.11 to show where the observed data indicate that mortality was actually
30%. These crosshatched "boxes" were constructed by drawing a line halfway
between grid points with observed mortalities >30% and those <30%. Thus, the
crosshatched box at the top of Figure 8.10 is a consequence of the observed
mortality at plot N-9 (Figure 8.4, 42%) and all other surrounding plots (P-9,
N-8, and M-9) exhibiting mortalities <30%. The >30% kriging mortality
contour does not include this area or a "hot spot" at L-6 (100% observed
mortality, Figure 8.4) because it is a weighted moving-average technique.
Figure 8.11 shows that the kriging results mimicked the observed data very
well. This observation is not surprising since point-kriging theory requires
a correspondence of observed and predicted values. The block-kriging
technique used here results in a less than one-to-one correspondence.
68
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90
80
< 70
z
V)
CD
~ 60
CD
50
o
X
tr
i 40
O
oc
u.
1 30
tu
u
z
20
10
CD
222
ACTUAL MORTALITY
LESS THAN 30%
ACTUAL MORTALITY
GREATER THAN 30%
KRIGING ESTIMATE >30% MORTALITY
15 30
DISTANCE (m) FROM NORTHEAST CORNER
o
D
Z
§
0.
45
FIGURE 8.10. Comparison of Lettuce Seed Mortality Predicted from
Kriging to Actual Lettuce Seed Mortality, 0- to 15-cm Soil
Fraction
69
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ACTUAL MORTALITY
LESS THAN 30%
ACTUAL MORTALITY
GREATER THAN 30%
KRIGING ESTIMATE > 30% MORTALITY
15 30
DISTANCE (m) FROM NORTHEAST CORNER
45
FIGURE 8.11.
Comparison of Lettuce Seed Mortality Predicted from
Kriging to Actual Seed Mortality, 15- to 30-cm Soil
Fraction
70
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8.2 SEDIMENT SAMPLING AT A WOOD TREATMENT SITE
The wood treatment site in Mississippi ceased operations in 1979. The
site was known to be contaminated with creosote and other wood-preserving
materials. The results discussed below are from an exploratory study to
determine whether bioassays can be used to detect creosote contamination in
stream sediments and water, and if feasible, to define the boundaries of
creosote-contaminated zones on the site. Samples were also subjected to
chemical analyses.
8.2.1 Assemble Information Relevant to the Problem
A preliminary site visit was made to obtain background information on
the history of creosote disposal, to determine the dimensions of the site,
and to define any special sampling problems. The site is bounded on the
south by Covington Avenue and on the east and north by a creek (Figure 8.12).
The creek is approximately 2 m wide at the widest point, with 2-m-high banks
on either side. The creek flows northwest (on the site), enters an open
concrete channel at the western site boundary, and then flows into a nearby
city.
Records obtained from the Mississippi Department of Natural Resources
(Bureau of Pollution Control) indicate that creosote and occasionally
pentachlorophenol were used for wood treatment on the site. From 1965 to
1979, the owner of the site permitted wastes from the treatment process to
flow overland to the creek, in violation of state pollution laws. Little
cleanup had been done when the site closed in 1979. However, it was clear
from the site visit that the site had been covered with fill material.
Creosote was still being emitted from the bank into the water along some
parts of the stream. Piles of creosote-contaminated material, as well as
pools of black sludge, were located immediately adjacent to the storage tanks
on the south side of the site (Figure 8.12).
8.2.2 Prepare a Statement of Study Objectives
The objectives of the study were to 1) determine if standard bioassays
could detect creosote contamination in water and sediments, and, if so, 2)
map the distribution of creosote contamination in the creek.
71
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Northern
Tributary
Girder
Bridge
Old
Railroad
Bridge
-N-
Creek Flow
Western
Tributary
Visibly
Contaminated
Area
Eastern
Tributary
£
o
Storage
Tanks
Covington Avenue
FIGURE 8.12. Wood Treatment Site in Mississippi
72
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8.2.3 Define the Data Evaluation Criteria and Reliability
Requirements
Since this was an exploratory study rather than one conducted 1n support
of regulatory activities, the data did not need to meet specific reliabilit.,
requirements. However, usual Quality Assurance procedures were observed ^t
the Corvallis Environmental Research Laboratory and the Pacific Northwest
Laboratory.
8.2.4 Determine What Is to Be Sampled
The primary interest in this study was creosote contamination of creek
sediments. Therefore, stream sediment samples comprised the bulk of the
samples taken. A small number of water samples were taken, as well as some
samples from the bank where creosote appeared to be entering the stream. In
addition, samples were taken from an upstream site (negative control) and
from the pile of creosote-contaminated sludge (positive control).
8.2.5 Choose Test Organisms for the Bioassay
An array of bioassays were used, including algae, Daphm'a, Microtox,
earthworm, Neubauer, and root elongation tests (Porcella 1983).
8.2.6 Define the Data Analysis Technique
A description of the spatial distribution of the contamination was one
objective of the study. Kriging was the first choice as a data analysis
technique because it permits generation of confidence intervals about
estimates of areal distribution. However, kriging generally requires a large
number of data points, and in the absence of a sufficient amount of data to
perform kriging, a simple linear interpolation could be substituted
(principally because the area of concern was a narrow stream channel).
8.2.7 Design the Field Sampling and Laboratory Studies
The field sampling scheme is diagrammed in Figure 8.13. The girder
bridge was used as the staging area and the starting point from which
distances to each sampling location were measured. One sediment sample was
collected at the point just before the stream entered the concrete channel at
the western end of the site, 660 m west of the initial sampling location at
the girder bridge. The next sample was collected at 420 m west of the
initial location, then every 40 m to the east until the visibly contaminated
73
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Northern
Tributary
Girder
Bridge
\
Old
Railroad
Bridge
Covington Avenue
IIIIMIIHIIIIIIIIIIIIIIIIIIIIfllllMIIIHIIIIIIIIIIIIIII IIIIIIIIIIH IllHlllllllllimilimiimiimi.
iilliMlliiMliillilllllllllll llliliUHIIIIIIiiliiNilll IIIIIIIIHIH Illlllllllillll
O 2 3 4 32J 7 911131517192123252728
"1 "
660
Western
Tributary
'30 56 8 10 12 14 16 182022 24 26f \
j 39 \
400 300 200 100 6 100 200 'm
Creek Flow Visibly
Contaminated
Area
Eastern «
Tributary £
Storage
Tanks
\^^^ O »33
iliMllil
Soil or Sediment Sample
Water Sample
Ai
FIGURE 8.13.
Location of Samples Collected from the Wood Treatment Site in
Mississippi (distances are in meters). Soil or sediment
samples are indicated by solid circles and water samples with
solid triangles.
74
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zone of the stream was reached. Samples were collected every 20 m in the
visibly contaminated zone, and beyond until a location 220 m east of the
starting point was reached. Three tributaries (Eastern, Western, and
Northern on Figure 8.13) drain into the creek. The last sample (220 m east
of the initial point) was collected upstream of the three tributaries. In
addition, one composite sample was collected from each of the three
tributaries. The negative control sediment sample was taken from the creek
south of Covington Avenue, upstream from the site. The positive control
sample was taken from the piles of creosote-contaminated sludge near the
storage tanks. This sludge was believed to be the same material that was
visibly contaminating the stream.
Water samples were collected at 660 m west of the initial point
(farthest downstream location), 380 m west of the initial point, 220 m east
of the initial point (farthest upstream location), and south of Covington
Avenue, where the negative control sediment sample was taken (Figure 8.13).
All samples were taken on the same day to maximize the comparability of
bioassay results and to minimize the sampling costs [samples were collected
from west to east (downstream to upstream) to minimize cross-contamination of
samples]. The laboratory analyses were completed in two phases. In phase 1,
only sediment samples from 660, 380, and 20 m west of the initial point, 120
and 220 m east of the initial point, the positive and negative controls, the
composite sample from the eastern tributary, and the water samples were
analyzed. The results of these bioassay tests were used to bracket the
contaminated zone. In phase 2, samples from 300, 140, and 60 m west of the
initial point were analyzed to aid in defining the contaminant boundaries.
8.2.8 Determine the Sample Collection Methods
The clay sediments lining the creek were very hard and were sampled
using a hand coring device. Where possible, surface sediment samples were
collected to a depth of 15 cm with hand trowels.
8.2.9 Define the Operational Procedures
Protective clothing and masks were worn by the field sampling personnel
during sample collection.
75
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8.2.10 Review the Design
The sampling design was reviewed by the project manager, a statistician,
the field sampling crew, and the biologist in charge of the laboratory
analysis before the fieldwork began.
8.2.11 Periodically Evaluate Progress
The bioassay results from phase 1 samples were evaluated and used as the
basis for selecting the samples to be analyzed in phase 2.
8.2.12 Analyze and Evaluate the Results
The results of bioassay analyses of phase 1 samples are shown in Table
8.3. The only locations where appreciable toxicity occurred were the
positive control and locations 660 and 380 m west of the initial sample
location. The water sample from the 380-m-west location was highly toxic,
while the sediments from that location were less toxic. At 660 m west,
however, the sediments were highly toxic to some organisms while the water
was not toxic.
Figure 8.14 contains toxicity maps of creek sediment elutriates from 660
m west to 220 m east of the initial point. These maps were based on bioassay
and chemical analyses from both phase 1 and phase 2 samples (distances to the
west of the initial point on Figure 8.14 are indicated by negative numbers).
The respective creosote concentrations determined for each sample by IR
(infrared spectroscopy) are expressed as a percentage of the highest creosote
value measured (9500 and 25 ppm for sediments and sediment elutriates,
respectively). We note that creosote is a complex mixture of organic
compounds. Since there were insufficient data to use the kriging technique
to devise maps, the maps were prepared using simple linear interpolation of
the results between the sampling points. Therefore, the precision of the
division locations between different zones of contaminant concentrations
cannot be estimated (as in kriging).
The top four bars on Figure 8.14 illustrate EC50s from the algal,
Daphnia, Microtox, and root elongation bioassays in which the test materials
were sediment elutriates. The results from different elutriate bioassays led
to different conclusions regarding the relative toxicities of different areas
of stream sediments. Such variable biological responses could result from
76
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TABLE 8.3 Bioassay Results from Phase 1 Samples Collected from the Wood Treatment
Plant in Mississippi
Sample Location
660 m west
380 m west
20 m west
120 m east
220 m east
Negative Control
Sample Type
Sediment
Water
Sediment
Water
Sediment
Sediment
Sediment
Water
Sediment
Water
Algae
63.7
NE(C)
73.7
6.6
NE
NE
NE
NE
NE
NE
(a)
Daphnla
73.6
NE
NE
0.2
NE
NE
NE
NE
NE
NE
EC50
Microtox
NE
29.9
9.6
NE
NE
NE
NE
NE
NE
Root . .
(a)
Elongation
100
NE
100
100
NE
NE
NE
NE
NE
NE
Neubauer
70.3
NR(d)
100
NR
NE
NE
NE
NR
NE
NR
Earthworm
27.9
NR
27.9
NR
NE
NE
NE
NR
NE
NR
Positive Control Sediment
0.6
6.9
8.5
8.1
0.9
3.9
Eastern Tributary Sediment
NE
NE
NE
NE
NE
NE
(a) Tests conducted with sediment elutriates.
(b) Tests conducted with sediment samples.
(c) NE - No effect.
(d) NR « Bioassay not required.
-------
N
Selenastrum (EC50)
EC50(%)
(Least Toxic)
75-NE EZ3
50-75
25-50
0-25
(Most Toxic)
Creosote (%)
0-25
25-50
50-75
75-100
Daphnia (EC50)
Microtox (EC50)
IlilllllllllllllllllllllllilllliltSSSS^IIIIIBS^^
Root Elongation (EC50)
Creosote {%)
-660
-380 -300 -140 -60 -20
Meters
120 140 220
FIGURE 8.14.
Bioassay Results from Sediment Elutriates at the Wood
Treatment Site in Mississippi. Negative numbers
represent samples collected downstream from the site.
78
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different organic compounds in creosote, which may bind differentially in
each area of stream sediments, or from in-stream seeps from the waste site.
Chemical analyses indicated that the most severe contamination occurred ir>
the extreme downstream portion of the creek study area. The algae, Daphnia.
and Microtox bioassays indicated that the most extreme toxicities actually
occur about 300 m west of the initial point. The Microtox bioassay was most
sensitive to the chemical contaminants in the downstream sediment elutriates.
The results from root elongation tests show a complete absence of a
detectable phytotoxic component. A comparison between relative creosote
amounts and algae, Daphnia, and Microtox response to sediment elutriates
collected between -140 and -400 m (Figure 8.14) suggests that contaminants
other than creosote caused the toxicity.
Figure 8.15 contains the bioassay results from creek sediments and the
respective chemical analyses. The highest creosote concentration measured in
sediments was 9500 ppm (two orders of magnitude greater than that in
elutriates). Again, the conclusions regarding contaminant distribution
differ depending on the bioassay used. Chemical analyses did not directly
correspond to toxicity (Figure 8.15). However, earthworm toxicity appeared
to track increasing creosote amounts more closely than the Neubauer plant
test.
It appears from a comparison of the results in Figures 8.14 and 8.15
that a significant fraction of the toxic compounds in sediments are water
soluble. In addition, stream cleanup decisions based on bioassay results are
simplified, because the stream is so narrow that extra sediment removal would
not involve much extra cost. Based on Porcella's (1983) criteria and the
Daphnia and Microtox sediment elutriate bioassay results, cleanup should
begin at about 220 m west of the initial point and continue to about the
660-m area.
The major conclusions from this study are 1) standard bioassay organisms
are sensitive to contaminants resulting from wood treatment operations,
2) different bioassay organisms have different sensitivities to the mixture
of contaminants resulting from wood treatment operations, 3) infrared
measurements of sediment contaminants resulting from wood treatment
operations are inaccurate predictors of biotoxicity, and 4) Daphnia^ and
Microtox bioassay results can be used to make cleanup decisions.
79
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EC50 (%)
(least toxic)
75-NE EZ3
50-75 B53
25-50 ^
°-25 trmn
(most toxic)
Creosote (%)
0-25
25-50
50-75
75-100
Neubauer (EC50)
Earthworm (EC50)
K$$$^$$$$^$$$$$$$$^^^
Creosote (%)
-660
-380 -300
-140 -60-20
120 140 220
Meters
FIGURE 8.15.
Bioassay Results from Sediments at the Wood Treatment Site in
Mississippi
80
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8.3 SOIL, SEDIMENT, AND SURFACE-WATER SAMPLING AT TWO HAZARDOUS WASTE
SITES
Two hazardous waste sites with different histories were sampled. Site 1
(Friedman, New Jersey) was initially ranked high as a potentially hazardous
site because of eyewitness reports that it had received drums and free-
flowing liquids during the late 1950s and early 1960s. A subsequent site
survey and detailed chemical study suggested that there would be few
environmental or human health problems at this site. Excavations conducted
during the survey revealed that the site had actually been used as a dumpsite
for household waste and building debris. However, analysis of site sediments
indicated the presence of very low levels of a few priority pollutants.
Because the site is near residential wells, an extensive bioassay study was
designed and carried out to assess toxicity.
Site 2 (Combe, New Jersey) is an abandoned commercial landfill situated
in a partially wooded rural residential area. The site is bordered on one
side by a hardwood wetland containing the headwaters of a brook that is the
source of the water for a fishery in a nearby state park. In addition, water
from the brook is the source for some residential ponds. The site contained
an old landfill, closed in 1972, that contains waste from the 1940s. A newer
landfill was closed in 1981. There were no records available to provide
information about the types or volumes of chemical or industrial wastes
deposited at the site. Because some offsite surface-water samples contained
>100 ppb of dichloromethane, carbon tetrachloride, trans-l,2-dichloroethene,
nonane, and 1,1-dichloroethane, and because similar concentrations had been
reported from two onsite seeps, this site was presumed to be contaminated.
8.3.1 Assemble Information Relevant to the Problem
Site 1 (Friedman, New Jersey, Figure 8.16) includes 0.68 ha located near
the intersection of two county highways. It is adjacent to an unnamed stream
that is tributary to a nearby river. Several residences and two trailer
parks are located within 402 m of the site, and all the residents obtain
water from private wells. In 1983 field investigations were begun on the
presence of hazardous substances and their area! extent and migration because
of public concern resulting from rumors that hazardous substances had been
dumped into the site. The site exists as an open vacant lot bordered to the
east by scrub vegetation running into a pine and hardwood grove. This grove
is nearly flat with a
-------
High Water
6 Lake Edge
Sample Point
Transect
Grid
Road Runoff
Depression
FIGURE 8.16. Seven Sampling Locations at or near Hazardous Waste Site
1. For clarity, individual sampling points are shown
for locations 4-6 ().
82
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A patch of dense, woody vegetation approximately 30.5 by 61.0 m is
located in the southwest corner of the property at the junction of the two
county roads. This dense vegetation covers a surface depression that is
approximately 2 m lower than the surrounding ground surface (location F-5,
Figure 8.16). The depression receives drainage from the west side of one
road and the south side of the other. Drainage water leaves the depression
via a 0.9-m-diameter corrugated metal culvert pipe that runs north-northeast
under the dump site at a slope of 2.3%. This culvert runs through the site
for 83.9 m to its discharge at a marshland, where it flows 30.5 m through a
small channel to the stream.
At the northeast and eastern portions of the site the slope of the land
increases (approximately 3.1 m of relief), forming a wooded escarpment with
slopes varying between 10% and 40%. This escarpment slopes into a marshland
formed by the tributary. The total relief through the site 1s about 6.2 m,
with an average slope of 3.3%.
Based on chemical concentrations from six shallow wells and eight local
domestic wells, the shallow ground water and deep aquifer were judged to be
either not contaminated or to contain inconsequential levels of chemicals.
Similarly, samples of site soils and actual waste (obtained from trenching)
revealed no pollution problems. However, stream sediment analysis indicated
that several priority pollutant organics and inorganics were present at very
low levels in sediments in and around the site. A comparison of the 1983
sediment concentrations with previous sampling results indicated that a group
of polynuclear aromatic hydrocarbons, most likely a result of previous road
maintenance operations, had been trapped in the surface drainage sediments.
The comparison also showed that the areas of highest concentration appeared
to be moving downstream. Lead, a common pollutant associated with roadway
runoff, was also found in the stream sediments. Overall, there did not
appear to be any significant contributions from organic or inorganic priority
pollutants in the ground water, surface water, or stream sediments that could
be attributed to site waste.
Site 2 (Combe, New Jersey) is a 40-ha inactive sanitary landfill
situated atop a hill in a partially wooded residential area. Portions of the
landfill appear to extend above the preexisting ground surface, with
elevations ranging from 244 to 268.4 m above mean sea level. The site is
83
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bordered to the east and south by a county road, to the north by private
properties on residential streets, and to the west-southwest by a 20-ha tract
described as a hardwood wetland (Figure 8.17). This wetland contains the
headwaters of a brook that is also a river tributary. Surface runoff from
the site drains to both the east and west branches of the brook.
The old landfill consists of two areas totaling approximately 12 ha.
These older landfill areas were filled and partially reclaimed in 1972 and
may contain refuse deposited during the 1940s. No record exists of the type
of wastes disposed in the old landfill. The present extent and configuration
of the old landfill areas are not yet confirmed.
The new landfill area is located to the south and west of the older
landfill and extends west to the wetland where some landfill ing operations
may have been conducted. The new landfill was closed and regraded in
September 1981. Existing cover material consists of coarse permeable local
soils and crushed bedrock. Severe sheet erosion has occurred on the steep
slopes a't the western and southern edges of the landfill where vegetation was
not established. Numerous brownish-black-stained seep areas are present both
at the base of the landfill and on the side slopes. The exact extent and
configuration of the new landfill are also unknown. In addition to the
landfill operation, several open fields near the site may be of importance.
Local residents have suggested that a field at the northwest corner may have
been used for unauthorized dumping of refuse, chemical wastes, and industrial
wastes.
A potential problem at the landfill site is the discharge of
contaminants into the local streams from surface runoff, ground-water
baseflow, and leachate seeps. All streams mentioned previously eventually
discharge into a source of public water supply. It is expected that the
wastes contained in eight identified seeps (location C-8, Figure 8.17) have
or will penetrate and contaminate site cover soils as well as offsite soils
in established flow paths. Eleven known and 24 unknown organics have been
found in the residential well samples near the site. Tetrachloroethylene and
chloroform were the most common. Both substances have been identified as
potential carcinogens by the EPA and were measured at levels that may
constitute a risk via water ingestion. Because of these identified priority
pollutants and the available information, this site was considered
contaminated.
84
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^C-11-4
Brook
To State Park
FIGURE 8.17. Sampling Locations at Landfill Site 2
85
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8.3.2 Prepare a Statement of Study Objectives
Based on chemical analyses of soil (including samples from trenches) and
surface- and ground-water samples, Site 1 was judged not to be an immediate
problem to either the environment or human health. Thus, the working
hypothesis for bioassay studies at this site was that the site did not
contain dangerous levels of priority pollutants.
There were two objectives in the bioassay program for this site:
1. To validate, insofar as possible, that samples from the same areas that
were previously declared to be chemically clean were also biologically
inactive
2. To evaluate the toxic potential of the site "bench" (area of streambed
periodically flooded) and the onsite stream area using results from
composited soil and water samples (see Figure 8.16).
Site 2 had both a prior history and outward signs of chemical
contamination. However, the extent of surface and subsurface contamination
was unknown because of intermittent ground-water seeps and the possibility of
undocumented dumping. For these reasons, bioassay studies at this site were
necessary to determine the degree and geographic extent of any offsite or
unidentified onsite chemical contamination and to assess the environmental
hazard. The objectives of the bioassay program for Site 2 were as follows:
1. To determine the toxicity of surface soils and seep areas in the new
landfill and open field area
2. To determine whether samples of sediment and water collected in the east
and west branches of the brook and the wetland to the southwest of the
landfill were toxic.
8.3.3 Define the Evaluation Criteria and Reliability Requirements for
the Results
Because samples from both sites were to be composited for screening
bioassays, the number of samples selected for compositing was calculated
using the method outlined in Section 5.2:
MDL
86
-------
where
n = the number of component samples to mix into a single composite
MAL = maximum acceptable limit. We selected 50% reduction in the
Selenastrum yield (relative to control) at an 80% elutriate
concentration, which is the highest sample strength that can be
bioassayed with algae. Such a limit would be liberal compared to
the arbitrary <20% EC50 standard in Porcella (1983).
MDL = minimum detectable limit. Previous results led us to believe that
control samples might cause up to 5% reduction in Selenastrum at an
80% elutriate concentration.
Thus, n _< 50 / 5 _< 10. According to these calculations, when 10
components are mixed and eluted with water (4:1 volume:weight) and the
resulting 80% concentration material causes a reduction in Selenastrum
numbers >50%, 1 or more of the 10 components is over the MAL, and each would
have cau'sed a reduction >50% if tested alone. It is possible that only one
sample might be over the MAL (50%) if it contained very high levels of
toxicant. However, testing individual components at full strength may not
identify this high concentration since percent reduction in Selenastrum
numbers (compared to control) cannot exceed 100%. Thus, EC50 determinations
on each component may be needed. If the elutriate causes a reduction of 5%
or less, then none of the 10 components would show a 50% reduction when
tested alone. However, the above calculations depend on the negative
linearity of the dose (units of toxicant extracted from the composite) versus
percent reduction function. Intermediate results (i.e., between 5% and 50%)
would require a statistical test such as given in Skalski and Thomas (1984).
Clearly, there are problems and research questions to resolve relative
to compositing samples from chemical waste sites. In order to use the
statistical test in Skalski and Thomas (1984), background parameters (e.g.,
based on the distribution of control effects) must be available. Since the
statistical test could not be used at either site, a three-stage laboratory
analysis strategy was used. Thus, intermediate and sometimes high reductions
in Selenastrum or Daphnia caused by composite elutriates triggered a final-
stage EC50 determination on all components. Often, a second-stage screening
using root elongation and Microtox was conducted. Moreover, when spatial
87
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patterns in final stage component EC50 values are found, kriging techniques
can be used to prepare a map (see Section 8.1.4 for an example).
8.3.4 Determine What Is to Be Sampled in the Field
To meet the objectives at Site 1, the following media and locations were
sampled (see Figure 8.16):
1. Soil and/or sediments in the wetland between the landfill and the stream
2. Sediments and water in the stream
3. Sediments and water at the juncture of the two county roads
4. Sediments and water upstream of the site.
Samples of the following media were collected from Site 2 (Figure 8.17):
1. Soil and/or water at all eight identified seep areas (C-8)
2. Soil/sediments/water in the wetlands southwest of the site
3. Sediment and water samples in the east and west branches of the brook
4. Neutral (control) soil and water samples collected north of the site
(C-7).
8.3.5 Choose Test Organisms for the Bioassays
The Selenastrum and Daphnia bioassays have been shown to respond to more
than 90% of the hazardous chemical wastes tested (Miller et al. 1985).
Therefore, to reduce costs, these bioassays were selected as a first-stage
screen to test the composited samples from Sites 1 and 2. EC50
determinations were not conducted in this stage. Instead, each composite was
tested using algae [three replicates each of two elutriate concentrations
(one at 80%, rather than 100%, because nutrients must be added to the growth
media, and a second concentration of 25%)]. Only one replicate of each of
two elutriate concentrations (100% and 25%) was tested using the Daphnia
bioassay. When results from the 80% concentration exceeded 50% reduction,
components were individually bioassayed as a second stage. Definitive EC50s
for Daphnia or algae were generally conducted as a final stage. Depending on
the second-stage results for the Microtox or root elongation bioassays, as
well as first-stage screening results, definitive EC50s for earthworms and
plants were conducted. The lower first-stage screening elutriate
-------
concentrations (25%) were included to evaluate multicomponent causes of
toxicity and/or to judge the existence of very toxic single components.
8.3.6 Define the Data Analysis Techniques
For Site 1 studies, objective 1 (see Section 8.3.2) simply required a
finding of no biological toxicity. For composite samples where results were
below 5% reduction in Selenastrum numbers, components were judged to be
biologically unaffected (see Section 8.3.3). Elutriate samples that
inhibited algae growth by more than 50% were reassayed to define their EC50
values (see Section 8.3.5). Samples with reductions between 50% and 75% were
usually rescreened with Microtox and root elongation bioassays.
To meet objective 2 at Site 1, two grids were established in the "bench"
region (see Section 8.3.7 and locations F-l and F-3; also see Figure 8.16).
If the composite components proved to be toxic, kriging could be used to
assess the areal extent of toxicity. Both objectives for Site 2 (see Section
8.3.2) could have been met using the methods for Site 1, objective 1.
However, as a precaution, several grid designs were used even though
questions about spatial distribution were not included in the objectives.
Rectangular grids ranged from 1.5 to 25 m on the longest side, depending on
2
the area targeted for sampling [e.g., a 3-m pond at the top of the landfill
or a 100- x 28-m area in the soybean field (Figure 8.17)]. These "extra"
samples allowed the compositing strategy to be used to assess areal toxicity
potential, and if toxicity was found, components were available to assess
either small- or large-scale patterns.
8.3.7 Design the Field Sampling and Laboratory Studies
Because there was very little chemical concentration data available for
either site, a varied field sampling protocol was developed. At each of the
sites an array of sampling schemes was used to collect soil/sediment and
surface-water samples. Rectangular grids were established at various
locations to systematically sample selected regions. In other instances
(i.e., along streams and apparent waste flows), samples were collected along
line transects or by opportunistic sampling of suspected contaminated areas.
Sample locations were mapped and identified using transit, compass, and meter
tapes. Soil and sediment samples were collected to a depth of 15 cm using a
metal hand spade. Water samples were obtained by submerging 1-L polyethylene
89
-------
bottles below the surface of water sources. Samples collected under these
protocols were evaluated using screening bioassays of composited samples (see
Section 8.3.3). A second, and sometimes third, laboratory analysis stage was
used to evaluate individual composite components when toxicity was found in
first-stage screening tests.
An example of a 25-point (component) rectangular grid from location F-3
at Site 1 (see Figure 8.16) is shown in Figure 8.18. A similar grid was used
at location F-l, while a 12-point grid was used at F-7. In Figure 8.19, an
example of a transect sampling approach at Site 1 is shown (location F-4,
Figure 8.16). Finally, in Figure 8.20, an example of opportunistic sampling
at location C-ll of Site 2 is shown (Figure 8.17). All other locations at
Sites 1 and 2 were sampled using variants of the methods shown in Figures
8.18 through 8.20 or in Section 8.1.
The compositing strategy used for the samples from the locations in
Figures 8.18 through 8.20 is shown in Table 8.4. Approximately 250 g of soil
or sediment yielded 1000 ml of elutriate for the bioassays (e.g., about 4:1
volumerweight). This quantity of material was more than adequate for
conducting three replicate Selanastrum tests at 80% and 25% dilutions each
(six tests) and one replicate Daphnia test at two dilutions, 100% and 25%
(two tests).
The basis for including up to 10 components in a single sample composite
was explained in Section 8.3.3. At location F-3 (Figure 8.18), samples from
two of the three bench locations with standing water (F-3-17 and~F-3-23) were
pooled and assayed to assess toxicity. The streamside soil samples (F-3-16
through F-3-25) and samples from farther away (F-3-1 to F-3-10) were
composited to examine the bench for toxicity (Figure 8.18). Location F-4
(Figure 8.19) provided the opportunity to assess toxicity in the stream water
and sediments from where the stream entered Site 1 (F-4-1) to beyond the site
boundary (F-4-7). To facilitate an upstream/downstream onsite comparison,
four water and sediment samples from two upstream (F-4-2 and F-4-3) and two
downstream stations (F-4-5 and F-4-6) were composited into samples F-4-C1 and
F-4-C2, respectively (Table 8.5). All water and sediment samples collected
at individual sites at location C-ll (Figure 8.20) were bioassayed to
determine if either branch of the brook or the unbranched brook itself in or
out of the state park contained toxic compounds.
90
-------
Composite 1
fas
J20
Stream
**->
23ft 22
21
Composite 2\
20
15
19
14
9
4
$ is
13
8
3
^ 17
^2
7
2
16
11
6
1
5 m
10m
FIGURE 8.18. Grid Sampling at Location F-3 of Site 1
F-4-7
Stream from
Culvert
-_
4° m
Rout* 537
----- Compoiittd Simples
FIGURE 8.19. Transect Sampling at Location F-4 of Site 1
91
-------
FIGURE 8.20. Opportunistic Sampling at Location C-11 of Site 2
92
-------
TABLE 8.4. Description of Composite Components Used in Screening Bioassays
of Samples Taken from the Locations Shown in Figures 8.18 to 8.20
Location
F-3
F-3
F-4
F-4
C-ll
C-ll
Samples to
Media be Composited
Water F-3-17 A & B
F-3-23 A & B
F-3-24 A & B
Soil F-3-1 to F-3-10
F-3-16 to F-3-25
Water F-4-2 A & B and
F-4-3 A & B
F-4-5 A & B and
F-4-6 A & B
Sediment F-4-2 A & B and
F-4-3 A & B
F-4-5 A & B and
F-4-6 A & B
Water C-ll-1 A & B
C-ll-3 A & B
C-ll-4 A & B
Sediment C-ll-1
C-ll-2
C-ll-3
C-ll-4
Sample Size from
Each Container
250 ml
250 ml
250 ml
100 g
100 g
125 ml
125 ml
100 g
100 g
250 ml
250 ml
250 ml
250 g
250 g
250 g
250 g
Total
Sample Size
500 ml
500 ml
500 ml
1000 g
1000 g
500 ml
500 ml
400 g
400 g
500 ml
500 ml
500 ml
250 g
250 g
250 g
250 g
93
-------
TABLE 8.5. Bloassay Results from Three Stages of Sample Analysis at Site 1. Results from other
samples that did not exhibit toxicity are not presented
Second-
First-Stage Screen Stage Screen
Algae
Site 1
Location
F-1
F-3
F-7
F-2
F-4
F-5
Sample
Number* ' Media
F-1-C1 Soil
F-3-C1 Soil
F-7-C1 Sol 1
F-7-1
F-7-1 0
F-7-1 1
F-2-C1 Sediment
F-4-C1 Sediment
F-4-C2
F-5-C1 Sediment
80%""
-33(f)
-91
NE
NE
NE
NE
-67
-97
-94
-100
25%
-25
-76
NE
NE
NE
NE
-48
-60
-67
-69
Daphnia
100%
NE<"
-71
NE
NE
-100
-100
NE
NE
NE
-100
25%
NE
NE
NE
NE
NE
NE
NE
NE
NE
-14
RE
Reduction lc)
NE
NE
-39
-69
NE
NE
NE
NE
NE
NE
Algae
EC50
NA(h)
20" >
NA
NA
NA
NA
22
33
36
20
Final -Stage Screen
Daphnia
EC50
NA
94
NA
NA
89
89
NA
NA
NA
31
RE
EC50
NA
NA
NA
28
NA
NA
NA
NA
NA
NA
NEU(d>
.NE
38
NE
35
74
63
27
NE
NE
NE
Earthworm
Loss(e)
NA
-20
(j)
-JJ>
__'k'
NE
..«>
..<">
_Jk)
_Jk)
(a) A "C" in the suffix indicates a composite.
(b) Percent dilution of elutriate.
(c) RE » Root Elongation.
(d) NEU * Neubauer Test.
(e) In 75% to 80% soil.
(f) Negative values are mortalities In 100% soil or
soil elutriate.
(g) NE - No Effect.
(h) NA * Not Applicable.
(i) Positive values are ECSOs.
(j) Analysis not yet completed.
(k) Insufficient sample.
-------
The transect sampling technique was to run transects down the length of
the stream starting at the west side of Route 537. Samples of sediment and
water were taken at 40-m intervals along the course of the stream. At each
sample location, one 2-kg sediment sample and two 1-L water samples were
collected. All samples (sediment and water) were similarly coded and
numbered F-4-1 to F-4-7.
8.3.8 Analyze and Evaluate the Results
The bioassay results from all locations (in addition to the examples
illustrated in Figures 8.18 and 8.19) at Site 1 that exhibited toxicity are
listed in Table 8.5. Composited sediments from locations F-2 (not
illustrated in detail; however, the composite contained four components),
F-4 (shown in Figure 8.19), and F-5 (not illustrated in detail, but it
contained two components) were toxic to algae in the first-stage screen and
very toxic to algae (low EC50 values) in the final screen. Further, the
sediment composite from location F-5 was also toxic to Daphnia, while the
F-2 composite was toxic to lettuce seeds; however, water samples from these
locations were not toxic. These toxic composite sediment samples were from
an area between the culvert outlet and the stream (F-2); instream, upstream
from the culvert entrance (F-4-C1); instream, below the culvert entrance
(F-4-C2); and in the depression where the two roads meet (F-5). Figure 8.16
shows the general locations. Previous chemical analyses of site sediments
(Section 8.3.1) indicated that polynuclear aromatic hydrocarbons had been
trapped in site surface sediments. This information, in concert with the
fact that no toxicity was found in sediments upstream (location F-6) or in
any of the water samples from the site, tends to support the existence of a
non-site-related toxicant, bound in sediments. The toxicity to lettuce
seeds of sediment composite F-2-C1 (Table 8.5) may indicate some other
toxicant is present in the bench area.
A 25-point rectangular sampling grid was set up on the east side of the
site between the landfill bank and the stream. Grid point F-3-5 was located
at well W-5. Two 1-L water samples (A and B) were collected on each of
three open-water patches in the grid near points F-3-17, F-3-23, and F-3-24.
One 2-kg soil sample was collected at each grid node (F-3-1 to 25 on Figure
8.18).
95
-------
Opportunistic sediment and water samples were collected at the road
intersections with the east and west branches of the brook (samples C-ll-1
and C-ll-2, respectively). Sample C-ll-3 was obtained beyond the confluence
of the two branches near State Park Road, and sample C-ll-4 was collected in
the park. Each sample consisted of two 2-kg sediment and four 1-L water
samples (except C-ll-2, where no standing water existed).
The 10 components contained within the soil composites that were taken
closest to the stream (see Figure 8.18 and Table 8.5) were slightly toxic
(F-1-C1) to very toxic (F-3-C1) for algae and Daphm'a in the first-stage
screen. Further analysis (final screen, Table 8.5) of sample F-3-C1
indicated that this upstream onsite location contained soils that were toxic
to algae and lettuce seeds and slightly toxic to earthworms. Moreover, both
composites were located closest to the stream where deposition of road runoff
would be likely during periods of high water. As with the instream water
samples, standing water from location F-3 was not toxic. However, this
sample also provides additional evidence for an insoluble phytotoxic
component in F-3 soils (sample F-3-C1) closest to the stream. Thus, there
may be a site area of localized phytotoxicity, but most biotoxicity appears
to have resulted from road maintenance.
The Neubauer phytoassay results from individual soil samples taken far
upstream from the site (principally samples F-7-1, F-7-10, and F-7-11)
provide evidence (EC50s range from 35% to 742 soil) that either a naturally
occurring phytotoxic component may exist in regional soils, or the component
is only bound in a few soils or sediments over the entire region'of the
stream. Thus, it appears that those areas of the site that were previously
shown to be chemically "clean" are also biologically inactive (objective 1,
Section 8.3.2). Results from onsite and offsite stream sediment and water
samples implicate road runoff as a likely source of the insoluble toxicants
detected. One onsite phytotoxic soil and one sediment composite sample were
found. Because there were three single phytotoxic soil samples far upstream,
it appears that site phytotoxicity may not be caused by site-related
chemicals.
96
-------
The results from locations C-8 to C-10 at Site 2 (location C-ll is shown
in Figure 8.20) that showed evidence of toxicity relevant to the study
objectives are presented in Table 8.6. The location of each sampling site is
shown in Figure 8.17.
For convenience, the bioassay results are discussed for the east branch
(location C-9 and sample C-11-2), west branch (location C-10 and sample
C-ll-1), and joint brook water and sediment samples, followed by the seep
(C-8) soil and water samples. A description of samples obtained from
locations C-8 to C-10 (for C-ll; see Figure 8.20) is given in Table 8.7.
There was no evidence of toxicity caused by water or sediments from the
east branch of the brook (including sample C-11-2; see Figure 8.17) since
only one sample from location C-9 was toxic (the root elongation bioassay
for sample C-9-5 showed some toxicity). However, several water and sediment
samples from the west branch were very toxic to algae in samples just below,
in, and just above the small residential pond (samples C-10-1 to C-10-3 for
water and sample C-10-9 for sediment). Evidence that the toxicity had
migrated downstream is provided by the sediment bioassay results from sample
C-ll-1 where algae, Daphm'a, and plant seed toxicity were fairly severe. In
addition, residential pond sediment caused earthworm toxicity (C-10-9; 100%)
and one of two sediment samples taken below the confluence of the branches
(C-ll-4; Figure 8.17) was also toxic to earthworms. Both mainstem brook
samples (including C-ll-4 in the park) indicate that the phytotoxic component
in the residential pond (see C-10-9; 6% EC50 for the Neubauer test) may also
have migrated. Finally, upstream water samples C-10-5 and C-10-7 exhibited
limited toxicity in the algal screen as well in the root elongation assay,
but samples taken further upstream (C-10-8 and C-10-10) were not toxic.
Thus, it appears that the toxic components may be emanating from the waste
site somewhere below location C-4 (Figure 8.17) and that the eastern area of
the site and the west branch of the brook either have been and/or are being
influenced by chemicals leaking from the waste site. It is probable that
these chemicals are being exported beyond the site boundaries (objective 2,
Section 8.2.3).
Bioassay results from soils in the six seep areas of location C-8
(Figure 8.17) show that soil from one seep (location C-8-3) caused major
biotoxicity to algae, Daphnia, lettuce seeds, and earthworms in the final-
97
-------
TABLE 8.6. Bioassay Results from Three Stages of Sample Analysis at Site 2.
other samples that did not exhibit toxicity are not presented
Results from
First-Stage Screen
Second-
Stage Screen Final -Stage Screen
Algae Daphnia
Site 2
Location
C-8
Sample
Number '
C-8-4
C-8-1
C-8-2
C-8-3
C-8-4
C-8-5
C-8-6
Media
Water
Soil
Soil
Soil
Soil
Soil
Soil
(b)
80% 25% 100%
-99 -25 -100
-I, .Je)
+159
-66 -56
+177
+187
+243
RE Microtox Algae Daphnia RE
25% Loss (%) X 30 EC50 EC50 EC50
-25 -23 4 7 14
-23
45 46
-33
-32
-34
Neubauer
Loss
(%)
66
25
31
73
-43
Earthworm
Loss
(%)
NE(f)
-100
NE
NE
"(g)
C-9 C-9-5
vo
oo
C-10
C-11
C-10-1
C-10-2
C-10-3
C-10-5
C-10-7
C-10-9
C-10-9
C-11-1
C-11-4
Water
Water
Water
Water
Water
Water
Water
Sediment
Sediment
-93
-95
-98
-39
-62
Sediment -100
-92
+20
-9
-64
-58
-71
-57
NE
-100
NE
-37
-32
-21
-45
-28
48
54
17
34
14
29
83
69
-47
-32
-100
NE
-20
(a) A "C" in the sample number suffix indicates a composite.
(b) Percent dilution of elutriate.
(c) Numbers with a minus sign are mortalities tn 100% soil.
Positive values are ECSOs. The screening result was at least
-70 in 100% soil.
(d) In 70% to 80% soil.
(e) In general, no test was done because of rules stated in text
or because positive results indicated no growth suppression
[all other blanks indicate (e)].
(f) NE « No effect.
(g) Insufficient sample.
-------
TABLE 8.7. Description of Samples Collected at Additional Site 2 Locations
Where Positive Bioassay Results Were Obtained
Location Description
C-8 C-8-1 Through C-8-6
Six individual seeps (contained in Area C-8, Figure 8.17)
on the west side of the landfill were sampled. Sample C-8-6
was the most northern, followed sequentially south to C-8-1.
At each sample location, two 2-kg soil samples were collected
and designated A and B. At C-8-4, four 1-L water samples
were collected. These water samples were collected at the
source of the seep, where water and gas were being emitted.
C-9 C-9-1 Through C-9-7
Starting 50 m upstream of the main road on the east branch of
Trout Brook, sediment and water samples were collected every
50 m. Sample C-9-1 was closest to the road while C-9-7 was
northernmost, with the rest sequentially located.
At each sample location, one 2-kg soil sample was collected.
When samples were collected, only occasional standing water
existed on the branch (no running water). At C-9-3 two 1-L
water samples were collected (C-9-3A and B); at C-9-5, four
1-L water samples were collected.
C-10 C-10-1 Through C-10-10
On the west branch of Trout Brook, samples of sediment and
water were collected every 50 m starting at the concrete
culvert on the south end of the pond on the site (C-10-1)
known as Beam's Pond. Sample C-10-9 was a sample in Beam's
Pond.
All except sample C-10-9 consisted of one 2-kg sediment
sample and two 1-L water samples; sample C-10-9 was two 2-kg
soil and four 1-L water samples.
99
-------
stage screen. These toxicities indicate that both soluble and insoluble
compounds are present at this seep. No toxicity was evident in soil from the
seep at location C-8-1. The positive results for algae in the first-stage
screen for soils from other seeps (locations C-8-2 and C-8-4 to 6) triggered
second-stage bioassays. An indication of soluble phytotoxic compounds was
evident from results of root elongation bioassays, while third-stage bioassay
results indicate potent insoluble phytotoxic compound(s) are also present.
The water collected from the seep at location C-8-4 was very toxic to algae
and Daphm'a, and also inhibited root elongation.
On the basis of these results, it appears that the chemicals that are or
have been emitted at each seep exhibit varying toxicities that may reflect
different waste compositions within the main site.
TOO
-------
9.0 NEEDED ENHANCEMENTS OR ADDITIONS TO BIOASSAY TECHNOLOGY FOR USE
AT HAZARDOUS WASTE SITES
Various aspects of the bioassay procedures, the field designs, the size
and adequacy of the data base from which specific chemical causation for an
observed toxic effect can or cannot be correlated, areal mapping of toxicity,
and hot spot detection can be improved by additional research, data, or
experience. Moreover, any procedure that can improve cost-effectiveness
(e.g., staged designs, compositing) may merit additional exploration. In
this section, some of the needed enhancements or improvements to bioassay
technology are discussed as well as cost-effective improvements in
procedures.
9.1 BIOASSAY STUDIES USING ADDITIONAL PURE CHEMICALS, MIXTURES, AND
CHEMICALLY CHARACTERIZED WASTE SITE SAMPLES
At .times, we have attempted to infer the chemical cause of an observed
bioassay result (Section 4.3). However, there are few data currently
available upon which to base such inferences. In fact, the data presented
in Section 4.3 represent the few bioassay results for pure chemicals and
waste site samples of known chemistry. There appear to be few, if any,
bioassay results available for mixtures of chemicals in the proportions found
in actual waste site samples. Because of this deficiency, not much is known
about the synergistic, antagonistic, or possible additive and/or
multiplicative effects of combinations of waste site chemicals. In addition,
little is known about bioassay results for classes of priority pollutants.
The availability of bioassay results for priority pollutants, alone and in a
few meaningful combinations, might be useful in interpreting the chemical
cause of observed toxicity for some waste site samples. Finally, a complete
chemical analysis of a large number of diverse waste site samples would allow
a toxicity prediction (based on chemical content) prior to bioassay. These
predictions could subsequently be compared to actual bioassay results. In
this way, the usefulness of bioassay results could be demonstrated and the
proportion of inaccurate predictions based on chemical analysis could be
documented.
101
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9.2 ADDITIONAL WASTE SITE STUDIES
In Sections 8.1 to 8.3, bioassay results are used to assess specific
objectives at four waste sites. Additional field studies are needed to
ascertain which of the statistical methods is most useful, to define the most
informative bioassays, and to rank the ease and expense with which questions
(objectives) can be addressed. Further, a comparison (based on actual site
experience) of bioassay and chemical analysis approaches to site
characterization and predicted toxicity is also needed to validate the
superiority of bioassay approaches for ecosystem risk assessment.
9.3 DECISION RULES TO RATE WASTE SITE SAMPLE TOXICITY
The only rule proposed to assess the seriousness of a laboratory-
derived EC50 (based on a waste site sample) 1s from Porcella (1983). In his
system, an EC50 < 20% elutriate is referred to as high, an EC50 > 20% or <
75% is moderate, and an EC50 > 75% is low. This system appears to be
arbitrary, and is not based on any predicted environmental risk or human
health considerations.
In terms of harm to the ecosystem, 50% mortality caused by 100%
elutriate might be catastrophic. However, laboratory results do not
generally translate directly to field observations (e.g., dilution by surface
or ground water would result in lower toxicity). Thus, a ranking system like
the one proposed by Porcella (1983), but with different and justified action
limits, is needed. Ranking the potential site toxiclty to resident organisms
may need to be based on a weighting scheme that downweights toxicity obtained
from elutriates, because resident organisms are exposed to actual site soils
and sediments. In contrast, an argument can be made for extra weighting for
very toxic elutriates. Thus, it is evident that additional thought,
experiments, and experience are needed to judge which values of toxic effects
should lead to remedial action.
9.4 SCREENING BIOASSAYS
In Section 8.3, screening bioassays were conducted using 80% and 25%
elutriates for algae and 100% and 25% elutriates for Daphnia (and limited
102
-------
replications of each level). As a first attempt at two-stage laboratory
designs, the screening procedure performed well, but there was no
quantitative measurement of performance. Thus, a more formal screening
procedure and rationale are needed, and results should be compared to a
series of samples run by the usual procedure(s). The usual and new methods
can be compared based on the cost per unit of information and the precision
and accuracy of each method. Such a structure could be developed and tested
at future sites or during the studies suggested in Section 9.2.
9.5 DEVELOPMENT AND ENHANCEMENT OF LABORATORY COMPOSITING STRATEGIES AND
CONCOMITANT FIELD DESIGNS FOR DETECTING MOVEMENT AND EXTENT OF
CONTAMINATION
The scope of a sampling program generally reflects a preconceived notion
of the number of samples that may be ultimately analyzed for contaminants.
Since the cost of complete priority chemical analysis is very high and some
bioassays are moderately expensive, generally few samples are collected from
individual sites. These relatively few samples may poorly represent the
extent of surface or subsurface contamination. However, by using group
testing methods, large numbers of samples (soil, water, or organism) may be
composited to minimize analytic or bioassessment costs without loss of
information, thereby permitting better characterization of the potential
hazards at a given site. The development of sampling methodology should
reflect these relationships between limitations in the bioassay analysis of
physical samples and the field sampling designs.
For this purpose, statistical techniques are needed to:
1. devise an optimal field sampling design based on several representative
site-specific scenarios (priors), and
2. devise an optimal compositing scheme based on both analytic limits for
chemistry and bioassays and a maximum acceptable limit for the
compound(s) or assay(s) in question.
Because these sampling purposes are related, they should not be addressed
separately.
103
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Earth, D. S., and B. J. Mason. 1984. Soil Sampling Quality Assurance User's
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Beckman Inc. 1982. Microtox System Operating Manual. Beckman Instruments,
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Clark, I. 1979. Practical Geostatistics. Applied Science, London.
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Gibb, J. P., R. M. Schuller, and R. A. Griffin. 1989. "Monitoring Well
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