United States
Environmental Protection
Agency
Corvallis Environmental
Research Laboratory
Corvallis, Oregon 97330
EPA
600283054
-------
PROTOCOL FOR BIOASSESSMENT OF
HAZARDOUS WASTE SITES
Prepared by
D.B. Porcella, Ph.D.
Tetra Tech, Incorporated
3746 Mt. Diablo Boulevard
Lafayette, California 94549
EPA-600/2-83-054
«f
-------
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.
-------
PREFACE
This bioassessment protocol was developed in steps. First, a set of
biological test procedures were defined for possible use. Then, the
conceptual basis and the specific tests as related to ecological needs and
current regulatory requirements were discussed at a workshop in Washington,
D.C. October 26-27, 1981. The attendees also considered the current status
of hazardous waste site prioritization and cleanup and statistical factors,
field application and evaluation procedures, and other possible biological
tests and procedures. The resulting protocol has been extensively reviewed,
and is being applied to existing sites to evaluate response levels in
relation to field studies. It should be noted that this protocol is not a
regulation, rather it is a set of tools for studying potential ecological
hazards at waste sites. The protocol relates to the National Contingency
Plan (Federal Register, 47(137):31180-31243, July 16, 1982) as published
pursuant to Section 311 of the Clean Water Act (CWA) and as revised under
Section 105 of the Comprehensive Environmental Response, Compensation and
Liability Act (CERCLA) of 1980.
-------
ACKNOWLEDGMENTS
The participants in the workshop to discuss this protocol are listed
below and represent federal and state agencies, industry, universities, and
consultants. This document was prepared and compiled by the author.
However, the workshop participants made substantial contributions and
subsequently reviewed the document, although they did not necessarily
endorse all of its contents. Their efforts are gratefully acknowledged.
Name
Karen Bergen
Albert Galli
Steven Gherini
Edwin Herricks
Don Huisingh
Don Klein
Conrad Kleveno
Richard Lee
Alfred Lindsey
Larry Marx
William Mason
James McKim
Gary McKown
William Miller
Tshwar Murarka
Royal Nadeau
Richard Peddicord
Spencer Peterson
Affiliation
USEPA
USEPA
Tetra Tech, Inc.
University of Illinois
State of North Carolina
Colorado State University
USEPA
Corps of Engineers
USEPA
Tetra Tech, Inc.
U.S. Fish and Wildlife Service
USEPA
Batelle PNL
USEPA
Electric Power Research Institute
USEPA
Corps of Engineers
USEPA
Location
Washington, D.C.
Washington, D.C.
Lafayette, CA
Urbana, IL
Raleigh, NC
Ft. Collins, CO
Washington, D.C.
Vicksburg, MS
Washington, D.C.
Bellevue, WA
Leetown, VA
Duluth, MN
Richland, WA
Corvallis, OR
Palo-Alto, CA
Edison, NJ
Vicksburg, MS
Corvallis, OR
ii
-------
Don Force!la Tetra Tech, Inc. Lafayette, CA
Alan Rohlik SOHIO Cleveland, OH
Eugene Shreckheise Batelle PNL Washington, D.C.
Richard Stanford USEPA Washington, D.C.
John Thomas Batelle PNL Rich!and, WA
John Wardell USEPA Denver, CO
Dan Weltering Procter and Gamble Cincinnati, OH
Robert Yelin Tetra Tech, Inc. Pasadena, CA
Introductory papers were presented by Peterson (Problem Description),
Kleveno (Problem Setting and Site Priority), Gherini (Chemistry), Herricks
(Biology), Murarka (Risk Analysis), Miller (Protocol Methods), Porcella
(Sampling Criteria), Thomas (Field Studies), and Klein (Microbial Tests).
Becky Boone of AWARE, Inc. provided invaluable coordination of the workshop
and subsequent mailing of the protocol.
Special thanks go to the Project Officers, Spencer A. Peterson and
William A. Miller of the USEPA, Corvallis. They provided overall guidance
and constructive criticism. Also, Clarence Callahan, Joseph Greene, and
Ibrahim Hindawi, USEPA, critiqued the procedures and approach. In addition
to others named, Alan Maki of Exxon and Pat Guiney of Gulf Research reviewed
the report.
Steven A. Gherini and Thomas M. Grieb of Tetra Tech, Inc. contributed
substantially to the report. However, the author is responsible for any
errors. Pencie Shrewsbury prepared the manuscript in its final "" form. The
work was done under a USEPA purchase order, P.O. 2B0177NALX.
m
-------
CONTENTS
Page
EXECUTIVE SUMMARY vi
INTRODUCTION 1
ASSESSING WASTE DISPOSAL SITES 1
PURPOSE OF THE BIOASSESSMENT PROTOCOL 3
A BIOASSESSMENT ANALOGY 4
PROTOCOL OBJECTIVES 5
BIOASSESSMENT PROTOCOL 7
PERSPECTIVE 7
APPLICATION OF BIOASSESSMENT PROTOCOL 18
BIOLOGICAL TESTS FOR THE BIOASSESSMENT PROTOCOL 20
POTENTIAL BIOASSESSMENT METHODS 22
EXPERIMENTAL DESIGN 25
SCOPE 25
RISK ASSESSMENT 25
STATISTICAL CONSIDERATIONS AND EXPERIMENTAL DESIGN 30
GUIDELINES FOR SAMPLING 37
Preliminary Assessment 38
Detailed Assessment 40
REFERENCES CITED 42
APPENDIX A - BIOLOGICAL TEST FOR BIOASSESSMENT OF HAZARDOUS
WASTE SITES A-l
APPENDIX B - GUIDELINES AND CONCEPTS OF SAFE PROCEDURES "- B-l
REFERENCES CITED
IV
-------
ILLUSTRATIONS
Figure Page
1. Evaluation of Environmental Testing Protocols for Hazardous
Waste Assessment 13
2. Site Response Management Plan 15
3. Matrix of Tests as Related to Types of Organisms and
Physiological Processes 23
4. Concentration-duration Results From an Acute Toxicity
Test Exposing Mysid Shrimp to a Simulated Refinery
Effluent 28
TABLES
Table Page
1. EPA Primary Industry Categories 8
2. EPA's Priority Toxic Pollutants 9
3. Primary Drinking Water Quality Standards 10
4. Definition of Toxicity Categories for Aquatic and
Terrestrial Ecological Assays 21
5. Duncan's Multiple Range Test of Complex Additions to
Scenedesmus 36
6. Minimal Experimental Design Showing The Maximum
Number of Samples and Tests for Preliminary
-Assessment of a Site 39
-------
EXECUTIVE SUMMARY
The bioassessment protocol is one of several tools, including chemical
analysis and field study, that can be used to characterize the potential
environmental risk associated with hazardous waste sites. The protocol can
be applied to priority ranking for deciding the need for cleanup of a site
compared to other sites, and to assess cleanup effectiveness by testing for
potential hazards at the site boundaries or along a sampling transect.
Bioassessment involves using defined biological tests to determine
biological response to concentrations of the biologically active components
of soil and water samples from a hazardous waste site. The tests are
described in Appendix A and include aquatic and terrestrial tests. The
algal, fish and Daphnia tests are used for water and soil extract samples,
and seed germination-root elongation, earthworm, and soil microorganism
tests are used for soil samples. The tests are standardized and each has a
background of literature citations which include some field evaluation.
Because of occupational risks, safe procedures must be used to minimize
hazard to staff during field sampling (USEPA, 1981) and during the
application of the protocol (Guidelines, Appendix B).
The key to defining site priority or cleanup effectiveness is in the
experimental sampling design. Careful definition of general and
site-specific issues is necessary.: With these issues carefully-in mind, the
design should be evaluated in terms of cost-benefit so that costly errors in
environmental risk and economic risk are minimized. Important points about
how these concepts relate to sampling design are discussed in the main text.
vi
-------
The bioassessment protocol is designed to be a set of tools that are applied
as appropriate to a specific site. Necessary samples are collected to
address the specific issues that occur at the site. Data from chemical
analyses and field studies may be available or may be required based on the
results obtained from bioassessment.
The bioassessment protocol will be improved for future use with field
application and with further research. It is promulgated at this time
because there is a present need for such a protocol, and ongoing field
application will lead directly to its improvement.
vii
-------
INTRODUCTION
ASSESSING WASTE DISPOSAL SITES
The potential hazard of planned, existing or abandoned waste disposal
sites depends on their risk to human health and the environment. Generally,
these hazards fall into four categories: toxicity, persistence,
bioaccumulation, and mobility. To minimize these hazards, cleanup and
control actions are taken based on data describing site characteristics.
The identification, characterization, and cleanup of hazardous
materials, sites and spills is a high priority of the U.S. Environmental
Protection Agency and society. Numerous potential cleanup sites have been
identified throughout the United States, and 115 priority sites have been
selected for cleanup (HMIR, 1981). Further characterization and cleanup of
approximately a dozen of the worst sites is expected to proceed shortly.
However it is not entirely clear how these characterizations and cleanups
will be conducted. The National Contingency Plan indicated that chemical
characterization of sites would have a high priority. It was proposed that
existing water and air quality criteria would be applied to chemical
characterizations to determine when cleanup was necessary, how much to
cleanup, and when to terminate the cleanup.
This approach has been criticized for various reasons. -/- Among the
reasons is that criteria applications would tend to be overly protective and
thus overly restrictive. Another criticism is that insufficient numbers of
adequate criteria exist. Yet another criticism is that single pollutant,
-------
constant concentration, laboratory derived criteria are not applicable to
environmentally released complex hazardous waste materials which may be
encountered at numerous disposal sites. If water and air quality criteria
are not applied then, what tools will be employed to assess the need for and
degree of cleanup required?
The multi-media biological testing protocol presented in this document
is an alternative or supplement to other assessment techniques. It is not
without problems, but currently it is felt that the advantages outweigh the
disadvantages.
The overall objective of this report is to describe a bioassessment
protocol that can be used immediately while being tested over a broad range
of pollutant and geoclimatic conditions. The protocol consists of sampling
environmental media followed by the application of specific biological
tests. The protocol is intended to be applied to existing or abandoned
waste disposal sites, but at least some of the procedures can be applied
during planning of new sites, to spills, or to new chemicals and materials
under appropriate conditions. Application of the data gained from these
tests will be on a case by case basis depending on the potential for
environmental impact and the intended use of the area in question.
The bioassessment protocol consists of a set of specifically defined
biological tests, and incorporate sampling design and statistical analysis
concepts. The protocol includes short term, acute tests for toxicity using
aquatic and terrestrial organisms. Except for the soil litter test, the
biological tests utilize only a single species but they do include plants,
invertebrates, vertebrates, and decomposers. The biological tests are
-------
presented in a stepwise manner in Appendix A while the protocol is presented
in the text.
PURPOSE OF THE BIOASSESSMENT PROTOCOL
The bioassessment protocol has the purpose of assessing the potential
for ecological harm from hazardous waste sites, and is one set of tools,
along with chemical and field studies, that can be used to minimize the risk
from hazardous materials. The responses of a range of test species to
exposure from water and soil samples are used to determine whether toxicity
exists at a site. The biological tests that make up the protocol cover a
range of biological taxa, are standardized, and have a history of use for a
variety of environmental assessments.
The bioassessment protocol is used for two purposes: site
prioritization and cleanup evaluation. Depending on a variety of factors
including concentration, type and availability of chemicals, organisms at
risk, exposure routes, and duration of exposure, certain sites have the
potential for more or less ecological risk. By incorporating these risk
factors, the test results can be used to rank sites in order of priority for
cleanup, isolation, or other action. Cleanup evaluation is the application
of the .biological tests to determine: "How clean is a site?" States,
industries, and federal agencies will be interested in using these tests for
these purposes. '-
-------
A BIOASSESSMENT ANALOGY
The ecosystem is an entity in which humanity and society are an
integral part. Protecting natural communities will, in most cases, afford
protection for human communities. The state of the natural community can be
viewed as an indicator of potential risk to society and effects on important
processes can be viewed as proxies for those processes that directly as well
as indirectly relate to society. The bioassessment protocol can be viewed
as a quantitative means of estimating biological impacts of hazardous waste
sites. The individual tests are bio-transducers for environmental
protection, providing an estimate of potential hazard to organisms caused by
chemicals that vary in their availability and toxicity in water or soil.
The potential for damage to human health and the ecological integrity
of hazardous waste sites can be perceived similarly to a dying canary in a
coal mine. The analogy applies to this protocol because bioassessment
methods are intended to provide a measure of potential acute biological
damage associated with samples from a particular site. A canary breathes
the mine air, responds to all biologically active components of the
atmosphere, and if an acute response occurs, dies, at which time the miners
flee. If the canary sickens over a long period of time, it would be
replaced and probably no human response would occur. Thus, chronic effects
are not assessed.
The bioassessment procedures -are similar to the canary in the mine and
provide a rapid screening of all of the biologically active c'omponents of
hazardous waste sites, and if an acute response is obtained, provide a
signal causing an appropriate response by society. The key points are:
-------
• Bioassessment provides a biological response which integrates
all of the biologically active components in a sample.
0 Bioassessment provides an estimate of the biologically
available forms of the sample components.
PROTOCOL OBJECTIVES
The overall objective of the bioassessment protocol is to provide a
more comprehensive measure of potential ecological hazard associated with
hazardous waste sites than chemical analyses and comparison to air and water
quality criteria can provide by themselves. Also, a preliminary assessment
can be obtained about sites where few data are available. Proper
application of the biological tests can improve the accuracy of the
assessment as well as improve cost-effectiveness by prioritizing hazardous
sites. Specific steps must be followed to achieve these objectives:
t Identify the waste disposal site.
• Define potential transport and fate of site materials and
populations at risk; list risk issues of concern.
• Define containment site boundaries.
• Design a sampling program to meet specified statistical
criteria.
-------
• Obtain appropriate soil and surface and ground water samples
at the site boundary.
• Obtain appropriate soil and surface and ground water samples
along a gradient of waste contamination.
• Select biological tests appropriate to answering the risk
issues defined previously.
• Perform necessary pretreatment of samples and the biological
tests.
Some of these steps may be repeated based on biological test results.
For example, containment site boundaries might be extended because protocol
results show that soil and ground water samples beyond the defined boundary
are excessively contaminated.
-------
BIOASSESSMENT PROTOCOL
PERSPECTIVE
Hazardous wastes have been characterized as wastes from a list of
specific industries (Table 1), wastes containing one or more components on
the priority pollutants list (Table 2), or a waste that is ignitable,
corrosive, reactive, or toxic. Also, wastes are hazardous if they exhibit
chemical measurements of a specified leachate that exceed the national
drinking water standards by a factor of 100 or more (Table 3). Concepts and
controversy relating to the characterization of hazardous wastes have been
discussed elsewhere (Anonymous, 1981). The tests described in this protocol
will be useful in refining these definitions.
To gain more perspective on the purpose of bioassessment, it is
instructive to ask which potential hazards are not being assessed with
biological tests. The bioassessment protocol only addresses the toxicity
issue. In addition, volatile materials will probably not be assessed
accurately. The problem of bioaccumuTation is not evaluated at present.
Thus, research is needed to develop procedures to evaluate volatile
materials and bioaccumulation. Chronic toxicity, carcinogenesis,
mutagenesis, and teratogenesis are not assessed using the bioassessment
protocol. A Level 2 protocol could be defined for assessing these hazards.
Although the bioassessment protocol does not measure alT facets of
hazardous waste site problems, it does provide a measure of those factors
that directly affect environmental processes, i.e. acute toxicity. In this
-------
Table 1
EPA PRIMARY INDUSTRY CATEGORIES
CO
Adheslves and sealants
Aluminum forming
Auto and other laundries
Battery manufacturing
Coal mining
Coil coating
Copper forming
Electric and electronic components
1 Electroplating
Explosives manufacturing
Foundries
Gum and wood chemicals
Inorganic chemicals manufacturing
Iron and steel manufacturing
Leather tanning and finishing
Mechanical products manufacturing
Nonferrous metals manufacturing
Ore mining
Organic chemicals manufacturing
Paint and ink formulation
Pesticides
Petroleum refining
Pharmaceutical preparations
Photographic equipment and supplies
Plastic and synthetic materials
manufacturing
Plastic processing
Porcelain enameling
Printing and publishing
Pulp and paperboard mills
Rubber processing
Soap and detergent manufacturing
Steam electric power plants
Textile mills
Timber products processing
-------
Table 2
ERA'S PRIORITY TOXIC POLLUTANTS
10.
11.
12.
13.
14.
15.
16.
17.
IB.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
•acenaphthene
•acrolein
•acrylonltrile
•benzene
•benzidine
•carbon tetrachlorlde
(let rachloromethane)
•chlorinated benzenes (other than dich-
lorobenzlenes)
chlorobenzene
1,2,3-trlchlorobeinzene
hexachlorobenzene
•chlorinated ethanes (including 1,2-dich-
loroethane, 1,1.1-trlchlorocthane and
hexachloroethane)
l,2-d1chloroethane
1,1.1-trichloroethane
hexachloroethane
1,1-dichloroethane
1,1,2-trfchloroethane
1.1.2,2-tetrachloroethane
chloroethane
•chloroaUyl ethers (chloronethyl, chlo-
roethyl and mixed ethers)
bis(chloromethyl) ether
bis(2-chloroethyl) ether
2-chloroethyl vinyl ether (nixed)
•chlorinated naphthalene
2-chloronaphthalene
•chlorinated phenols (other than those
listed elsewhere; includes trlchloro-
phenols and chlorinated cresols)
2,4,6-trichlorophenol
parachlorometa cresol
•chloroform (tr)chloromethane)
•2-chlorophenol
•dichlorobenzenes
1,2-dichlorobenzene
1,3-dichlorobenzene
1,4-dichlorobenzene
•dlchlprobenzldine
3,3-dichlorobenzidine
•dichloroethylenes (1,1-dichloroethylene
and 1,2-dichloroethylene)
. 1.1-dichloroethylene
1,2-trans-dichloroethylene
•1,3-dichlorophenol
•djchloropropane and dichloropropene
oprc
Ehh
1,2-dichloropropane
1,2-dichloropropylene (1,3-dlchloro-
propene)
•2,4-dimethylphenol
•dinitrotoluene
2,4-dinitrotoluene
2,6-dinitrotoluene
•1.2-diphenylhydrazine
•ethyl benzene
39. *fluoranthene S3.
•haloethers (other than those listed else-
where)84.
40. 4-chlorophenyl phenyl ether 85.
41. 4-bromophenyl phenyl ether 86.
42. bts(2-chloroisopropyl) ether 87.
43. bis(Z-chloroethoxy) methane 88.
•halomethanes (other than those listed
elsewhere) 89.
44. methylene chloride (dichloromethane) 90.
45. methyl chloride (chloromethane) 91.
46. methyl bromide (bromomethane)
47. bromoform (trtbromomethane)
48. dichlorobromomethane 92.
49. trlchlorofluoromethane 93.
50. dichlorodifluoromethane 94.
51. chlorodtbromomethane
52. 'hexachlorobutadlene 95.
53. *hexach1orocyc1opentad1ene 96.
54. *1sophorone 97.
55. 'naphthalene
56. 'nitrobenzene 98.
•nltrophenols (Including 2,4- 99.
dinitrophenol and dinltrocresol)
57. 2-nitrophenol 100.
58. 4-nitrophenol 101.
59. 2.4-dinitrophenol
60. 4,6-dinItro-o-cresol 102.
*nitrosamines 103.
61. N-n1trosod1methylamine 104.
62. N-nltrosodiphenylamlne 105.
63. N-nitrosodi-n-propylaminc
64. *pentachlorophenol 106.
65. 'phenol 107.
•phthalate esters 108.
66. bis(2-ethylhexy1) phthalate 109.
67. butyl benzyl phthalate 110.
68. dl-n-butyl phthalate 111.
69. dl-n-octyl phthalate 112.
70. dlethyl phthalate 113.
71. dimethyl phthalate 114.
•polynuclear aromatic hydrocarbons 115.
72. benzo(a)anthracene (1.2-benzanthra- 116.
cene) 117.
73. benzo(a)pyrene (3,4-benzopyrene) 118.
74. 3.4-benzofluoranthene 119.
75. benzo(k)f!uoranthane (11,12-benzofluor- 120.
anthene 121.
76. chrysene 122.
77. acenaphthylene 123.
78. anthracene 124.
79. benzo(ghi)perylene (1,12-benzopery- 125.
lene) 126.
80. fluorene 127.
81. phenanthrene 128.
82. dibenzo (a,h| anthracene (1,2,5.6-dibenz- 129.
anthracene)
tndeno (l,2.3-cd)pyrtni (2.3-o-phenyt-
enepyrene)
pyrene
•tetrachloroethylene
•toluene
•trichloroethylene
•vinyl chloride (chloroethylene)
pesticides and metabolites
•aldrin
•dieldrtn
•chlordane (technical mixture S meta-
bolltes)
•DOT and metabolites
4.4'-DDT
4.4'-DDE (p.p'-ODX)
4,4'-DDD (p.p'-TDE)
•endosulfan and metabolites
a-endosulfan-Alpha
b-endosulfan-Beta
endosulfan sulfate
•endrln and metabolites
endrln
endrln aldehyde
•heptachlpr and metabolites
hepUchlor
heptachlor epoxtde
•hexachloroeyclohexane (all isomers)
a-BHC-Alpha
b-BHC-Beta
r-BHC (lindane) -Gamna
g-BHC-Delta
•polychlorinated biphenyls (PCB's)
PCB-1242 (Arochlor 1242)
PCB-1254 (Arochlor 1254)
PCB-1221 (Arochlor 1221
PCB-1232 (Arochlor 1232
PCS-1248 (Arochlor 1248
PCB-1260 (Arochlor 1260
PCB-1016 (Arochlor 1016)
•toxaphene
•antimony (total)
•arsenic (total)
•asbestos (fibrous)
•beryllium (total)
•cadmium (total)
•chromium (total)
•copper (total)
•cyanide (total)
•lead (total)
•mercury (total)
•nickel (total)
•selenium (total)
•silver (total)
•thallium (total)
•zinc (total)
•*2,3.7,8-tetrachlorod1benzo-p-diox1n
(TCDD)
Specific compounds and chemical classes listed In the NRDC
consent decree and referenced in the Clean Hater Act.
** This compound Mas specifically listed In the consent decree; however,
due to Its extreme toxfclty EPA recommends that laboratories not ac-
quire an analytical standard for this compound.
-------
Table 3
PRIMARY DRINKING WATER- DUALITY STANDARDS
Annul! Average
Maximum Daily
Air Temperature Maximum
Parameters V. • ^C Level*
Inorganic Chemicals
Arsenic 0.05
Barium 1.
Cadmium 0.010
Chromium 0.05
Lead 0.05
Mercury 0.002
Nitrate (as N) 10.
Selenium 0.01
Silver 0.05
Fluoride
53.7 and below 12.0 and below 2.4
53.8 to 58.3 12.1 to 14.6 2.2
58.4 to 63.8 14.7 to 17.6 2.0
63.9 to 70.6 17.7 to 21.4 1.8
70.7 to 79.2 21.5 to 26.2 1.6
79.3 to 90.5 26.3 to 32.5 1.4
Chlorinated Hydrocarbons
Endrin (1, 2. 3, 4. 10, 10-hexachloro-6. 7-epoxy-l, 4, 4i. 5 0.0002
6, 7, 8, Sa-octahydro-l, 4-endo-5, 8-dimethano naphthalene)
Lindane (1, 2. 3. 4. 5, 6-hexachlorocyclohexane, gamma 1 sorer) 0.004
Methoxychlor (1. 1. l-Tr1chloroethane) 1. 1-bts (p-methoxyphenyl) 0.1
Toxaphene (C..H1nCl,-Techn1ca1 chlorinated camphene, 67-68 percent 0.005
chlorine) lu lu °
Chlcrophenoxys: 2.4-D, (2, 4-D1chlorophenoxyacet1c acid) 0.1
2. 4. 5-TP Silvex (2, 4, 5-Trichlorophenoxy- 0.01
propionic acid)
b
Turbidity (for surface water sources) 1 TU up to 5 TU
Coliform Bacteria
Membrane filter technique: 1/100 ml mean/month
4/100 ml in one sample if <20 samples/month
4/100 ml in more than 51 if >20 samples/month
Fermentation tube with 10 ml portions: no conforms In lot of portions/month
no conforms In >3 portions/sample if <20 samples/month
no conforms in >3 portions of 51 of samples if >20 samples/month
Fermentation tube with 100 ml portions: no col 1 form bacteria in >601 of portions/month
no conform in 5 portions in one sample 1f <5 samples/month
no conform in 5 portions in 20* of samples if >5 samples/month
Radioactive Material ' Level
Combined radium 226 and radium 228 5 pCi/1
Gross alpha particle activ1tyc 15 pC1/1
Beta particle and photon radioactivity from man-nade radionuclides 4 •illirem/year
Tritium for total body 20,000 pCi/1
Strontium-90 in bone narrow 8 pC1/l
"mg/1 unless otherwise stated.
blf meet special requirement.
Includes Ra226 excludes Radon, Uranium.
Source USEPA (1977)
10
-------
regard the biological tests are useful for assessing the potential
ecological hazard of hazardous wastes. Ignitability, corrosivity and
reactivity have impacts that are catastrophic and overshadow long-range
environmental concerns, and therefore, require more immediate action.
However, the bioassessment tests provide responses to the toxicity of such
materials.
An accurate perspective on bioassessment tests requires consideration
of the role of chemical analysis and field ecological studies in evaluating
hazardous waste sites. Chemical analysis is important for actual cleanup
procedures and for evaluating the fate and effects of materials from
hazardous waste sites. An extensive list of chemicals such as those in
Table 2 and 3 can be measured in water, soil, and soil leachate samples.
The measured concentrations can be compared to standards or criteria
extrapolated from laboratory bioassay procedures. Then, appropriate actions
are taken according to published regulations; however, considerable
uncertainty remains. The decision maker is never certain whether all toxic
chemicals are on the list, whether they are measured adequately, or whether
the mixture exerts different toxicity than the sum of the individual
chemicals. Also, not all of the chemicals are equally available to
organisms under a particular set of conditions. Although chemical results
are administratively easy to use, they are not as meaningful in an
ecological context as biological measurements. Although the relative
precision of biological tests compared to chemical tests is often
questioned, biological tests have teen found to be as precise -tfs chemical
tests if proper procedures are followed. Moreover, the "question of
precision is less important than accuracy, that is, integrating the effects
and bioavailability of compounds to organisms.
11
-------
The most direct approach for evaluating the ecological hazard of an
hazardous waste site is to observe effects on the ecosystem by field
studies. However, several disadvantages for this approach exist. To
observe effects, damage to the ecosystem must have occurred already, and
that is what should be avoided. In addition, field ecological studies are
often expensive and time consuming, are not predictive and in fact are
retrospective. On the other hand, field studies are necessary for many
purposes because they directly measure ecological harm.
We conclude from the above discussion that biological tests are
necessary for evaluating potential ecological effects of hazardous waste
sites. However, the biological tests must undergo testing by comparing
biological test results to chemical and field study data. This process is
being conducted by the U.S. EPA, Corvallis Environmental Research
Laboratory, Oregon (Figure 1). It is expected that others will use the
tests and report their results. As experience is gained in applying the
bioassessment protocol, it will be improved.
For regulatory reasons, the bioassessment protocol cannot supplant all
chemical and field data. Many of these data will be available at the
initiation of site studies or are obtained by observation and measurement at
that time. Coordination of this information with the bioassessment protocol
results is an important need in protocol development.
As an example of how the bioassessment protocol can be - -incorporated
into assessment of a hazardous waste site, we have included exderpts from a
site response management plan from USEPA's Region IV. The following
discussion is taken directly from Mathis (1981) with minor modification
12
-------
Fate and Transport of
Chemical Constituents
Field Evaluation
and Assessment
Data Base:
Waste Materials
Management Practices
Geochemlcal Mitigation
Ecological Studies
Bioassay Tests
Level 1 Bloassays*
of Complex Hazardous
Wastes in Terrestrial
and Aquatic Systems
I
Environmental Risk
Assessment Procedure
Decision Criteria and
Technical Assistance:
• Siting
• Cleanup Alternatives
t Protection of Critical
Environments
• Extent of Cleanup
Required
Application of Bloassessment Protocol.
Figure 1. Evaluation of Environmental Testing Protocols for Hazardous Waste Assessment
(Peterson, 1982)
-------
including provision for applying the bioassessment protocol. The EPA Site
Tracking System is portrayed in a flow chart (Figure 2). Circled numbers in
the flow chart correspond approximately to the numbered sections in the
text.
A principal investigator with responsibility for site evaluation is
assigned. The major milestones are as follows:
1. SITE_IDENIIF1CATION This represents the entry of a potential
into the system, and may be initiated
many methods of information gathering.
is that in the absence of an affirmative
al1 reported
uncontrolled site
through any of the
General guidance
showing that no hazardous material is involved,
potential sites will be entered into the system.
PRELIMINARY ASSESSMENT At this point the investigator
completes a search of available files in federal and state
agencies, usually accomplished by telephone contact outside
the agency, and will also complete telephone interviews with
identified persons having knowledge of this site. The purpose
of this action is to discern possible releases which would
require emergency containment action to mitigate an imminent
hazard to health or the environment, with immediate
investigation (response time in hours); and to permit a
subjective evaluation of the degree of hazard of other sites
as HIGH MEDIUM, LOW, NONE, or UNKNOWN. This judgment becomes
the basis for prioritizing the site for the next level of
investigation. Resources expended at this point generally
range from 0.5 to 1.5 person-days.
SITE INSPECTION This activity involves a visit to the site by
a team of at least two investigators. Their function is to
observe the potential site prior to entry, assess risk for
site entry, interview knowledgeable indigenous personnel,
appraise the population at risk, identify potential exposure
routes, and if justified, enter the site to observe and
subjectively evaluate topography, geology, quantity and type
of material present, conditions of storage or disposal,
evidence or probability of release or migration, and resources
needed to quantify or objectively measure these parameters.
Due to safety considerations, no sampling is conducted at this
time; however, considerable information is collected and
recorded in the form'- of observations and photographs.
Resources expended in this activity range from 2.0 -to 4.0
work-days exclusive of travel. The purpose of this activity
is to produce a more certain evaluation of the potential
hazard as HIGH, MEDIUM, LOW, or NONE, to prioritize the site
for field investigation as needed and to permit preparation of
a TENTATIVE DISPOSITION.
14
-------
en
I
-W Implementation
Figure 2. Site Response Management Plan (from Mathis, 1981),
Approximately Correspond to Text Numbers.
Circled numbers
-------
4. TENTATIVE DISPOSITION This activity comprises a decision
pointfromwhichthe site is tentatively classified as most
appropriate for one of four courses of action. The decision
is recommended by the principal investigator, and is reviewed
by appropriate section leaders for concurrance. Basis for
this recommended decision is a review of all assembled
information on the site. Resources required are approximately
1.0 work-days plus review. The four alternative decisions
possible are:
a. Enforcement Action by State or Federal Agency.
This implies that a viable defendant and an imminent
hazard are both present.
b. Remedial Action using Federal, State or Other Resources.
This implies that either a responsible party may be
willing to undertake necessary action, or that no viable
defendant is apparent and direct action using the
authority of CERCLA seems appropriate. It also implies
that an imminent hazard is present.
c. Further Investigation Needed.
This indicates that collection of field data through
sampling, geophysical studies, the bioassessment
protocol, or other means is required to ascertain the
presence or absence of an imminent hazard, or to quantify
and delineate the extent of that hazard. This mandates a
resource-intensive investigation, and requires a review
to set the priority for this effort. Prioritization may
be aided by an initial preliminary assessment using the
bioassessment protocol.
d. No Further Action Required.
This implies that uncontrolled hazardous material is not
present at this time, and no significant hazard exists.
5. FINAL STRATEGY DETERMINATION This Activity represents the
coordinated timetable for a recommended Enforcement Action or
Remedial Action, the timetable for the required
investigations, or the final concurrance in a finding of No
Further Action Required. Final Strategy Determinations are
tracked, and are amended as progress is made on the respective
timetable. When a Final Strategy Determination of No Further
Action is reached the site may be placed in the inactive file
of the system. Resource requirements in this stage are highly
variable. The effectiveness of cleanup procedures can be
evaluated using the bioassessment protocol. It is possible
that litigation may take several years to complete, at great
- cost, and remedial measures may entail costs in the millions.
Extensive investigation effort may cost hundreds of thousands
of dollars and involve many months of effort. In contrast, a
determination of no further action may be processed in a
single work-day.
It is noteworthy that at each milestone in the process there
is an opportunity to reappraise the priority of each site for the
next level of activity, and that at each milestone, limited
resources are focused on those sites which are most significant in
terms of:
16
-------
1. Seriousness of hazard to health.
2. Seriousness of hazard to the environment.
3. Presence of a viable defendant or responsible party.
4. Existence of a technically feasible remedy.
5. Availability of uncommitted and appropriate resources.
6. Other factors which tend to raise priority; EG: State
Opinion.
Generally, as a high priority site advances through the
process, increasing amounts of money and resources are required.
This situation has resulted in the identification of several
rate-limiting steps in the processing of potential uncontrolled
hazardous material sites.
The first of these limitations is the availability of
analytical support for the program. Analytical capability, and
the associated quality assurance support, is a finite resource due
to the high cost, and is also finite in terms of physical
capacity. Furthermore, the pursuit of Enforcement Options places
an even greater demand on this resource than investigation or
remedial design, due to the need for elaborate amounts of data of
unimpeachable quality. It may also be projected that where
remedial actions are conducted in anticipation of a possible legal
action to recover costs, documenting the findings, the action
taken, and the benefits accrued thereby^ will all necessitate a
significant increase in analytical effort beyond that minimum
necessary to design and implement the remedy. Proper application
of the bioassessment protocol may allow better definition of the
priority of a site and thus minimize the number of sites requiring
such detail.
The second limitation which has emerged is the need for
intensive expenditures of manpower to perform on-site tasks in a
safe manner. To approach a totally unknown site in what is
generally agreed to be a conservative, safety conscious method
requires a team of 5 to 6 persons in order to cope with all
contingencies. Furthermore, this team requires elaborate and
costly equipment to detect possible agents in real time, while
providing protection against a wide spectrum of toxicants. This
equipment, in turn, requires recurrent training of personnel and
periodic maintenance if it is to be used effectively. The
bioassessment protocol could provide an excellent solution for
this problem by showing those sites and/or locations where acute
biological risks are present.
Finally, the team and its special equipment are not readily
transportable by many commercial carriers when prepared for
operation. While many regulatory agencies and private
laboratories acknowledge similar safety criteria for' site
investigation, very few of them are staffed or funded to allow
operation in accordance with recommended procedures. ' Thus,
specialized contractors may often be the most feasible choice for
site sampling. Although the bioassessment protocol may not be
directly applicable here, it can be used to minimize needs for
outside contractors by a prioritization of sites based on
biological responses.
17
-------
APPLICATION OF BIOASSESSMENT PROTOCOL
At least three scenarios regarding hazardous waste sites in relation to
the potential application of bioassessment can be defined:
• The site is uncharacterized and unprioritized. In this case
an assessment of potential hazard to site workers,
neighborhood public, downstream users, and the natural
ecological community is needed. First, the Hazard Ranking
System (Federal Register, 47 FR 10972, March 12, 1982) should
be applied (e.g., Caldwell et.al_., 1981) and then, if the
potential hazard is high enough, a rapid physical-chemical
screening approach should be taken (e.g. Turpin et_al_., 1981).
Other data would be obtained as discussed in the previous
section (Mathis, 1981). The bioassessment protocol would be
used to provide an initial assessment of acute toxicity and to
prioritize the site relative to other sites.
• The site is characterized but the extent of contamination is
unknown. In this case the waste site is not well defined and
it is probable that the bioassessment protocol would be used
to screen for the extent of contamination and provide input to
the experimental design for more detailed assessment. -
• The site is characterized and cleanup or other remedial action
is being taken. In this case bioassessment would provide an
18
-------
estimate of remaining hazard, monitoring of incipient hazard,
and would help in establishing and monitoring the boundaries
of a required containment zone. Criteria related to
bioassessment results would be based on samples collected at
the boundaries of the containment zone or, for groundwater, as
appropriate to projecting the effects of flow beyond the
containment zone boundaries.
The first and second scenarios lead to the site priority or screening
line of the bioassessment protocol. These data are needed before proceeding
to the more detailed assessment. The screening assessment has the intent of
providing a rapid survey of potential problems. The third scenario leads to
the detailed assessment. The key step is to develop a sampling design
appropriate to the site and potential hazards of the site.
Samples are collected at essentially three stations for the preliminary
assessment: The core station (most impacted station), the site containment
boundary, and a reference station (off site). Appropriate surface or ground
water and soil samples are collected at each site for bioassessment.
Bioassessment results for the core and boundary stations are compared to the
reference site. Three replicate samples, randomly collected at each site,
are composited for bioassessment.
Detailed assessment consists of transects and multiple stations
designed to evaluate questions concerning the spread of toxic materials and
the relative hazard associated with the samples. Generally, the transect
would begin at the core station. Multiple stations along the containment
boundary would be required. Optimum allocation of manpower and laboratory
19
-------
resources must be based on the Experimental Design and related concepts as
discussed under that heading.
Response levels for each organism: high, intermediate, low or none,
can be obtained for each sample. Points can be assigned for the response
levels, summed, and priorities assigned according to relative biological
hazard. Then, if appropriate, the sites can be further ranked according to
other risk criteria having to do with fate and human effects considerations.
BIOLOGICAL TESTS FOR THE BIOASSESSMENT PROTOCOL
Two types of samples are tested, water and soil. Water samples are
collected from surface and ground water sites or obtained as extracts from
soil samples. Soil samples are collected from appropriate sites using grab
sample apparatus. Soil core samples are not required but may be used.
Descriptive data, methods of collection, and sample treatment and storage
requirements for water and soil samples are described in the sampling
chapter.
Organisms for use with water samples include algae, daphnids, and fish.
Organisms for use with soil samples include decomposers, plant seeds and
earthworms. These tests are summarized in Table 4, along with the target
variables, appropriate sample type, and the response levels. These response
levels are provisional at this time and provide guidance on tHe relative
hazard associated with a given sample. The response levels will be refined
as more data are obtained. Results are reported as actual measured EC™ or
LCj-g and then the samples are categorized into the appropriate response
20
-------
t\J
Table 4
DEFINITION OF TOXICITY CATEGORIES FOR AQUATIC AND TERRESTRIAL ECOLOGICAL ASSAYS
Response Levels for LC™ or EC™ Concentrations'
Assay
Freshwater
Fish
Freshwater
Invertebrate
Freshwater
Algae
Activity Measured
96-hr LC5Q
(lethality)
48-hr EC,n
DU
(immobilization)
96-hr EC5Q
f nffltalfh inh4 hi -Hi-inA
oatuf i e
Type3
S
L
S
L
S
L
MADb
1
100
1
100
1
100
Units
9/L
percent
g/L
percent
g/L
percent
High
<0.01
<20
<0.01
<20
<0.01
<20
Moderate
0.01-0.1
20-75
0.01-0.1
20-75
0.1-0.1
20-75
Low or Not Detectable
0.1-1
75-100
0.1-1
75-100
0.1-1
75-100
Seed Germination and
Root Elongation
Earthworm Test
115-hr EC5Q
(inhibit root
elongation)
100 percent <20
20-75
75-100
336-hr LC
50
500 g/kg
<50
Soil Respiration Test 336-hr EC
50
S
L
500 g/kg <50
100 percent <20
50-500
50-500
20-75
500
500
75-100
S = solid, L = aqueous liquid, includes water samples and elutiate or leachate. Nonaqueous liquids are evaluated
on an individual basis due to variations in samples such as vehicle, percent organic vehicle, and percent solids.
MAD = Maximum applicable dose.
LCrQ = Calculated concentration expected to kill 50 percent of population within the specified time interval.
ECgg = Calculated concentration expected to produce effect in 50 percent of population within the specified time
interval.
-------
level category.
The tests included in the bioassessment protocol were selected based on
the validity of tests and their feasibility for use. Validity factors are
the accuracy, precision, and sensitivity (precision at low concentration) of
a test. Accuracy concerns how well the results can be extrapolated to a
natural system. Precision and sensitivity are statistical factors and vary
with the organisms and the typefs) of toxicants. Feasibility factors
concern the cost of the test, the degree of expertise needed by the
operator, the convenience of performing the test, and the speed with which a
result is obtained. Although these data are available for some of the
tests, ongoing research will provide a more complete compilation of these
data. This information will be necessary in order to design sampling
programs with the optimal allocation of sampling and testing resources.
POTENTIAL BIOASSESSMENT METHODS
A matrix of general organism types and major physiological processes
versus soil and water is shown in Figure 3. The protocol methods cover a
wide spectrum of types and processes but there are obvious gaps. An
important gap, which is not shown, concerns bioaccumulation. Since
bioaccumulation requires a long-term exposure, it is not included in the
protocol. A possible research effort on octanolrwater coefficients related
to microbial responses, or direct fliicrobial bioaccumulation tests" could be
useful for assessing bioaccumulation.
22
-------
Organism
Type
Physiological
Process
Laboratory Biological Test6
Soil
Water1
Plant
Invertebrate
Vertebrate
Decomposer
Photosynthesis
Growth
Genetic
Respiration
Lethality
Growth
Genetic
Respiration
Lethality
Growth
Genetic
Enzyme
Growth
Genetic
Germination, Root Elongation
(Tradescantia)
Earthworm
(Earthworm)
Litter Decomposition
(Ames Test)
Selenastrum
Daphnids
Fathead Minnow
(Microtox)
(Ames Test)
Parentheses indicate test needs more study before broad use; dash
indicates no candidate test; otherwise the tests are in the protocol,
Appendix A.
Water includes surface and ground water samples or soil leachates.
Figure 3. Matrix of Tests as Related to Types of Organisms and
Physiological Processes-.
23
-------
Although some specific processes, photosynthesis and respiration, are
not included in the protocol, it would be feasible to develop rapid tests
for evaluating them. Other gaps such as a vertebrate soil test should be
disregarded because the benefits are small compared to the costs of
developing and performing such tests. Existing tests such as mutagenetic
response of the plant Tradescantia, the Ames test, and Microtox (Patent
Pending) may have application as part of a protocol but more development of
these techniques is required. Microbial enzyme tests exist that are rapid,
inexpensive, and may provide ecologically accurate and meaningful results.
Adaptation of these tests to evaluate hazardous waste sites could be a
cost-effective study goal.
24
-------
EXPERIMENTAL DESIGN
SCOPE
The sampling program must be designed so that answers to the two
issues—site prioritization and cleanup evaluation—are obtained. These
issues can be phrased as questions: In terms of relative toxicity how does
the site rank compared to other sites in the state for other defined
region)? Are levels of toxic materials in soil or water samples
sufficiently low that potential environmental hazard beyond the site
boundary is minimal?
To begin answering these questions, risk, statistical design, sampling
constraints, and the characteristics of the tests themselves must be
considered. Each of these topics will be briefly discussed before providing
a step-by-step sampling protocol.
RISK ASSESSMENT
Some element of risk assessment is involved in all decision making. To
evaluate the potential risk to ecosystems from hazardous waste sites, a
basic understanding of the concepts of risk assessment can help in defining
the issues and significance of the1 method. In this section,-" the major
concepts of risk assessment are discussed in general and then addressed
specifically to the bioassessment protocol.
25
-------
There are three broad steps in makinq a risk assessment: analyzing the
system, determining the "dose-response" relation, and integrating these
factors to estimate the risk. Note that defining the level of acceptable
risk is essentially a political decision. Acceptable risk may be more a
question of perception. Thus, decisions may be left either to the
individual, for example, cigarette smoking, or to society, for example,
nuclear radiation.
In analyzing the hazardous waste system, the composition and quantity
of material released to the environment need to be estimated. Then,
transport and migration should be defined for materials including any
environmental transformations of compounds that would occur under the
specific conditions at the site.
The dose-response relationship refers to the concept of relating some
response to the concentration of material and, in most cases, the duration
of exposure. A target variable must first be defined that bears a
functional relationship to exposure. This is a key step since knowing the
amount of material that is present is not meaningful without knowing that
there is an effect, that a quantitative relation exists between
concentration and effect, and that the effect is important ecologically and
on a significant scale. The biological tests play an important role in
developing these relationships.
After the above two steps are1 accomplished within reasonable" confidence
limits, the risk can be assessed by determining the quantity o'f material,
the exposure to key parts of the system, and estimating the risk using the
concentration-duration-effect relationship. This integration step requires
26
-------
many assumptions which must be clearly identified. For example, extension
of results to low doses is difficult, inaccurate, and for statistical
reasons, most costly to obtain. Thus, extrapolation is often practiced, and
it may be based on assumptions that are incorrect.
The bioassessment protocol as it presently exists in this report does
not deal explicitly with specific hazardous materials, their quantity, fate,
or transformation. Instead, the biological tests measure responses to the
mixture of materials obtained in a sample. The responses are directed at
the concentrations available to organisms and at the mixture which may be
different than the sum of the individual effects because of interactions.
By analyzing transect samples, a response gradient can be determined that
could be related to transport and migration processes.
The biological tests were developed using a concentration-duration-
effect relationship (for example, Figure 4). Initially, this relationship
was developed using one toxicant at a time. However, studies using multiple
pollutants and complex waste mixtures as in Figure 4 have shown the
concentration-duration-effect concept to exist. Thus, the concept of using
dilutions of a complex waste in a synthetic or natural receiving medium
appears valid.
There is one other issue of risk that must be considered. What are the
effects of error? Two kinds of error exist; saying that there is a certain
level of risk when in fact there is none and saying that there is none, when
in fact a risk exists. In the first case, significant ' unnecessary
expenditure might occur. In the second, considerable environmental damage
might occur. It is important to evaluate both errors in a cost-benefit
27
-------
i-
B _
t
0)
u
Q)
o_
48 hour
exposure
4-
24 hour exposure
-a
I I I I
0 20 M0 60
Percent Effluent
B0
100
Figure 4. Concentration-duration Results From an Acute Toxicity
Test Exposing Mysid Shrimp to a Simulated Refinery
Effluent (data from Buikema, et a]_., 1982)
28
-------
manner before defining acceptable risk. For example, if there is
significant additional cleanup cost with little environmental benefit, it
may be possible to accept a higher level of test response at the containment
boundary. Conversely, the no-effect risk level might be appropriate for a
cleanup cost associated with high hazard. These questions must be explored
in more detail at specific sites.
The variance in the biological test results needs to be known as well
as the variance in sampling. This information is used to allocate sampling
and test resources. Without proper design, money is wasted and proper
results may not be obtained. These factors are discussed qualitatively in
the next section.
Some general approaches to risk analysis that provide further
information on risk assessment have recently been discussed (Ricci and
Molton, 1981; Starr and Whipple, 1980; Squire, 1981). A detailed review
of the literature on carcinogenic risk assessment is contained in Krewski
and Brown (1981) which has general applicability in the context of this
report.
29
-------
STATISTICAL CONSIDERATIONS AND EXPERIMENTAL DESIGN
The goal of using bioassessment procedures as a tool for the evaluation
of the potential environmental hazards among existing sites is to detect
differences within and among sites and to distinguish among groups of sites
on the basis of this potential. To achieve these objectives, appropriate
test procedures must be identified and experiments must be designed in such
a manner that differences in the test results, which reflect the potential
hazards, will be identified. Three main categories of statistical
considerations which relate to these requirements are discussed herein.
These are: the location of sample collection sites, experimental procedure
specifications, and analytical techniques. Although discussed separately,
these topics are not independent and decisions concerning each of these
aspects affect the options available in the other levels of the sampling
design.
The selection of sampling locations within the individual hazardous
waste sites will be a function of the type of biological test to be
performed, as well as uniformity of the site with respect to edaphic
characteristics and habitat type. It is known that the relationship between
hazardous waste concentrations and morphological, chemical and biological
sampling area determinants will vary widely within the sampling sites. Yet
at the same time, it is essential to the overall study design for the
comparison among sites, that these variations be kept at a minimum. In this
manner the effect of the individua-1 hazardous waste sites is isolated and
can be identified.
30
-------
The feasibility of two procedures which will minimize the influence of
within site variability on the estimated site effect should be evaluated.
The first is the characterization of sample site-sample type relationships.
The ability to specify sampling site selection criteria based on
morphological, edaphic or biological characteristics for each proposed type
of biological sample should be addressed. Certainly, tradeoffs must be made
in the process of the specification of sampling area characteristics. This
is because the very criteria which narrow the possibilities of sampling
locations within waste sites can, at the same time, restrict the number of
wastes sites throughout the United States where the biological test can be
applied. However, for the purpose of reducing the sample variance within
sites and equilibrating sample variances among sites, site-type
characterization must be discussed.
Secondly, the feasibility of random sampling within the acceptable
sampling area should be addressed. Random sampling is essential to the
assumptions of many appropriate analytical techniques and, as discussed
below, the larger the number of replicates the greater is the ability to
distinguish among differences in the target variable or variables among
sampling (hazardous waste) sites. In the overall study design, a stratified
random sampling plan should be adopted with the strata identified on the
basis of the set of descriptive criteria discussed above and random sampling
within .the strata.
Related to the question of the selection of sample location within
individual waste sites is the specification of control samples. Each
bioassessment procedure selected should be conducted with control or
reference stations at each site. These samples will provide the means to
31
-------
assess the effects of the hazardous waste within the sites and will serve as
quality control checks for the bioassessment program. Procedures for
control samples (experiments) should be designed such that they can be
repeated in the same manner at each site.
The second major area of statistical considerations which must be
addressed is the appropriate level of sampling effort. Decisions on the
level of sample replication or sampling effort in general cannot be made
independently of careful consideration of the minimum level of difference in
selected biological parameters that it is desired to detect and the
precision with which differences should be detected. It is crucial to
carefully plan field experiments in order to define these levels, to
establish the number of samples required, and to specify the appropriate
analytical approach.
Taking the analysis of variance (ANOVA) technique as an example of the
type of statistical technique that might be used to identify differences in
the mean values of specified measurements among the waste sites, sampling
specifications and their implications to the overall study objectives will
be discussed in the following paragraphs.
In considering the use of the ANOVA model, two significant criteria are
specified. These are the probability of rejecting the null hypothesis when
it is true (alpha probability, or Type I error) and the probability of
accepting a null hypothesis when i-t is false (beta probability,-"or Type II
error). Respectively, these errors are the probability of concluding there
is a risk when no risk exists (Type I) and no risk when a risk does exist
(Type II).
32
-------
Results of statistical tests are often summarized by stating that no
significant difference among stations was found at the 0.05 significance
level. This refers to the alpha probability criterion which embodies the
risk of mistakenly rejecting a null hypothesis that no differences exist.
At a particular site having low potential for harm, one might select a less
restrictive alpha criterion to minimize costs. The significance level of
the beta parameter should also be specified, especially in the case of
comparisons to determine the relative environmental risk from hazardous
waste sites, because decision makers are also interested in the probability
that the test was unable to detect a difference that did exist. Another
statistical parameter, referred to as the power of a test (1-beta) is
important in this context since it defines the probability of correctly
detecting experimental effects (e.g. differences among waste sites) in a
particular bioassessment procedure.
Closer examination of the beta probability and its complement, the
power of a test, is instructive since these probabilities can be defined as
a function of sample size. In this manner, the probability of the level of
difference that can be reliably detected with alternative allocations of
sampling resources (stations and replication) can be determined.
In addition to the value of such investigations in a^ posteriori
analyses of results, the probability of detecting differences in the
selected biological parameters between waste sites and the levels of
differences which can be detected with proposed sampling designs should be
determined, and this requires that the population variance of selected
bioassessment parameters must be determined. This will be accomplished
during the ongoing research being performed by the USEPA's Corvallis
33
-------
Environmental Research Laboratory.
Besides the probability criteria, consideration must be given to the
methods used to distinguish among waste sites or to categorize them
according to enviromental risk. Quantitative methods are recommended, and a
large array of parametric and nonparametric statistical techniques are
available for this purpose. As indicated above in the discussion of sample
replication, the allocation of sampling resources should be made so as to
optimize the level of difference that can be reliably detected among sites.
Before the application of the parametric models, it must be determined
if the underlying assumptions (i.e., homogeneity of variances, independence,
and normality) can be met. In the case that the assumptions of parametric
tests cannot be maintained by data transformations, nonparametric tests can
be substituted. Although nonparametric tests lack the distributional
assumptions of parametric tests, they have less power and they lack the
ability to evaluate interactive effects.
The analysis of variance (ANOVA) is an example of a parametric
statistical model which can be used to make univariate comparisons among
waste-site samples. The purpose of the statistical model is to estimate
true differences among the sample means. To describe possible effects of
the waste site, a simple linear model is proposed by which any single
observation can be decomposed as follows:
ai
34
-------
where:
Y = biological observation for site i and replicate j
u - mean value over all sites and relicates
Of = effect of site i on Y
£4.5 = random deviation of the observation from its expectation
' J
' U+ £•()•
The ANOVA is used to test the hypothesis that there is no difference in
the biological observations made at different sites, i.e. that differences
do not exist in the component due to site location (a. = a2 =....= an).
In cases where significant differences are found to exist among
sampling sites, multiple range tests (such as the Student-Neumann-KeuIs or
Duncan's) can be used to identify subsets of samples (sites) having equal
mean values for the variable under examination. An example of the use of a
multiple range test to demonstrate treatment differences among algal
bioassays and to identify subsets having equivalent mean values is shown in
Table 5.
In conclusion, decisions concerning the methods which will be used to
distinguish among the waste sites sampled must be made in advance of the
adoption of any bloassessment procedure. In this manner the bioassessment
program can be optimized in order to provide the level of precision to
assess potential hazards for the nvinimum costs. -<~
35
-------
00
Table 5
DUNCAN'S MULTIPLE RANGE TEST OF COMPLEX ADDITIONS
TO SCENEDESMUSa (From Cleave, et al., 1980)
IMI M«n«nf ercp, X
bricfi frMrth rM*. A»
IrMt. concn.
1 20
25 20
27 10
42
44
45
26 15
21 20
15 10
7 10
22 15
32 5
20 5
4 5
28 S
24 5
30 15
43
33 20
10 IS
6 15
9 20
23 10
31 10
3 10
39 10
5 20
14 15
8 5
12 5
2 15
41
16 . 5
38 15
29 20
35 10
13 20
19 10
18 15
37 20
36 5
17 20
34 15
40 5
11 10
"Treatments ere
other are connecte
AR elutriate
BP elutriate
BP elutriate
control
control
control
BP elutriate
BR salts
AP salts
AR salts
BR salts
BP salts
BR elutriate
AR elutriate
BP elutriate
BR salts <
BP salts * <
BR elutriate * * * •
AP column salts » » *
AP column leachate * *
BR elutriate * *
AP column leachate *
AP column salts *
AP elutriate *
ranked from the lowest value at the top of the listing to
d by a line of stars to the riqht of the ranking list.
tract.
no.
* 1
* 41
* 32
* 15
36
45
44
16
* 20
* 6
* * 31
26
18
17
24
14
27
28
to
8
35
19
38
33
39
11
22
37
2
3
the hiqhest value at the bottom of the lit
COftCft)
mL
20
5
10
5
5
5
15
10
5
5
20
20
20
15
15
20
20
20
10
10
5
IS
15
20
5
15
10
5
15
5
10
10
15
20
10
10
15
20
15
10
ting. Any
AR elutriate
control
BP salts
AP salts
AP column leachate
control
control
AP salts
BR elutriate
AR salts
BP salts
AP column salts
control
AP elutriate
BR salts
BP elutriate
AP salts
BR elutriate * * *
AP column salts * *
AP column leachate * *
AP column salts *
AP elutriate * *
BR salts * *
AP column salts *
AR elutriate *
AR elutriate *
groups of treatments that are not significantly different
*
* *
* *
* *
* *
*
(P < 0.05) from each
-------
GUIDELINES FOR SAMPLING
The primary reason for a sampling design is to allocate sampling and
testing resources in an optimal manner. In the preliminary assessment, a
minimal design of three replicate samples composited for analysis at each of
three stations (core, boundary, reference stations) is specified, obviating
the necessity for a statistical design. However, if resources permit and
potential hazard is great enough, a statistical design should be used. For
the detailed assessment, the number of stations and the number of sample
replicates are defined during statistical design.
Three types of samples are obtained: soil samples, ground water, and
surface water. A preliminary survey to observe physical-chemical-biological
factors such as surface waters and flow directions, topography, types of
habitats, site boundary conditions, aquifer locations and underground flow
nets, and other ecological variables is an invaluable aid to selecting
stations.
Each site will have specific characteristics that will cause a specific
set of stations to be selected. The most samples and stations would arise
from a situation where a groundwater aquifer underlies a disposal site,
surface water is within the site containment boundary, and the area of
direct disposal is large. Thus, the reference station, the core station,
and the site boundary station would each require three types of samples:
surface water, ground water, and soil. A total of 18 water samples and 9
soil samples would be obtained at that site using three replicate samples at
each of the three stations. In addition, extracts from the soil would be
tested. To minimize the number of tests, the samples would be composited
37
-------
for the biological testing. Such a hazardous waste site would have the
experimental design shown in Table 6.
The final selection of sampling stations depends on the user-defined
heirarchy of needed information related to the available resources. The
methods for selecting stations and criteria for defining the number of
replicates are based on the statistical analysis to be performed. Selected
Type I error (alpha) levels and the desired power of the test (1-beta) then
fix the number of stations and replicates required. Needed resources should
be based on potential risk not on a budget figure. For example, if
potential risk to ecosystems or society is relatively large or the cost of
cleanup is relatively large, greater testing resources should be made
available to insure that cleanup will be effective.
Preliminary Assessment
The preliminary assessment is used for initial prioritization and to
determine the range of probable responses to be obtained with the biological
tests. A minimum program consisting of the reference station, core (most
impacted) station, and the site containment boundary station should be
sampled. At least 3 replicate randomly selected samples should be obtained
at each station and composited for testing.
If the biological tests uniformly give low or nondetectabTe response
levels (Table 4), it is assumed that the site will be relatively risk free.
Any test that shows a response at the intermediate or high level for a
particular sample is cause for review and, probably, further analysis.
38
-------
Table 6
MINIMAL EXPERIMENTAL DESIGN SHOWING
THE MAXIMUM NUMBER OF SAMPLES (9) AND TESTS (36)
FOR PRELIMINARY ASSESSMENT OF A SITE
Tests and Samples to be Collected
Station
Reference
Core
Boundary
Water
Surface
Alga
Daphnid
Fish .
1 sample
Alga
Daphnid
Fish .
1 sample
Alga
Daphnid
Fish .
Samples
Ground
Alga
Daphnid
Fish .
1 sample
Alga
Daphnid
Fish
1 sample
Alga
Daphnid
Fish .
Soil
Soil
Seeds
Worms
Microbes.
I sample
Seeds
Worms
Microbes.
1 sample
Seeds
Worms
Microbes.
Leachates8
Alga
Daphnid
Fish
Alga
Daphnid
Fish
Alga
Daphnid
Fish
1 sample
1 sample
1 sample
Additional samples would not be collected because extracts would be
obtained from soil samples.
Composite sample from three random grab samples obtained from each
station.
39
-------
Then, the appropriate steps and decision points shown in Figure 2 would be
followed.
It may be desirable to perform a statistically based sampling program
(e.g., ANOVA) using the uncomposited samples to analyze the data. The
variance of each biological test should be known. Expected variance in a
particular biological test might be relatively high (coefficient of
variation = 50 percent). Specifying appropriate levels of alpha (0.05 to
0.2) and the power of the test (1-beta = 0.8), one might wish to determine
whether a difference could be shown at the intermediate response level
(Table 4) for the site containment boundary sample compared to a reference
station.
Detailed Assessment
Statistical methods should be used to evaluate the differences in the
results of the bioassessment tests among the sampling stations. The
analytical procedures must be specified in advance so that adequate sampling
is conducted. Guidelines for selecting sampling locations within the
individual sites should be developed. For example, attempts should be made
to characterize optimal locations for individual sample types. In this
manner sample variances will be reduced. Transects along major exposure
routes or along site containment boundaries could be used.
A pilot or feasibility study for the purpose of determining'the number
and types of samples required and optimum allocation of resources should be
performed. The preliminary assessment might serve for this purpose.
40
-------
The error levels would be more conservative than those described for
the preliminary assessment. For example, based on potential risk the
following level could be selected, alpha = 0.1. Then the sampling stations
and number of replicates would be specified according to resources needed to
characterize the site and the expected results from the biological tests.
The power of the test (1-beta) should be estimated and reported.
41
-------
REFERENCES CITED
Anonymous. 1981. Is this waste hazardous? Not always an easy answer.
Civil Engineering. Amer. Soc. Civ. Engr., N.Y. September, p 81.
Buikema, Jr., A.L., B.R. Niederlehner, and J. Cairns, Jr. 1982. Biological
Monitoring. Part IV - toxicity testing. Water Res. 16:239-262.
Caldwell, S., K.W. Barret, and S.S. Chang. 1981. Ranking system for
releases of hazardous substances. In "Management of uncontrolled
hazardous waste sites". Haz. Mat. Contr. Res. Inst., Silver
Spring, MD. pp 14-20.
Cleave, M.L., D.B. Porcella, and V.D. Adams. 1980. Potential for changing
phytoplankton growth in Lake Powell due to oil shale development. Env.
Sci. Tech. 14:683-690.
HMIR. 1981. Hazardous Materials Intelligence Report. 23, October, 1981.
Supplement. World Information Services, Cambridge, Massachusetts.
Krewski, D. and C. Brown. 1981. Carcinogenic risk assessment: A guide to
the literature. Biometrics. 37:353-366.
Mathis, W.R. 1981. Identification and assessment of uncontrolled hazardous
material disposal sites. (Draft). Region IV. USEPA. Atlanta, GA,
30308.
Peterson, S.A. 1982. Draft hazardous materials research plan. (Drafts.
USEPA. Corvallis, OR, 97330.
Ricci, P.P. and L.S. Molton. 1981. Risk and benefit in environmental law.
Science. 214:1096-1100.
Squire, R.A. 1981. Ranking animal carcinogens: A proposed regulatory
approach. Science. 214:877-880.
Starr, C. and C. Whipple. 1980. Risks of risk decisions. Science.
208:1114-1119.
Turpin, R.D., J.P. LaFornara, H.L. Allen, and U. Frank. 1981.
Compatibiltiy field testing procedures for unidentified hazardous
wastes. In "Management of uncontrolled hazardous waste sites". Haz.
Mat. Contr. Res. Inst. Silver Spring, MD. pp 110-113.
USEPA. : 1977. National Interim Primary Drinking Water Regulations.
U.S.G.P.O. Washington, D.C.
USEPA. 1981. Interim Standard Operating Safety Procedures.-/- Emergency
Response Division. (Draft). USEPA Headquarters. Washington, D.C.
42
-------
APPENDIX A
BIOLOGICAL TESTS FOR
BIOASSESSMENT OF HAZARDOUS
WASTE SITES
-------
CONTENTS OF APPENDIX A
Page
OVERVIEW OF BIOASSESSMENT PROTOCOL A-l
TEST PROCEDURES A-l
GLOSSARY A-3
Response Variables A-3
Controls A-3
Sample Descriptions A-4
Test Descriptions A-5
TEST SAMPLES A-7
REPORTING A-7
RESPONSE LEVELS A-9
GENERAL MATERIALS AND METHODS FOR BIOLOGICAL TESTS
OF WATER SAMPLES A-ll
GENERAL INSTRUCTIONS A-ll
Setup and Preparation A-ll
Facilities A-12
Construction Materials A-12
Test Containers A-13
Cleaning and Preparation of Glassware A-13
Receipt and Quarantine for Fish A-14
Disease Treatment for Fish A-15
Performing the Tests A-15
Test Material A-15
Sample Test Concentrations A-18
Preparation of Toxicant A-18
Dissolved Oxygen Concentration A-19
FRESHWATER ALGAE 96-HOUR TEST A-19
Introduction and Rationale A-19
Materials and Methods A-20
Equipment A-20
Freshwater Algal Nutrient Medium A-21
Test Organisms and Culture Maintenance A-21
Test Procedure „ A-23
Response Monitoring A-25
Electronic Particle Counting A-25
Biomass (dry weight) - A-26
Absorbance A-26
Microscopic Counting A-27
Results and Data Interpretation A-27
Calculations A-27
A-i
-------
CONTENTS (continued!
Page
STATIC ACUTE TOXICITY TESTS WITH FRESHWATER FISH
AND DAPHNIA A-29
Introduction and Rationale A-29
Materials and Methods A-29
Dilution Water A-29
Species A-31
Source A-32
Sizes, Life Stages A-32
Culturing, Care, and Handling A-32
Holding and Acclimation A-34
Test Procedures A-34
Results and Data Interpretation A-37
GENERAL MATERIALS AND METHODS FOR BIOLOGICAL TESTS
OF SOIL SAMPLES A-40
GENERAL INSTRUCTIONS A-40
Setup and Preparation A-40
Containers, Cleaning, and Preparation A-40
Sampling and Sample Preparation A-41
ROOT ELONGATION TEST A-43
Introduction and Rationale A-43
Materials and Methods A-45
Facilities A-45
Test Containers A-45
Equipment A-47
Test Organisms A-47
Size Grading of Seed A-47
Preparation of Glassware A-49
Tissue Paper Precleaning A-49
Test Procedures A-49
Test Medium A-49
Procedure for Planting Seed A-50
Incubation A-52
Measurement of Root Length A-52
Range-finding Test A-54
Definitive Test A-54
Results and Data Interpretation A-55
Assay Acceptance Criteria A-55
Calculations and Reporting A-56
EARTHWORM TEST A-57
Introduction and Rationale " A-57
Materials and Methods A-59
Test Organisms A-59
Breeding of Test Organisms A-59
Test Procedures A-59
Range-Finding: Contact Test A-59
Definitive Test: Artificial Soil Test A-61
A-ii
-------
CONTENTS (continued)
Page
Test Conditions for Artificial Soil and
Soil Sample Extracts A-63
Test Conditions with Artificial and
Sample Soils A-64
Results and Data Interpretation A-66
SOIL RESPIRATION A-67
Introduction and Rationale A-67
Materials and Methods A-67
Test Procedure A-68
Results and Data Interpretation A-69
REFERENCES CITED A-73
A-iii
-------
OVERVIEW OF
BIOASSESSMENT PROTOCOL
TEST PROCEDURES
A set of aquatic and terrestrial biological tests have been compiled to
aid in assessing potential environmental hazard of hazardous waste sites.
Users would select all tests or those appropriate to their needs and use
them according to the procedures contained in this report.
The three tests directed at aquatic ecosystems are the algal bioassay,
and the fish and Daphnia toxicity tests. The three terrestrial tests are
the root elongation test, the earthworm acute toxicity test, and the soil
litter microorganism test.
The aquatic tests are applied as appropriate to surface or ground water
samples, extracts of soil samples, and nonaqueous samples where appropriate.
The terrestrial tests are applied to soil extracts and soil samples.
Two levels of testing are defined, range finding and definitive, but
more detailed sampling and testing designs can be devised for specific
needs. . Range finding tests are a geometric series of dilutions by factors
of 10, for example, 1.0, 0.1, 0.01, 0.001. At least three dilutions should
be performed. Definitive tests are a geometric series of dilution by
factors of 2, for example, 1., 0.5, 0.25, 0.125, 0.0625, 0.03125. At least
six dilutions should be performed. For the definitive test, concentrations
usually bracket the intermediate value of the range-finding test or utilize
A-l
-------
the most effective value as the highest concentration. Soil or water are
diluted in synthetic media (algal assay medium (AAM), reconstituted water,
artificial soil) and, if appropriate, in unimpacted natural waters or soils
from the site environs.
Control biological tests should always be performed. These include,
where appropriate, negative, positive, solvent and reference controls
(defined in the following section). The tests have been designed to be as
reproducible as possible using carefully standardized test media and
organisms. Quality assurance should follow the "Guidelines and
Specifications for Implementing Quality Assurance Requirements" (USEPA,
1980). Safety procedures should be practiced to prevent exposure to staff
(Appendix B).
The tests described in this appendix were taken from several important
sources and readers should be aware of the references. The three aquatic
tests and the root elongation test are from Brusick and Young, 1982, and the
earthworm test is in development (contact C. Callahan, Con/all is ERL). The
soil respiration test was largely provided by Lighthart and Bond (1976).
Other major sources include the Committee on Methods for Toxicity Tests with
Aquatic Organisms (1975) and ASTM (1980), USEPA Methods (1979), Standard
Methods (APHA, 1981), Miller et.ll- (1978), and a review of statistical
methods by Stephan (1977).
A-2
-------
GLOSSARY
To insure a common understanding of terms in this report, a glossary
has been included.
Response Variables
Effective Concentration (EC) - concentration that produces the desired
effect at a specified level in a percent of the exposed population
within a specified time.
96 hour ECgQ - concentration that produces the desired effect in 50 percent
of the population within 96 hours of exposure. Typical levels are 10,
20, 50, 100 percent.
Lethal Concentration (LC) - concentration that produces death as the effect
at a specified level in a specified time.
Stimulating Concentration (SC) - concentration that causes growth to
increase at a specified level in a specified time.
Controls
Negative Control - a test in which no toxicant is added to 100 percent
dilution medium.
A-3
-------
Positive Control - a test in which an effective concentration of a known
toxicant is added to 100 percent dilution medium.
Solvent Control - a test in which the solvent used to extract the toxicant
from a water or soil sample is evaluated. Then the solvent is added to
100 percent dilution medium at the same concentration as would occur
with the extract.
Reference Controls - tests using natural water or soil samples collected
from unimpacted areas of the site environs.
Sample Descriptions
Aqueous Sample - a receiving water (surface or ground water) or a soil
extract obtained by extracting with water.
Non-Aqueous Sample - a water sample with more than 0.1 percent organic
material (>1000 ppm), or any soil extract obtained by extraction with
organic solvents.
Solid Sample - any solid phase material; generally, a soil sample.
Leachate - a sample of water that has percolated through a column of soil or
other material such as waste. "
Elutriate (extract) - a sample of water obtained by mixing a solid sample
with a specified weight ratio of solvent, usually water, for a specified
A-4
-------
time and then separating from the solid phase by centrifugation and/or
filtration.
Dilutions of Samples - a solid, aqueous or non-aqueous sample which is mixed
homogeneously with natural soils or waters (receiving system) or
standard soils or waters (artificial or reconstituted, reference or
benchmark).
Standard Sample - any sample of constant or defined composition, e.g.,
synthetic water samples, soil conservation service regional soil sample,
artificial soil.
Test Sample - site samples purposely contaminated with a known toxicant
substance or mixture.
Contaminated Sample - site sample contaminated during site operation.
Usually collected at boundary, or along a gradient.
Test Descriptions
Definitive Test - test used to establish the effective concentration of a
substance or material. As used herein, the concentrations are in a
geometric series with a ratio of 2.0.
Range-Finding Test - test used to determine the appropriate range of
concentrations in which to apply the definitive test. The range-finding
test can be entirely different from the definitive test, a slight
A-5
-------
modification of the definitive test, or an application of the definitive
test over a broader range of concentrations. Generally, as used herein,
the concentrations are in a geometric series with a ratio of 10.0.
Bioassessment Protocol - a combination of bioassessment tests for assessing
potential environmental hazards at a site.
Bioassessment Procedure - a bioassessment test applied to a sample, for
example, methods of evaluating algal growth in a soil extract.
Bioassessment Test - a specific biological population for assessing a
biological response to a mixture of toxicants or a single toxicant. For
example, the canary in the coal mine and the fish toxicity tests are
Bioassessment Tests.
Bioaccumulation - uptake and retention of environmental substances by an
organism from all sources (Veith jst _§]_., 1979).
Bioconcentration - uptake and retention of environmental substances by an
organism from water (Veith et_al_., 1979). A bioconcentration factor
(BCF) can be calculated as the quotient of the concentration of chemical
in the tissue (or whole) of an aquatic organism divided by the
concentration in the water in which the organism resides.
A-6
-------
TEST SAMPLES
x.
Tests are conducted on water, soil extracts or extracts, and soil
samples collected as appropriate to the experimental design from the
hazardous waste site. Dilutions are made into standard or reference water
and soil samples. Sampling design should follow guidelines discussed in the
text. In all cases, including actual sampling, transportation of samples,
storage, pretreatment, dilution, and actual testing, procedures designed to
provide protection of personnel safety and safety of the general public and
of the environment must be carefully followed. Safety guidelines are
discussed in Appendix B.
REPORTING
The test record should include where applicable:
• name and address of test laboratory
0 date or period of testing
• name of person responsible for testing
t number of tests carried out (rangefinding and definitive
tests)
0 exact description of test conditions
A-7
-------
• details of any variation of test materials and conditions from
protocol
• details of test organism (age, maintenance and breeding
conditions, source of supply)
• average live weight and range and number of organisms per dose
at start and end of test
t description of obvious physical or pathological symptoms or
distinct changes in behavior observed in test results
0 graph showing concentration/effect curve
• mortality and changes in weight for control animals
• where appropriate, mortality and changes in weight for animals
used as control, reference, or test animals
• date and signature of the person performing the test
• any other documents on test conditions
A-8
-------
RESPONSE LEVELS
Generalized response levels for the 3 aquatic and 3 terrestrial tests
are summarized in Table A-l. The qualitatively defined levels are presented
as guidance. They are intended to help users to evaluate relative toxicity
of specific samples. However, the response levels are not fixed values and
further results will be incorporated to obtain better qualitative estimates
of toxicity. Furthermore, the low or not detectable levels may be
misleading since a lack of strong toxicity does not necessarily mean a
sample is "safe"; it only is an indication of the immediate potential
hazard due to toxicity.
A-9
-------
Table A-l
DEFINITION OF TOXICITY CATEGORIES FOR AQUATIC AND TERRESTRIAL ECOLOGICAL ASSAYS
Response Levels for LC5Q or EC5Q Concentrations0
Assay
Freshwater
Fish
Freshwater
Invertebrate
Freshwater
Algae
3> Seed Germination and
,1. Root Elongation
o
Earthworm Test
Soil Respiration Test
Activity Measured
96-hr LC5Q
(lethality)
48-hr EC,n
ou
(immobilization)
96-hr EC5Q
(growth inhibition)
115-hr EC5Q
(inhibit root
elongation)
336-hr LC5Q
336-hr EC5Q
bamp i e
Type9
S
L
S
L
S
L
L
S
S
L
MADb
1
100
1
100
1
100
100
500
500
100
Units
g/L
percent
g/L
percent
g/L
percent
percent
g/kg
g/kg
percent
High
<0.01
<20
<0.01
<20
<0.01
<20
<20
<50
<50
<20
Moderate
0.01-0.1
20-75
0.01-0.1
20-75
0.1-0.1
20-75
20-75
50-500
50-500
20-75
Low or Not Detectable
0.1-1
75-100
0.1-1
75-100
0.1-1
75-100
75-100
500
500
75-100
aS = solid, L =, aqueous liquid, includes water samples and elutiate or leachate. Nonaqueous liquids are evaluated
on an individual basis due to variations in samples such as vehicle, percent organic vehicle, and percent solids.
MAD = Maximum applicable dose.
= Calculated concentratio
= Calculated concentration expected to produce effect in 50 percent of population within the specified time
cLCrn = Calculated concentration expected to kill 50 percent of population within the specified time interval.
™
interval.
-------
GENERAL MATERIALS AND METHODS FOR BIOLOGICAL
TESTS OF WATER SAMPLES
GENERAL INSTRUCTIONS
Setup and Preparation
The recommended test organisms in freshwater tests are the algae,
Selenastrum capricornutum. the juvenile fathead minnow, Pimephales promelas,
and early instars of Daphm'a magna. The recommended test period is 96 hours
for the algal test, 96 hours for the fish test, and 48 hours for the daphnid
test. Thus, the principal finding obtained from an algal study is the
96-hour EC5Q, ECgQ or SC2Q, from the fish study the 96-hour LC5Q, and from
the daphnid study the 48-hour EC50.
/
The procedures for the fresh water tests have been developed largely
from previous work (Brusick and Young, 1982). Modifications to the original
protocols have been made where necessary to adapt tests to the requirements
of the Bioassessment Protocol.
Materials and methods that are common to all, or nearly all, aquatic
tests are presented in this section. The section for each specific test
discusses materials and methods unique for that test and identifies which of
the general materials and methods are applicable.
A-ll
-------
Facilities
The facilities should include tanks equipped for temperature control
and aeration for holding and acclimating test organisms, and a constant
temperature area or recirculating water bath for the test vessels. If the
use of reconstituted dilution water is necessary, there should be a tank for
its preparation. If air is used for aeration, it must be free of oil and
fumes. The test facility must be well ventilated and free of fumes.
Illumination should be provided of an intensity and duration that is
specified in the Materials and Methods section for each test.
Construction Materials
Materials that come in contact with samples, stock solutions, or test
solutions should minimize sorption of any constituents of the test material
and not contain any substances that can be leached or dissolved by the
water. Glass, #316 stainless steel, and perfluorocarbon plastics must be
used whenever possible to minimize leaching, dissolution, and sorption.
Unplasticized plastics may be used for holding and acclimation tanks and in
the water supply system. Rubber, copper, brass, galvanized, and lead must
be avoided. If stainless steel is used it must be welded, never soldered.
Silicone adhesive used to cement glass containers sorbs some organochlorine
and organophosphorus compounds which are difficult to remove; therefore, as
little adhesive as possible should be in contact with test material
solutions and extra beads of adhesive should be on the outside, not the
inside, of the containers.
A-12
-------
Test Containers
Fish tests should be conducted in 20-liter wide-mouth soft-glass jars
or in all-glass containers 30 cm wide, 60 cm long and 30 cm high. Daphnids
should be exposed in 4-liter wide-mouth soft-glass bottles, in 3.3-liter
battery jars or in 250-miHi liter beakers. Algal tests should be conducted
in Erlenmeyer culture flasks of Pyrex or Kimax type of glass. The flask
size is not critical, but due to COp limitations the volume-to-volume ratio
is. The recommended contents-to-flask-volume ratios for hand shaken flasks
are:
25 ml in 125 ml flask
50 ml in 250 ml flask
100 ml in 500 ml flask
Maximum permissible contents-to-volume ratios in continously shaken flasks
should not exceed 50 percent.
Cleaning and Preparation of Glassware
Each testing container must be cleaned before use. A new container
must be (1) washed with non-phosphate detergent, (2) rinsed with 100 percent
acetone, (3) rinsed with water, (4) rinsed with 10 percent nitric acid, (5)
rinsed thoroughly with tap or other clean water, and (6) a final rinse with
distilled or deionized water (3 volumes). After testing, each container
should be cleaned as above unless the container is discarded.
For fish bioassays, disinfect test containers for 1 hour with an
iodophor, 200 mg hypochlorite per' liter, or a quaternary ammonium salt such
as 800 ppm Roccal II (National Laboratories, Montvale, New Jersey 07645)
with at least one thorough scrubbing during the hour, then rinse thoroughly.
For safety, do not use acid and hypochlorite together.
A-13
-------
All glassware used in algal testing is prepared as above. Flasks are
dried in an oven at 50° to 70°C. Demonstrably nontoxic plugs (for example,
Gaymar white, polyurethane or equivalent, Gaymar Industries, Orchard Park,
New York 14127) are inserted and the glassware is autoclaved for 20 minutes
at 1.1 kg/cm2 (15 psi) and 121°C. Cooled flasks are stored in closed
cabinets.
Receipt and Quarantine for Fish
Stock fish shipped from outside sources may have been subjected to
changes in temperature, dissolved oxygen and pH, handling disturbances, and
other stresses, and should be examined carefully for health and vigor.
Introduce holding water gradually into the shipping baas, observing the fish
for abnormal behavior. When the difference in water temperature between the
bag and holding tank is 2° or less, fish from one bag should be introduced
into the tank and observed for five minutes for acute stress. If acute
stress is not seen, the remaining fish may be introduced into the tank in a
similar manner.
To prevent spread of disease, incoming fish for stock should be
quarantined for at least 2 weeks and observed for abnormal behavior and
parasites. The quarantine tanks should be prepared in advance by thorough
scrubbing and cleaning with an industrial cleaner, rinsing with water,
sterilizing with a quaternary ammonium salt such as 800 ppm Roccal II, and
rinsing with at least three changes of water before filling with dilution
water. If after 2-weeks' quarantine they show no signs of infection or
abnormal behavior they are transferred to stock holding tanks, otherwise,
they are either discarded or treated as described in Disease Treatment for
Fish, below.
A-14
-------
To prevent initiation and spread of disease, nets, buckets, fish
graders, and hands should be routinely disinfected with 200 ppm Roccal II
before being placed in the water.
Disease Treatment for Fish
Freshwater fish may be chemically treated to cure or prevent diseases
by using the treatments recommended in Table A-2. However, if a group of
fish is severely diseased, the entire lot should be destroyed. Generally,
the fish should not be treated during the first 16 hours after arrival at
the facility because they may be stressed due to collection or
transportation and some may have been treated just prior to transit. Tests
must not begin with treated fish for at least 4 days after treatment. Tanks
and test chambers which may be contaminated with undesirable microorganisms
should be disinfected following the procedures outlined in Cleaning and
Preparation of Glassware, above in this section.
Performing the Tests
Test Material
All samples and test materials must be handled according to safe
procedures that protect the workers, society, and the ecosystem. These
procedures are described in Appendix B. The test material may be a solid,
aqueous liquid, or nonaqueous liquid. For the quantity of sample required
to run each test, see Table A-3.' Samples are usually tested directly
without preparation, however, some test materials require pretest
preparation. Except for the algal test, the aqueous sample (extract or
water sample) should be run directly in the dilution water and must not be
A-15
-------
Table A-2
RECOMMENDED PROPHYLACTIC AND THERAPEUTIC TREATMENTS
FOR FRESHWATER FISH (from Brusick and Young, 1982)a
Disease
External
bacteria
Monogenetic
trematodes
fungi, and
external .
protozoa
Parasitic
copepods
Chemical
Benzalkonium chloride
(Hyamine 1622®)
Nitrofurazone (water mix)
Neomycin sulfate
Oxytetracycline hydrochloride
(water soluble)
Formalin plus zinc-free
malachite green oxalate
Formalin
Potassium permanganate
Sodium chloride
Dexon (3555 AI)
Trichlorfon
(Masolen®)
Cone., mg/1
1-2 AIb
3-5 AI
25
25 AI
25
0.1
150-250
2-6
15,000-30,000
2000-4000
20
0.25 AI
Application
30-60 minc
30-60 minc
30-60 minc
30-60 minc
1-2 hoursc
30-60 minc
30-60 min
5-10 min dip
c,e
30-60 min1-
f
These recommendations do not imply that these treatments have been cleared or
registered for these uses. Appropriate State and Federal regulatory agencies should
be consulted to determine if the treatment in question can be used and under what
conditions the uses are permitted. These treatments should be used only on fish
intended for research. They have been found dependable, but efficacy against
diseases and toxicity to fish may be altered by temperature or water quality.
Researchers are cautioned to test treatments on small lots of fish before making
large-scale applications. Prevention of disease is preferred, and newly acquired
fish should be treated with the formalin-malachite qreen combination on three
alternate days if possible. However, in general, fish should not be treated on the
first day they are in the facility. This table is merely an attempt to indicate the
order or preference of treatments that have been reported to be effective. Before a
treatment is used, additional information should be obtained from such sources as
Davis (1953), Hoffman and Meyer (1974), Reichenback-Klinke and Elkan (1965) Snieszko
. (1970), and van Duijn (1973).
AI - active ingredient.
Treatment may be accomplished by (1) transferring the fish to a static treatment
tank and back to a holding tank; (2) temporarily stopping the flow in a
flow-through system, treating the fish in a static manner, and then resuming the
flow to .flush out the chemical, or (3) continuously adding a stock solution of the
chemical to a flow-through system by means of a metered flow or the technique of
. Mount and Brungs (1967).
One treatment is usually sufficient except for "Ich", which must be treated daily or
every other day until no sign of the protozoan remains. This may take 4 to 5 weeks
at 5 to 10°C and 11 to 13 days at 15 to 21°C. A temperature of 32°C is lethal to
Ich in 1 week.
f Minimum of 24 hours, but may be continued indefinitely.
Continuous treatment should be employed in static or flow-through systems until no
copepods remain, except that treatment should not be continued for over 4 weeks and
should not be used above 27 C.
A-16
-------
Table A-3
SAMPLE SIZE REQUIREMENTS FOR AQUATIC ECOLOGICAL ASSAYS
(From Brusick and Young, 1982)
Solid Liquid (liters)
Type of Test (grams) Aqueous Nonaqueous9
Freshwater Fish 100 (75)b 100L (75L) 0.100 (0.075)
Freshwater Invertebrate 10 (4) 10L (4L) 0.010 (0.004)
Freshwater Algae 2 (1) 1L (0.06L) 0.010 (0.005)
Nonaqueous liquids include aqueous samples with greater than 0.2% organics,
nonaqueous liquids, solvent exchange samples, and extracts or leachates in
a nonaqueous (organic) vehicle.
The first value given is the requested sample size for routing testing.
The value in parentheses is the minimum feasible sample size to conduct the
test.
aerated or altered in any way, except that it may be filtered through a
sieve or screen with holes 2mm or larger to remove large particles.
Aqueous samples should be filtered (0.45 micrometer cellulose acetate
filters) to remove indigenous algae for the algal assay. This should be
done as soon after collection as possible (on site is preferable) and must
be done before sample storage. Solid and nonaqueous samples may be added
directly by weight or volume respectively, diluted with dilution water and
small subsamples of equal volume added to each test container. Samples
must be covered at all times' and violent agitation must "be avoided.
Undissolved materials must be uniformly dispersed by gentle agitation
immediately before a portion of the sample is taken for use.
A-17
-------
If testing is to be done on-site, the tests should begin within 8
hours of collection. If testing is to be done at a laboratory, the samples
should be placed on ice for preservation during transportation. Testing
should be performed as soon as possible after laboratory receipt of the
samples. Samples should be stored at 4°C if testing is not initiated upon
sample receipt. The temperature of the sample should be adjusted to that
of the test (+2°C) before portions are added to the dilution water. Solid
materials may be added directly to dilution water.
When diluting samples containing highly volatile substances, it may be
desirable to add the test sample below the surface of the dilution water.
Complete and accurate records of collection methods, treatments, and
addition techniques must be maintained.
Sample Test Concentrations
Preparation of Toxicant. Depending on its nature, the test material
is prepared by one of two methods. In the first method, solids or
non-aqueous liquid materials may be added directly by weight or volume
respectively to a stock solution or to the dilution water. The stock
solution may be deionized water or a solvent and then equal volume
subsamples of a small size are added to each treatment. If it is not
possible to prepare a homogenous solution of the toxicant, it must be added
directly to the dilution water in each replicate flask or tank.
The second method is for aqueous samples and allows testing by percent
volume (volume/volume). Up to 100 percent of the sample with
filter-sterilization if required, is used in the test. Additional test
concentrations are prepared on a volume-percent basis by mixing appropriate
A-18
-------
volumes of sample with appropriate dilution water or medium.
Controls should consist of the dilution water or nutrient medium, and
a receiving water sample if appropriate. These are called negative
controls. It may be necessary to perform a range finding test with broad
dilution limits (factors of 10, 100, 1000 > before performing the final
tests. Specific requirements are listed in the Section on Test Procedure
for each test.
Dissolved Oxygen Concentration. Aeration of test solutions during the
test should be avoided to minimize loss of highly volatile materials.
Dissolved oxygen must be brought at least to minimum standards (40 percent
of saturation) by dilution. If the dissolved oxygen concentration is less
than 40 percent saturation in any test chamber for fish or Daphnia tests,
this should be noted in the final report. Algal tests do not have defined
dissolved oxygen concentration requirements.
FRESHWATER ALGAE 96-HOUR TEST
Introduction and Rationale
Unicellular algae are important producers of oxygen and form the basis
of the food web in aquatic ecosystems. Since algal species and communities
are sensitive to environmental 'changes, growth may be inhibited or
stimulated by the presence of pollutants. Therefore, the response of algae
must be considered when assessing the potential ecological effects of
industrial or municipal discharges on aquatic ecosystems.
A-19
-------
A simple screening test for toxicity to algae can be conducted in 96
hours. Algae are exposed to various concentrations of the test material
and growth is measured at 96 hours. Results are expressed in terms of the
ECgg (the lowest test concentration causing inhibition of growth by equal
or greater than 90 percent relative to the control), and EC5Q (the lowest
test concentration causing inhibition of growth by equal or greater than 50
percent relative to the control). Stimulatory effects, if any, should be
noted and expressed mathematically in terms of SCon and used for estimation
of bioactivity of the sample.
Materials and Methods
General procedures listed for all aquatic tests in the GENERAL
INSTRUCTIONS are applicable to the static acute toxicity test with
freshwater algae. Specific areas discussed in the GENERAL INSTRUCTIONS
that should be followed are: facilities, construction materials, test
containers, cleaning and preparation of glassware, and test material.
Materials and methods unique to freshwater algal tests are included below.
Equipment
Equipment should include a constant-temperature room or incubator
capable of providing temperature control of 24 +_2°C. Daily hand shaking or
a gyrotary shaking apparatus capable of 100 oscillations per minute should
be used for test culture flasks. " Continuous illumination of~ 4300 +430
o
lumens/m (400 ft-c) is required for freshwater green algae. Overhead
cool-white fluorescent bulbs should be used. Light intensity is measured
adjacent to the flask at liquid level using a light meter capable of being
A-20
-------
calibrated against National Bureau of Standards lamps. Culture containers
for this and other aquatic tests are discussed in the Test Container
section.
Freshwater Algal Nutrient Medium
Algal Assay Medium (AAM) is prepared by adding 1.0 ml of each of the
macronutrient and micronutrient stock solutions, in the order listed in
Table A-4, to 900-ml of filter-sterilized deionized water, with mixing
after each addition. Then the final volume is brought to 1 liter with
filter-sterilized deionized water. Deionized water is filter-sterilized by
passing through a 0.45 micrometer porosity cellulose acetate membrane
filter (pre-rinsed with 100-ml deionized water) into a sterile container.
Medium should be constituted as needed but can be stored in the dark at 4°C
to reduce possible photochemical changes and bacterial growth for periods
up to one month.
Test Organisms and Culture Maintenance
For freshwater algal assays, the recommended test organism is
Selenastrum capricornutum, a unicellular non-motile chlorophyte that is
easily maintained in laboratory cultures. Obtain algal cultures (Culture
No. ATCC 22662) from the American Type Culture Collection, 12301 Parklawn
Drive, Rockville, Maryland, 20852.
Upon receipt of the algal culture, approximately 1.0 ml should be
aseptically transferred to the" AAM. The rest of the culture can be
maintained up to six months in a dark refrigerator at 4°C. Weekly aseptic
routine stock transfer is recommended to maintain a continuous supply of
"healthy" cells for experimental work. To retain a unialgal culture over a
A-21
-------
Table A-4
COMPOSITION OF ALGAL ASSAY MEDIUM (AAM)
(From Miller et aj.., 1978)
Macronutrients
Stock Solutions
Concentration
Compound (g/1)
Nutrient Composition
Prepared Medium
Concentration
Element (mg/1)
NaN03
NaHCO,
K2HP04
MgS04'7H20
MgCl2*6H20
CaCl2'2H20
25.500
15.000
1.044
14.700
12.164
4.410
N
Na
C
K
P
S
Mg
Ca
4.200
11.001
2.143
0.469
0.186
1.911
2.904
1.202
Micronutrients
Stock Solutions
Concentration
Compound (mg/1)
Nutrient Composition
Prepared Medium
Concentration
Element (uq/1)
H3B03
MnCl2'4H20
ZnCl2
CoCl2'6H20
Na2Mo04'2H20
FeCl3*6H20
Na2EDTA'2H20
185.520
415.610
3.271
1.428
0.012
7.250
160.000
300.000
B
Mn
Zn
Co
Cu
Mo
Fe
32.460
115.374
1.570
0.354
0.004
2.878
33.051
Other forms of the salts may be used as long as the resulting
concentrations of elements are the same.
A-22
-------
long period of time it is advantageous to prepare a semi-solid medium
containing 1.0 percent agar made up with AAM, autoclaved and cooled to
45°C, and placed in sterile Petri plates. A portion of a liquid algal
culture is streaked onto it and incubated under standard conditions. Algae
should be transferred onto fresh plates every four weeks. Fresh liquid
cultures should be started by transfer of a single algal colony to liquid
medium at four week intervals. For test inoculation, liquid cultures are
used.
A 6- to 8-day-old stock culture is used as the inoculum source.
Population density in the stock culture is determined by direct counting or
spectrophotometry with a standard curve. The culture should always be
checked microscopically to insure that it is unialgal and healthy. A
volume of inoculum calculated to provide a concentration of 10,000 cells/ml
in the test concentration at the start of the test is aseptically added to
each test flask. The volume of inoculum added should be between 0.1 and
1.0 ml. See the section on Response Monitoring to determine cell counts.
Test Procedure
Three replicates of each test concentration and control are tested.
Prior.to conducting the rangefinding or definitive 96 hr ECcn test, the 100
percent sample should be assayed with and without the addition of stock
nutrient solutions equivalent to ' full-strength algal assay medium. If
inhibition in the 100 percent sample plus AAM is less than 50 percent of
the control, no further testing is necessary. Samples causing greater than
50 percent inhibition should be assayed by diluting the 100 percent sample
A-23
-------
(without nutrient addition) with algal assay medium to prepare each
dilution series. This assures known nutrient availability in the test
dilutions to calculate algal yield. Stock nutrient solutions are added to
the 100 percent sample as in making up AAM to insure nutrient salts are
equivalent to 100 percent AAM. Full strength AAM is used to dilute samples
for the dilution series.
A range finding test will probably be necessary before running the
actual test. A control plus concentrations of 80 percent, 10 percent, 1
percent and 0.1 percent (W/V or V/V) are usually necessary using 3
replicates each. The addition of AAM to make the 80 percent samples
insures adequate nutrients for the test, while avoiding chemical effects or
nutrient inavailability which occurs when using 100 percent AAM in test
samples. The definitive test will span the moderate response
concentration(s) using a geometric series. For example, if 1 percent
(0.01) and 10 percent (0.10) gave toxic responses (EC,-Q), a test series
would include: 0.1, 0.05, 0.025, 0,0125, 0.00625, 0.003125. solutions
(W/V or V/V).
Controls include the AAM (negative control) to check standard organism
response and receiving water if applicable (reference control), a solvent
control if applicable (dilution water plus solvent). The positive control
is applied with ZnCl2 in AAM at a concentration of 80 ug Zn++/l to give a
range of inhibition of 51-66 percent (long term mean = 58.8).
A-24
-------
Response Monitoring
After 96 hours of exposure, algal growth is measured by any of the
following methods: (a) electronic particle counting, (b) biomass (dry
weight), (c) absorbance, or (d) microscopic counts. Cursory microscopic
observation is desirable to reveal and record any abnormalities in cell
shape or condition. Because the algal test is designed to provide a
comparative response to varying dilutions of sample dilute, it is better to
use an electronic particle counter to measure growth. Other techniques can
be used (Brusick and Young, 1982; Miller et£]_., 1978; American Public
Health Association, 1981; Porcella and Cleave, 1981) but results must be
reported along with conversion equations as mg/1 dry weight.
Electronic Particle Counting
A Model ZBI Coulter Counter with Mean Cell Volume (MCV or MHR)
Computer is used. The particle counter offers the greatest precision and
accuracy and is the preferred method if equipment is available and samples
are suitable. The MHR Computer must be calibrated with the organic
calibration material; biomass may be determined indirectly by the
following equation:
Cell counts (cells/ml) x MCV ( m3) x (2.9 X 10"7)* =
mg dry weight ^. capricornutum/1iter
*Note that the conversion factor of 2.9 x 10 may differ between
laboratories and should be determined by each investigator. The standard
reference particle (Part Number 1607081) can be obtained from Coulter
Electronics, Inc., Hialeah, Florida.
A-25
-------
If there are particles in the test material, it is possible to
eliminate counts contributed by other particles. Uninoculated flasks are
counted and these counts subtracted from the total counts. Then dry
weights of cells can be calculated with the above formula. Particles may
clog the aperture, and in such cases, another method should be used. The
advantage of this method is that it allows for determination of biomass
produced in addition to cell numbers.
Biomass (dry weight)
For this method, a measured portion of algal suspension is filtered
through a tared 0.6 micrometer PVC membrane filter. The filters are
prepared as follows: Dry for two hours at 70°C in an oven. Place filters
in folded sheets of paper or aluminum weighting dishes on which the weights
or codes are written. Cool in a desiccator for at lease one hour and
weigh. Filter a suitable portion of culture (50 ml or less as the cell
density dictates) under a vacuum of 51 kPa. Rinse filter funnel with 50 ml
distilled water using a wash bottle and let the rinsings pass through the
filter. Dry at 70°C, cool in desiccator, and weigh. Subtract tare weight,
divide by volume (liters) of culture filtered and express as mg/1 dry
weight.
Absorbance
Measure absorbance with a spectrophotometer or colorimeter at a
wavelength of 750 nm. Report instrument make and model, geometry and path
length of the cuvette, wavelength' used, and the equivalence "to biomass
(absorbance units per milligram dry weight per liter).
A-26
-------
Limit photometric measurement of absorbance to a range of 0.05
-------
C = maximum standing crop fmg/1) obtained in the AAM control.
T = maximum standing crop (mg/1) obtained in the test sample.
IN = dry weight (mg/1) of inoculum used at start of test.
Four toxic concentrations must be tested in the definitive test. They
are: the concentrations which inhibit less than or equal to 25 percent;
from 25 percent up to 50 percent; from 50 percent up to 75 percent; and
the lowest concentration which inhibits more than 75 but less than or equal
to 90 percent of the test algae. Three endpoints may be calculated from
the percent response vs. concentration data. For samples which are
inhibitory, an ECgQ (defined as the lowest test concentration causing
growth inhibition of 90 percent relative to control) and an EC™ (defined
as the lowest test concentration causing growth inhibition of 50 percent
relative control) are calculated. For samples which are stimulatory, an
SC£Q (defined as the lowest concentration causing growth stimulation of 20
percent relative to control) is calculated. For all samples, the EC™,
ECgg, and SC2Q are calculated using any of several statistical methods.
The 96-hour ECgQ results are evaluated according to criteria defined
in Table A-l which will permit the test material to be ranked by toxicity
category. While the ECgQ endpoint may be the most meaningful biological
effect for long-term impact on the environment, the more sensitive EC™ is
used in this assay to rank samples.
A-28
-------
STATIC ACUTE TOXICITY TESTS WITH FRESHWATER FISH AND DAPHNIA
Introduction and Rationale
The static toxicity tests with freshwater fish and Daphnia utilize
juvenile fathead minnows, Pimephales promelas, and early instars of Daphnia
magna. The static acute exposure period is 96 hours for the fathead minnow
and 48 hours for the daphnid study. The 96-hour mean lethal concentration
(96-hour LCgg) is calculated for the fathead minnow. Because death is not
always easily determined in Daphnia. the 48-hour effective concentration
(48-hour EC5Q) is calculated for Daphnia.
Materials and Methods
Procedures listed for all aquatic tests under GENERAL INSTRUCTIONS are
applicable to the static acute toxicity tests with freshwater fish and
Daphnia. Sections that should be followed are: Facilities, Construction
Materials, Test Containers, Cleaning and Preparation of Glassware, Receipt
and Quarantine for Fish. Disease Treatment for Fish, Test Material, and
Dissolved Oxygen Concentration. Materials and methods unique to freshwater
fish and Daphnia tests are included below.
Dilution Water
Dilution water can be from the site (upstream of possible
contamination) local dechlorinated tap water, or reconstituted water. A
minimal criterion for an acceptable dilution water is that healthy
organisms will survive in it for the duration of acclimation and testing
A-29
-------
without showing signs of stress such as discoloration or unusual behavior.
Water in which daphnids survive and reproduce satisfactorily should be an
acceptable dilution water for tests with freshwater organisms.
The dilution water should be of constant quality and should be
analyzed by accepted methods (Durrant et al_., 1974; Imai and Siegel, 1973;
Santelman, 1972; Walley eta/L, 1974) to ascertain that none of the
following substances exceeds the maximum allowable concentration shown:
Maximum
Pollutants Concentration
Suspended solids 20 mg/1
Total organic carbon 10 mg/1
Un-ionized ammonia 20 ug/1
Residual chlorine 3 ug/1
Total organophosphorus pesticides 50 ng/1
Total organochlorine pesticides plus PCB's 50 ng/1
The dilution water is considered to be of constant quality if the
monthly ranges of the hardness, alkalinity, and conductivity are within 10
percent of their respective means and if the monthly range of pH is less
than 0.4 units. Reconstituted dilution water may be prepared according to
the method shown in Table A-5. For comparability of results between tests,
the hardness should be as close as possible to 100 mg/1 as CaCO,.
•3
A-30
-------
Table A-5
RECOMMENDED COMPOSITION FOR RECONSTITUTED
FRESH WATER THAT IS MODERATELY HARD (calculated from ASTM, 1980)
Salts Added to
Distilled Water*, mg/1 Water Quality
CaS04*2pH20 70 H (air equilibrated) 8.3
MgS04 70 Hardness, mg/1 as CaC03 100
KC1 4.5 Alkalinity, mg/1 as CaC03 100
NaHCO- 168 Total dissolved solids 250
* Stock solutions of individual salts can be prepared so that 10 ml in one
liter produces the desired final concentration. Store stock solutions in
the dark at 4°C.
Species
The juvenile fathead minnow, Pimephales promelas. and early instars of
Daphnia magna are the species to be used in Level 1 freshwater static acute
toxicity tests. The fathead minnow is a warm-water fish of ponds, lakes,
and sluggish streams. Daphnids occur in nearly all types of freshwater
habitats. Both species, have been recommended as bioassay orqanisms by the
Committee on Methods for Toxicity Tests with Aquatic Organisms (1975;
ASTM, 1980) because of their wide geographic distribution, important role
in the aquatic food web, temperature requirements, wide pH tolerance, ready
availability, and ease of culture.
A-31
-------
Source
Fathead minnows may be obtained from private, state, or federal fish
hatcheries, or captured from wild populations in relatively unpolluted
areas. However, collecting permits may be required by local and state
agencies. Fish collected by electroshocking should not be used. Daphnia
should be reared in the testing facility from laboratory cultures.
Sizes, Life Stages
Fathead minnows used in testing should weigh between 0.5 and 1.0 g
each. All fish in each test should be from the same year class, and the
standard length (tip of snout to end of caudal peduncle) of the longest
fish should be no more than twice that of the shortest fish. Weights and
lengths should be determined by measuring representative specimens before
the test or the control fish after the test. Very young fish (not yet
actively feeding), spawning fish, and spent fish should not be used.
Daphnia magna used in testing should be in the early instar stages
(stages 2-4) of their life cycle. All organisms in a test must be from the
same source and as healthy and uniform in size and age as possible.
Culturing. Care, and Handling
Fathead minnows are maintained at 20-22°C in a flow-through system
with a turnover of at least two volumes daily, or in a recirculatinq system
in which the water is passed through a carbon filter and an ultraviolet
sterilizer (ASTM, 1980).
Daphnia magna are maintained in a static system at 19-22°C. Tanks
must be cleaned with a siphon periodically to remove debris, and water
A-32
-------
should be added as necessary to maintain volume. Cultures must be
maintained under optimum conditions at all times to prevent formation of
ephippial eggs; daphnids from cultures in which ephippia are being
produced must not be used in testing. Generally, periodic subculturing of
cultures, elimination of crowding, and adequate food prevent problems in
Daphnia cultures.
Both species should be fed at least once a day, at which time careful
observations should also be made for mortality and for signs of disease,
stress, and injury. Dead and abnormal individuals should be removed as
soon as they are observed.
Water quality should be held constant as described above and
temperature changes should not exceed 3°C in any 12-hour period. Fish
tanks should be scrubbed at least twice a week.
The organisms should be handled as little as possible. When handling
is necessary, it should be done as gently, carefully, and quickly as
possible so that the organisms are not needlessly stressed. Small dip nets
are best for handling fish and wide bore pipettes (0.5cm) for Daphnia.
Organisms that touch dry surfaces or are dropped or injured during handling
should be discarded.
Test organisms should always be shielded from disturbances, and
overcrowding should be avoided. '
A-33
-------
Holding and Acclimation
After collection or transportation, the fish should be held in and
acclimated to the dilution water for at least 2 days before beginning a
test under the same holding conditions as described in the Care and
Handling, section.
A group of animals must not be used for a test if individuals appear
to be diseased or otherwise stressed or if more than 5 percent die within
48 hours prior to beginning the test. If a group fails to meet these
criteria, they must be discarded or treated and held an additional 4 days.
Fathead minnows should not be fed for 48 hours prior to the beginning
of a test. However, the Daphnia may be fed up to the beginning of the
test.
Test Procedures
Unless the approximate toxicity of the sample is already known, at
least six concentrations of test material should be prepared. The highest
dose should be at the maximum applicable dose (MAD) for that sample type
(see Table A-l) unless physical characteristics of the sample or other
previously gathered toxicity data contravenes this.
In fathead minnow tests, at least 20 fish must be exposed to each test
concentrations per replicate with'two replicates per concentration used in
the test. For Daphnia maqna tests, five organisms per replicate with three
replicates per concentration should be used. The use of more organisms and
replicate test containers and random assignments of test organisms to
A-34
-------
containers is desirable.
The fathead minnow tests should be conducted at 22 +2°C, and those
with Daphnia at 19 +2°C. A photo period of 16 hours light and 8 hours dark
is used for both tests. Neither type of test animal should be fed during
exposure. The test conditions are summarized in Table A-6.
Table A-6
SUMMARY OF TEST CONDITIONS
(from Brusick and Young, 1982)
Fathead Minnow,
Pimephales promelas Daphnia maqna
Temperature, °C 22 + 2 19 + 2
Photoperiod, hours 16:8 16:8
light:dark
Water quality, hardness3 100 100
mg/1 as CaCOj
Container size 20 liters 250 ml
Test volume 15 liters 200 ml
Organisms per container 10 5
Replicates 2 3
Feed No No
Duration, hours 96 48
Measurements of D.O. 0, 24, 48, 72, 96 0, 48
and pH, hours
a For dilution water only; the investigators add salts in Table A-5 as
appropriate to obtain 100 ug/1 as CaCO-.
A-35
-------
In the fathead minnow test there should be 15 liters of test solution
or control water in each 20-liter jar. If 30 x 30 x 60 centimeter
containers are used, the solution should be between 15 and 20 centimeters
deep (about 30-35 liters).
In the daphnid test there should be 2 to 3 liters of solution or
control water in each 4 liter wide-mouth bottle or 3 to 4-liter battery
jar, or 200 mi Hi liters in each 250-mi Hi liter beaker.
Test organisms should be placed in the test and control vessels not
more than 30 minutes after the test solutions are prepared. Ten fish in
each vessel and five daphnids in each replicate are recommended. Chemical,
physical, and biological data are taken and recorded as described below for
the duration of the test.
If no toxicity is detected at any concentration and the MAD dose was
tested, then no further testing is required. The test material may be
reported as having no detectable toxicity. Test materials that kill or
immobilize all or nearly all the test organisms at all dilutions should be
retested with a lower dose range.
The biological loading in each test and control vessel should not
exceed 0.8 g of test organis'm per liter or be so high as ta fl) reduce
dissolved oxygen concentration in the control tanks below acceptable
levels, (2) raise the concentration of metabolic products above acceptable
levels, or (3) stress the organisms by overcrowding, any of which may
A-36
-------
invalidate the test results.
Results and Data Interpretation
In the fathead minnow test, dissolved oxygen concentration, and pH
should be measured for each replicate at the beginning of the test and
every 24 hours thereafter in the controls and in the high, medium, and low
concentrations. Conductivity and hardness should be measured at the
beginning of the test in the control and each test concentration for each
replicate. Meters can be used but must be standardized. Temperature of
the water bath or controlled-temperature area should be recorded
continuously or every 24 hours.
In the Daphnia test, temperature, dissolved oxygen, pH, hardness, and
conductivity on the high, medium and low concentrations, should be recorded
initially and at 48 hours.
Mortality is the effect most often used to define acute toxicity to
aquatic organisms. Criteria for death are usually lack of movement,
especially of gill movement in fish, and lack of reaction to gentle
prodding.
Because death is not always easily determined with some invertebrates,
an ECgQ may be calculated rather than an l-C^Q. The principal criterion for
effect on Daphnia is immobilization, defined as lack of movement except for
minor activity of appendages.
A-37
-------
Mortality, immobilization, and abnormal behavior should be recorded.
Dead or immobilized organisms should be removed as soon as they are
observed. For definitions of fish behavior terms, and suggested code for
recording and reporting, see Table A-7. If more than 10 percent of test
organisms in any control die or are immobilized, the entire test is
unacceptable.
The concentration of test material lethal to 50 percent of the
population (LC50) and 95 percent confidence limits should be determined at
24-, 48-, 72-, and 96-hour exposures for fish tests, and the EC5Q and 95
percent confidence limits at 24- and 48-hour exposures for Daphnia maqna
tests. Any of several methods including moving average, Spearman Karber,
Litchfield-Wilcoxin, probit, or binomial may be used. For a discussion of
the above methods, refer to the review article by Stephan (1977). The
results (96 hours for fish and 48 hours for Daphnia) are evaluated
according to Table A-l which defines the toxicity categories.
A-38
-------
Table A-7
DEFINITION OF FISH BEHAVIOR TERMS
(From Brusick and Young, 1980)
Definition
Observable responses to the test fish. Individually or 1n groups, to the range of factors constituting
their environment.
Marked by a state of Inactivity or abnormally low activity; motionless or nearly so.
Reacting to stimuli with substantially greater Intensity than control fish.
Exhibiting more or less continuous hyperactivlty.
Rising and remaining unusually long at the surface.
Diving suddenly straight to the bottom; remaining unusually long at the bottom.
Moving the body or parts of the body with sudden jerky movements.
In a state of tetany; narked by Intermittent tonic spasms of the voluntary muscles.
Lacking tone, resilience or firmness; weak and enfeebled; flabby.
Unaffected by or not exposed to a particular experimental treatment; conforming to the usual behavioral
characteristics of the species.
Progressive self-propulsion 1n water by coordinated movement of tall, body, fins.
Broken off or tapered off to a stop.
Characterized by lack of consistency, regularity, or uniformity; fluctuating, uneven; eccentric.
Revolving around a central point; moving spirally about an axis.
Skimming hurriedly alonq the surface with rapid body movements.
Turned upside down, or approximately so.
Turned 90° laterally, more or less, from the normal body orientation.
Color of skin due to deposition or distribution of pigment.
Color appearance lighter than usual for the species.
Color appearance darker than usual for the species.
Color appearance abnormally varied; mottled.
The skin.
Observably losing mucous skin coating to an abnormal degree.
Showing observable clumping or clotting of the mucous skin coating, especially at the gills.
Visibly bleeding as from gills, eyes, anal opening.
Physical action of pumping water into mouth and out throuoh pills, so as to absorb oxygen.
Observably faster than normal to a significant degree.
Observably slower than normal to a significant decree.
Falling to occur at reoular or normal Intervals.
Broken off or tapered off to a stop.
Swimming at surface with mouth open and laboriously pumping surface water and air through gills.
Performed with apparent abnormally great difficulty and effort.
No Observed Effect Concentration: The highest test concentration 1n which fish experience no mortality and exhibit no observable
behavioralabnormalitiesatany time during a specified period of exposure to the test material. Ordinarily determined for periods
from the start of testing to the end of each successive ?4 hours.
Code
1.
a.
b.
c.
d.
e.
f.
9-
h.
1.
2.
a.
b.
c.
d.
e.
f.
3.
a.
b.
c.
4.
a.
b.
c.
5.
a.
b.
c.
! d.
e.
f-
i
Term
General Behavior Ob:
ttv
Quiescent: Mai
Hyperexci table: Rei
Irritated: Exl
Surfacing: Ri!
Sounding: Oil
Twitching: Moi
Tetanous: In
Flaccid: Lai
Normal : Uni
chi
Swimming. Pn
Ceased: Bri
Erratic: Chi
Gyrating: Rei
Skittering: Sk-
Inverted: Tui
On side: Tui
Pigmentation. Co'
Light discolored: Col
Dark discolored: Col
Varldiscolored: Col
Integument. Th«
Mucus shedding: Ob!
Mucus coagulation: She
Hemorrhagic: Vis
Respiration. : Ph>
Rapid: Obs
Slow: Ob:
Irregular: Fa1
Ceased: Brc
Gulolng air: Sw1
Labored: Per
No Observed Effect Concentration:
A-39
-------
MATERIALS AND METHODS FOR BIOLOGICAL
TESTS OF SOIL SAMPLES
GENERAL INSTRUCTIONS
Setup and Preparation
The recommended test organisms in terrestrial tests are seeds from
various angiosperms used in the root elongation test (RE test), earthworms
Eisenia foetida, and soil litter microorganisms. The recommended test
period is 115 hours for the RE test, 14 days for the worms, and 14 days for
the soil litter test. The principal findings are EC5Q for the seeds
measured by percent germination and root elongation, EC™ for the worms, and
ECgQ for the soil litter test. Although inhibition of seed germination and
root elongation are observable toxic responses and are reported, root
elongation inhibition is the preferred endpoint for the RE test. The
concentration which inhibits root elongation by 50 percent of the control
) is estimated and used to rank samples.
Containers. Cleaning, and Preparation
Required containers are discussed under the appropriate test. Cleaning
and preparation of test containers" should follow procedures described in the
GENERAL INSTRUCTIONS for the aquatic tests.
A-40
-------
Sampling and Sample Preparation
Samples should be collected randomly at the site boundary or other
critical location or along a suspected gradient (identified by any of
several methods) such that the most impacted soil can be identified on one
end of the gradient and the non-impacted soil on the other end. An example
experimental design is shown in the following diagram where A can be a
lagoon, an area where leaky drums are stored, or other situation:
A grid at each sampling point is set up, and the surface soil is sampled
from a randomly selected quadrant. The points can be uniformly or
logarithmically spaced depending on objectives.
Soil samples are returned to the laboratory, and should be analyzed as
soon as possible. Storage at 4°C can be used. When tests are to be run,
the samples are air dried and ground. If extracts are to be assessed in any
of the aquatic or terrestrial tests, they should be prepared all at one time
using the procedures in Table A-8. The reason for splitting the extract
sample for the various tests is to standardize the procedures so that
results may be more comparable among the different tests. The split will
then be diluted to yield various concentrations of the extracts for the
test. Concentrations should be related to the soil extracted.
A-41
-------
Table A-8
METHODS FOR PREPARING SOIL EXTRACT
1) Weigh an adequate amount of air dried soil sample for all desired
tests.
2) Add a weight of distilled water equal to four times the soil weight.
3) Shake for 48 hours (150 rpm) at constant temperature (20 +2°) in the
dark.
4) Allow to settle, decant and filter with 0.45 urn membrane to obtain
the extract. Soil sample extracts with high clay content will have
to be centrifuged and decanted prior to filtration.
5) Relate all extracts to the original weight of soil. Measure volume
of extract and relate to initial soil weight. For example, if 3100
ml of extract is obtained from a 1000 gram of air dried soil, there
are 3.1 ml/gram. Then, if 25 ml of extract are added to 100 gram of
soil for a test, this would be equivalent to 8 gram of soil (25/3.1)
or a 7.4 percent soil (8/108). This would be the highest
concentration and for a geometric series of tests subsequent samples
would be decreased by halves. For example, for 7.4, 3.7, 1.85
percent, extract plus sample volumes would be: 25 + 0, 12.5 + 12.5,
6.25 + 18.75,
6) Do not concentrate extracts; extracts should be prepared within 24
hours of collection. Extracts should be checked for salinity using
conductivity.
A-42
-------
To perform the soil tests with the earthworms or litter decomposition,
the soil samples will be diluted (percent W/W) with artificial soil to
produce the desired tests concentrations.
To perform the terrestrial tests, the following soil samples are needed
(minimum in parentheses):
Soil, kg extract, 1
RE Test 2.5 (1.25) 10 (5)
Earthworm Test 4.0 (2.0)
Soil Respiration Test 2.0 (1.0)
These samples would provide sufficient material to perform a second test.
If range-finding tests or further repeat assays are performed, additional
samples should be collected.
ROOT ELONGATION TEST
Introduction and Rationale
. The development of a seed into a mature plant is a series of complex
processes. To assess toxic effects requires the selection of a stage in
plant development that is sensitive to a broad range of toxicants and is
important physiologically. Seed germination and root elongation are
critical links in plant development beginning with a dormant embryo and
during a period of rapid growth when essential plant structures are formed.
A-43
-------
Toxic substances that prevent or reduce germination or root elongation
will decrease plant populations and can reduce crop yields. In natural
systems those species affected are less able to compete with other species
and tolerant species may be selected, resulting in changes in species
diversity, numbers, and population dynamics.
The inhibition of seed germination and root elongation has been used in
determining selective toxicities of herbicides (Horowlitz, 1976; Santelman,
1972)), screening plants for heavy metal (Imai and Siegel, 1973; Walley
etaj.., 1974) and salinity tolerance (Durrant et ^1_., 1974; Neiman and
Poulsen, 1971), and evaluating toxic chemicals (Hikino, 1978; Rubinstein,
et al_, 1975) and allelopathic substances (Asplund, 1969; Muller, 1965).
However, many toxicants apparently do not affect plants significantly
(Kenaga, 1981), but samples must still be evaluated. The root
elongation/seed germination bioassay has several advantages. It is a rapid
test germination and root elongation can be observed after 115 hours of
incubation. It is a simple test that does not require large expenditures
for equipment and facilities or complicated techniques. Personnel required
for performing the bioassay do not need to be highly skilled.
The same chemical may cause responses at different doses in different
plant species (Rubinstein et _al_., 1973). To detect an effect from chemicals
of. unknown toxicity, several plant species should be selected. The species
used in this test -- lettuce (butter crunch), Lactuca sativa L.; cucumber
(hybrid Spartan valor), Cucumis s'ativa L.; red clover (Kenland), Trifolium
pratense L.; wheat (Stephens), Triticum aestivum L.; and radish (Cherry
Belle), Raphanus sativa L. -- are representative of economically important
plants and different plant families. Seeds of the selected species should
A-44
-------
germinate, grow rapidly, contain no natural inhibitors, and require no
special pretreatment. All test organisms are grown under identical
environmental conditions (constant temperature, 25°C, constant dark, and
enclosed to maintain uniformly high relative humidity).
Although inhibition of root elongation and germination are observable
toxic responses, root elongation inhibition is the preferred endpoint in
this bioassay. Usually, elongation is inhibited at lower concentrations of
toxic substances than is seed germination.
Materials and Methods
Facilities
The facilities must include work areas for planting seed and for
measurements, preferably isolated from other activities. There should be a
fume hood, a distilled water source and refrigeration available at 4°C. The
test facility must have a controlled environmental chamber capable of
maintaining a uniform temperature at 25°C within +2.0^C.
Test Containers
One-piece molded-glass tanks (for example, Anchor-Hocking Glass Co.,
Lancaster, Ohio 43130), with a 6-liter capacity (approximately 24 cm (L) x
16 cm (W) x 18 cm (H)) are used for dosing seeds. Glass plates (13 cm x
15 cm) of single-strength window glass are prepared with polished edges.
The glass plates are supported at a 67° angle in the tank with glass pegs.
The pegs are 2 to 3 cm long and 5 mm in diameter. Twenty pegs are attached
with epoxy to the inside of each glass tank (Figure A-l). An alternative is
A-45
-------
I
-e»
CT>
en
01
Ol
3
V>
X
3
VI
n>
-*
ri-
ft)
CL
UQ
_^
Oi
CD
O)
V)
to
n>
n>
3
O
ft)
o.
t»
•o
o>
(A
r*
—i.
O
CT
O»
ua
3"
IQ
W)
in
TJ
O>
to
U)
3
VI
(D
-J
rt-
n>
o.
tr
(D
rf
•x.
(D
ft)
3
O
o.
tn
Figure A-l. Example Presentation of Setup of Glass Tanks for
RE Test,
-------
a glass rack (for example, Shamrock Scientific Glassware, Little Rock,
Arkansas 72205) constructed from two glass rods (approximately 23 cm long)
and six half-circles (12 cm O.D) of glass tubing connected to the rods at
right angles at 35 mm intervals.
Equipment
Items specifically needed include a spray bottle with a fog or mist
nozzle, metric ruler, forceps, Soxhlet extraction apparatus, triple beam
balance, pH meter, storage bottles, and plastic bags (minimum of 60 cm x
20 cm x 36 cm). An illuminated magnifier may be helpful for planting,
seedling examination, and root measurement.
Test Organisms
The seeds used in the test are available from commercial seed
companies, State Agricultural Experiment Stations, and laboratories of the
U.S. Department of Agriculture. Seed from one seed lot for each species
should be purchased in amounts adequate for 1-year's testing. Information
on seed lot, the seed year or growing season collected and germination
percentage should be provided by the source of seed. Only untreated (not
treated with fungicides, repellants, etc.) seed is acceptable for the
bioassessment protocol.
Size Grading of Seed
After purchase, size grading is carried out on the entire seed lot for
each kind of seed. Small samples of 100-150 g are sized at a time. The
seed lot is inspected and trash, empty hulls, and damaged seed are removed.
Depending on species, select a series of four screens to separate sample
into size classes (see Table A-9). The four screens are nested with the
A-47
-------
Table A-9
HAND SCREENS FOR SIZING SEEDS
(From Brusick and Young, 1982)
Perforated Metal Sheet
Species
Red Clover
Radish
Wheat
Cucumber
Round Holes
1/19, 1/18, 1/17, 1/16
(Fractions of an inch)
6-1/2, 7, 7-1/2, 8
(64ths of an inch)
9, 9-1/2, 10, 10-1/2
(64ths of an inch)
Oblong Holes
Wire Mesh
1/13 x 1/2
1/14 x 1/2
1/15 x 1/2
1/16 x 1/2
(fractions of an inch)
Lettuce
1/6 x 1/28
1/6 x 1/30
1/6 x 1/32
1/6 x 1/34
(fractions
of an inch)
a Supplied by (for example), A.T. Ferrell and Company, Saginaw, Michigan
48601, or Seedburo Equipment Company, Chicago, Illinois 60607.
screen containing the largest holes on top and screens with successively
smaller holes in sequence below. A blank or bottom pan collects the
fraction that passes through all screens. Seed is poured onto the top
screen and the whole set of nested screens are shaken (by hand or with a
vibrator) until all the seed remains on one screen or reaches the bottom
pan. The separated fractions are collected and saved. The procedure is
repeated until all the seed in the lot is sized. That size class which
contains the most seed is selected and used exclusively for the duration of
the tests. The seeds in the size class are divided into small lots, placed
A-48
-------
in separate envelopes or sacks, and stored in moisture-proof sealed
containers in a refrigerator at 5°C.
Preparation of Glassware
The glass tanks (fitted with glass pegs or tanks with glass racks) and
glass plates are thoroughly washed as described under Cleaning and
Preparation of Glassware in the aquatic section.
Tissue Paper Precleaning
Eight to 10 sheets of single-ply cellulose tissue (for example,
Kimwipes) are placed in a Soxhlet Extractor and extracted using standard
chemical procedures with distilled water for a minimum of 24 hours (4
cycles/hour). After extraction, the tissues are removed, air dried, and
stored in a dry glass container.
Test Procedures
Test Medium
The test medium is an aqueous extract of a particulate or solid sample.
Aqueous extracts of solids are prepared using the procedure outlined in
Table A-8. Aqueous extractions of solid samples should be tested as soon as
possible or the solid sample must be stored in closed polyethylene
containers until extraction can be made. Dilutions of the extract or
aqueous extractions should be made without use of solvents or additives
except for distilled water, which is used as a negative control. Acids and
salinity will cause toxicity in some cases. Generally, extracts with pH
greater than 6.5 and salinity lower than 0.01 N salt will not be toxic.
A-49
-------
Acid and saline soils may exert seed elongation toxicity even without
toxicants and it may be necessary to compare results to a artificial
control.
Procedure for Planting Seed
Whatman No. 3MM chromatography filter paper rectangles (13 x 15 cm) are
soaked in the test solution in a shallow tray for a minimum of 5 minutes to
saturate. One sheet of filter paper is removed from the test solution,
allowed to drain, and placed on a glass plate to which the paper adheres.
Trapping air bubbles between the filter paper and the glass plate should be
avoided. Using forceps, 15 seeds from one species are placed on the filter
paper substrate in a row, equally spaced, across the top of the plate 2.5 cm
down from the top edge. Seeds are placed with the radicle end (embryo or
germ) toward the bottom of the plate (Figure A-2) and, in the case of wheat,
with embryo side of the seed up. A narrow strip, (1/2 cm wide) of
previously cleaned (Soxhlet Extraction) single-ply tissue is placed over the
row of seeds to hold them in place and, if necessary, sprayed with just
enough fine distilled water mist to cause the tissue to cling to the seeds
and filter paper. Test solution, usually 500 ml, is poured into the
rectangular glass tank fitted with glass peg guides (empty tank if glass
rack is used). The glass plate holding seed and substrate is inserted in
the glass tank between the glass peg guides or in the glass rack to support
the plates at a 67° angle with the horizontal (Figure A-l). The lower end
of the plate opposite the seeds should be immersed in the test solution with
a minimum of 2 cm, but not more than 3 cm, of the plate and filter paper in
the solution. Solution volumes smaller than 500 ml can be used if clean
inert glass beads are added to the solution to displace and raise the liquid
level. This procedure is repeated for each seed type (lettuce, radish,
A-50
-------
Ul
I
*.«t.n ma Marved) «* r.dfe1. end town. th. botto, of .,.»..
Seed being covered "with natron strips o'f tissue paper.
etow seed (enlarged) wlth.radlcli end tomrd bottae of plate.
Fine distilled water Mist causes tissue to dine to seed and filter paper.
Figure A-2. Examples of Preparing and Orienting the Seeds for the RE Test.
-------
wheat, cucumber, red clover).
Incubation
The glass tank containing 5 plates with 15 seeds each and the test
solution is enclosed in a heavy plastic bag and tied shut (Figure A-l). The
enclosed tank is placed in the dark, 25+2°C controlled chamber. A tank is
prepared for each test solution of sample solution, the positive controls,
and the negative (distilled water) controls. The concentration range of NaF
for the positive controls which causes an EC5Q for each seed species is:
radish 400-500 mg NaF/1; wheat 300-400 mg NaF/1; lettuce 100-200 mg NaF/1;
cucumber 150-200 mg NaF/1; and red clover 80-100 mg NaF/1.
Measurement of Root Length
Measurement of root length is made at 115 hours from the start of dark
incubation. It is important to measure each plate as nearly as possible to
115 hours (not to exceed +30 minutes). To measure root length, remove a
plate from the tank and place it on a flat surface. The lengths of all
roots are measured to the nearest millimeter and entered on the data sheet.
Measure from the transition point between hypocotyl and root to the tip of
the root (Figure A-3). At the transition between the hypocotyl and the
primary root the axis may be slightly swollen, contain a slight crook, or
change noticeably in size (radish, lettuce, cucumber, red clover). In
wheat,.the single longest primary or seminal root is measured from the point
of attachment to the root tip. For additional descriptions and photographs
helpful in making root measurement's, see USDA (1952) and Wellington (1961).
A-52
-------
U1
UI
II
i
r*
5
I
1
•*
g
n
3
o
3
a.
Figure A-3. Examples of Measuring Root Elongations 1n the RE Test (Bruslck et_al_'»
-------
Range-finding Test
The purposes of the range-finding test are to determine if definitive
testing is necessary, and to aid in the selection of concentrations to be
used in the definitive test when needed. The range-finding test consists of
one control tank, and one tank each of 100, 10, 1, 0.1, and 0.01 percent
extract.
A species need not be included in the definitive test if the tank
containing 100 percent extract had mean root lengths of at least 65 percent
of control and at least 10 of 15 seeds germinated. Also, in this situation
it is not necessary to examine the plates containing this species in the 10
to 0.01 percent tanks. If one or more of the species show mean root lengths
less than 50 percent of the control at even the most dilute concentrations,
it is advisable to extend the range and repeat the range-finding test before
proceeding to the definitive test.
Definitive Test
Estimation of an EC^Q in this test will require a control and at least
six extract concentrations chosen in a geometric series. The highest
concentration in the definitive test should be the next concentration
greater than the range-finding concentration which reduces mean root length
to less than 50 percent of the control. For example, if the range-finding
test shows that 1 percent extract causes mean root lengths less than 50
percent of the control, then the definitive test would begin at 10 percent
extract. In a geometric series, ihe ratio of one concentration-to the next
is the same: for the above example, 10, 5, 2.5, 1.25, 0.615, and 0.312
percent. If more than six concentrations are used, not all species must be
tested at all concentrations. However, each species must be tested with at
A-54
-------
least six concentrations and those concentrations must be in a geometric
series.
Results and Data Interpretation
Assay Acceptance Criteria
To accurately estimate the EC™, specific criteria must be met for each
of the test species. For the definitive test, criteria 1, 2, and 3 must be
met:
1. At least 10 of 15 seeds on the control plate must germinate.
2. Each test concentration in a series must be at least 50
percent as strong as the next concentration, except for the
control.
3. Four toxic concentrations must be defined in the definitive
test for which mean root length is inhibited in the following
ranges: less than or equal to 25 percent, less than or equal
to 50 but more than 25 percent, more than 50 but less than or
equal to 75 percent, and the lowest concentration which is
more than 75 but less than or equal to 90 percent.
Since most toxicants affect root elongation at lower concentrations
than germination, criterion 3 must be met to satisfy the requirements of the
definitive test in addition to criteria 1 and 2. If a species fails to
satisfy criteria 1, 2, and 3, the definitive test must be repeated for that
species.
A-55
-------
Calculations and Reporting
Provided criteria 1, 2, and 3 are met in the definitive test, the EC5Q
is estimated in the following manner. For each species which satisfied
these three criteria, plot on semi-log paper sample concentration on the
logarithmic axis and percent control mean root length on the arithmetic
axis. Draw a straight line between the four test sample concentrations used
to satisfy criterion 3. The concentration at which this line crosses the 50
percent point for the control root length is the EC50 for root elongation.
If no effects were seen with 100 percent sample, or if criterion 3 could not
be met due to germination inhibition, it is not possible to estimate an EC50
for root elongation.
For each of the species tested either the concentration in fa) £r (b)
must be calculated and reported.
(a) If the species satisfied criteria 1, 2, and 3, report an
estimated ECgg for root elongation. Use graphical
interpolation to estimate the EC5Q and rank the test sample
using the evaluation criteria in Table A-l.
(b) If the species satisfied criteria 1 and 2 but not criterion 3
(criterion 4 or 5 used instead), report the lowest
concentration for which fewer than 10 of 15 seeds germinated.
The ECgQ cannot be estimated for root elongation or
inhibition of seed germination from data in this category.
Currently, test samples are not ranked from data of this
type.
A-56
-------
EARTHWORM TEST
Introduction and Rationale
Earthworms have been selected as an indicator species because they are
representative of the terrestrial environment and are of considerable
importance in improving soil aeration, drainage and fertility (Edwards and
Lofty, 1972). The tests developed in this protocol were taken from the
European Economic Council Guideline for Testing of Chemical Toxicity to
Earthworms (OECD, 1981).
Earthworms differ from aquatic organisms in that they may be exposed to
toxic chemicals in the aqueous phase via soil moisture, in the vapor phase,
or by coming into contact with particulate matter on the surface of soil
constituents. Moreover, they may be protected in soil because many
chemicals become tightly adsorbed onto soil fractions, particularly organic
matter, and the soil colloids making up the clay fraction.
Hence, a simple immersion test, which yielded consistent and
reproducible results for relatively soluble chemicals or formulated
pesticides, was rejected because it would not provide information on
comparatively insoluble compounds which affect the worms only when they are
In direct contact, or on compounds'"which affect the worms only as a vapor.
There are tests which involve the injection of test chemicals either
into the pharynx or body of the worms and although these give reproducible
A-57
-------
results, they require considerable expertise and have the drawback that it
1s difficult to relate the results of such tests to field conditions.
The test method is proposed as a two-stage test. The first stage would
be a relatively simple contact toxicity test involving exposure of the worms
to extracts on filter paper to examine potential toxicity. Toxic samples
would then be tested further using soils or applications of extracts in a
defined soil medium. The contact test was chosen because the exposure of
the worms in such a test more closely resembles the natural situation.
To provide a routine test for the protocol, a commonly used test
species was selected. Eisenia foetida is not a common species in soil
although it does occur in soils with considerable organic matter. It is
common in sewage beds, particularly in trickling filters, where it is
exposed to industrial chemicals. It is a species with a short life cycle,
reaching maturity in seven to eight weeks at 15-20°C. It is prolific; a
single worm produces 2-5 cocoons per week each of which will give several
worms. It can be bred readily in a wide range of organic wastes. This
means that laboratories could easily breed their own stock if supplied with
cocoons from a central source, and a standard strain could be used.
A-58
-------
Materials and Methods
Test Organisms
Test organisms should be adult Eisenia foetida (at least 2 months old
with a clitellum) of weight 400 - 800 mg. All worms for a specific test
should be from the same breeding box. Individual worms are used (1/vial) in
the range-finding test; ten individual worms (about 4 to 8 g) should be
added to each test container for the definitive test.
Breeding of Test Organisms
Eisenia foetida can be bred in a wide range of animal wastes. The
recommended breeding medium is a 50:50 mixture of horse manure and peat, but
other animal wastes are also suitable. The medium should be of pH about
7.0, have low conductivity (less than 6.0 umho/cm) and not be contaminated
excessively with ammonia or animal urine. Wooden boxes 500 x 500 x 15 cm
with tightly fitting lids are ideal for large-scale breeding and should
produce more than 1000 worms in six weeks. To produce sufficient worms,
such a medium will support 1 kg worms in 20 kg waste and each worm will
weigh up to 1 g. To obtain worms of standard age and weight it is best to
start the culture with cocoons which take three weeks to hatch and seven
weeks to become mature worms at 20°C.
Test Procedures
Range-Finding; Contact Test
Glass vials, 8 cm long x 3 cm diameter are recommended. The sides of
these are lined with a strip of filter paper 9.5 x 6.7 cm (Whatman Grade 1).
A-59
-------
The extract is applied in water as appropriate, to give a range of known
concentrations. The control should be treated with distilled water only.
It is recommended that the toxic dose ranqe be established in a
preliminary test after which a more precise test may be made with a
restricted dose range. The doses are calculated in terms of volume of
extract diluted with distilled water to give the following concentrations:
100 percent, 10 percent, 1 percent extract.
For a more precise contact test, five doses in a geometric series
(e.g. in the ratio 100, 50, 25, 12.5, 6.25) should be used. For each test,
ten replicates per dose, of one worm per vial, would be the minimum
requirement. Do not use more than one worm per vial.
In each test, a range of doses of extract plus a positive control using
0.354 mg Cu/1 copper sulfate. This concentration will provide a response
range of 0.9 to 1.1 of the LCcn. A negative control should be used.
Vials should be laid on their sides for the duration of the test.
Test temperature = 20 +2°C.
Test in continuous dark.
Test duration = 48 hours.
Worms should be classed as dead when they do not respond to a gentle
mechanical stimulus to the anterior end.
Discard vials after the test.
A-60
-------
Definitive Test: Artificial Soil Test
In this test, worm survival is evaluated after 14 days in a mixture of
an artificial soil (Table A-10) and soil samples or extracts of soils from
the site. Extracts can be added to the artificial soil to aid in defining
concentrations to test using artificial soil plus soil samples. Otherwise
the definitive test can directly be applied using mixtures of site soil and
artificial soil. Soil is a variable medium so for this test a carefully
defined artificial loam soil is used. It was developed specifically as a
growth medium for the earthworm test. The medium for the definitive test
should be based upon the three general constituents listed in Table A-10;
sieve and chemical analyses are not required. This artificial soil mixes
well and Eisenia foetida will survive in it for long periods. Its
absorptive capacity is similar to that of a typical arable soil.
The dry constituents are blended in the correct proportions and
thoroughly mixed mechanically in either a large-scale laboratory mixer or
small electric cement mixer. The peat is finely ground in a laboratory
mill, and the pH is adjusted to 7.0 by addition of appropriate amounts of
calcium carbonate. Moisture content is then determined by drying a small
sample at 80°C and reweighing. From these data, the amount of deionized
water required to achieve a moisture content of 20 percent of dry weight is
calculated (25 g water per 100 g of dry soil). This is added and the medium
remixed before use.
A-61
-------
Table A-10
COMPONENTS OF ARTIFICIAL SOIL
General Composition by Weight
1. 70% Industrial Sand
2. 20% Kaolinite Clay
3. 10% Sphagnum Peat
Specific Composition
1. Industrial Sand
Diameter in Microns
45
45
63
90
125
180
250 & greater
Percent
1.7
9.3
29.0
34.3
20.8
4.0
0.8
2. Kaolinite Clay
Composition
S102
T102
A12°3
Fe2°3
MgO
CaO
Na20
loss on ignition
Percent
58.5
1.3
28.0
1.0
0.3
0.2
2.0
0.3
8.4
A-62
-------
Test Conditions for Artificial Soil and Soil Sample Extracts
A range of dosage levels identified from the contact test results will
be used in these tests. As far as possible, the range of doses selected
should be chosen so that some are on either side of the estimated LCgQ.
This is expected to bracket the middle concentration (i.e. LCgg) of the
definitive test and other concentrations in the series would be selected
accordingly. For example, if the contact test indicated that the median
lethal concentration was in the area of 40 percent of the extract (4 parts
extract to 6 parts deionized water), the range used would be 0 (negative
control), 20 percent, 40 percent, 60 percent and 80 percent. For a total of
100 parts, the sum of extract and deionized water (V/V) would be 0 + 100;
20 + 80, 40 + 60, 60 + 40, and 80 + 20, respectively. The test containers
are 500 ml crystallizing dishes, containing 400 g of the artificial soil,
covered with plastic lids, petri dishes, or plastic film.
In each test, one positive -control with copper sulphate at a
concentration of 600 mg Cu/kg of prepared soil and four control containers
treated with solvent blank (distilled water) should be used. Mortality
should be assessed by emptying the soil into a tray, sorting out the worms
and testing their reaction to a mechanical stimulus to the anterior end.
For each test, four replicates per dose with ten test worms should be used.
The average weight of test and control worms should be calculated at the
beginning and end of the test.
A-63
-------
Test Conditions With Artificial and Sample Soils
Soil samples collected from the site must be prepared for mixing and
"dilution" with the artificial soil. Soil is prepared by the procedures
outlined in steps in Table A-ll. Then, usina a top loading balance,
appropriate amounts of site soil and artificial soil are weighed to prepare
the amount of soil needed for the appropriate tests.
Test containers are 500 ml crystallizing dishes covered with plastic
lids, petri dishes, or plastic film. In each dish 400 g of the moist test
medium is used. For each test dose, a 1600 g mixture of the moist,
prepared, artificial soil and freshly sampled soil is prepared. For
example, the test concentration desired is 75 percent. Therefore 400 g of
moist artificial soil is added to 1200 g of sample soil and thoroughly
mixed. Then, four 400 g aliquots are weighed out and placed in each 500 ml
crystallizing dish test container. For practical reasons, the sample soil
concentration should never exceed 75 percent. Range finding test
concentrations could be 0.75, 0.15, 0.075. Definitive test concentrations
could be 0.75 0.5, 0.25, 0.125, 0.0625, 0.03125.
Copper sulfate is the positive control and should be included in the
soil assay by adding a total of 0.96 g Cu with the deionized water to
achieve 600 mg Cu/kg of Moist Soil. The main purpose of the positive
control is to account for variability in the test organisms.
For each test, four replicates of 400 g, each containing ten test worms
should be used. A positive and negative control (100 percent artificial
soil), each with four containers, should be used. Mortality should be
A-64
-------
Table A-ll
PROCEDURE FOR HOMOGENIZING SOIL SAMPLES
(Lighthart, 1980; Unpublished Procedure)
1. Air dry soil to be tested. (Air drying is considered completed when an
aliquot of soil has no more weight loss.)
2. Add 25 burundum cylinders and about 2 liters of air dried soil to a
ball mill.
3. Mill to coffee ground size (ca. 5 minutes) then sieve through a 2mm
mesh sieve.
4. Return larger particles back to the ball mill and repeat steps 2
through 4 until the sample is completely ground with the exception of
rocks. Discard rocks.
5. Homogenize soil using a laboratory or small cement mixer thoroughly
before use.
6. Clean the ball mill by adding 1 quart of silica sand and 10 burundum
cylinders. Mill for 15 minutes, discard, and then brush out mill.
A-65
-------
assessed by emptying the soil into a tray, sorting out the worms and testing
their reaction to a mechanical anterior stimulus.
The average weight of the test and control worms should be determined
at the beginning and end of the test. Environmental conditions should be:
The test temperature - 20 +!°C.
The soil moisture should be 20 +5 percent
(add 25 ml deionized water/100 g of air-dry soil).
Test in continuous light.
Test duration = 14 days.
An assessment of mortality at 7 days and continuation of test to 28 days is
optional. If more than one mortality assessment is made it may be necessary
to adjust the moisture content of the soil due to losses during sorting.
Results and Data Interpretation
The mortality/dose data should be plotted on log probit graph paper and
the median lethal concentration (LC5Q) and its confidence limits estimated.
If the LCgQ cannot be established the LCQ and LCj00 values should be given.
Mortality in negative controls should not exceed 10 percent. If there
is some mortality a correction based on Abbott's formula can be made:
Corrected mortality % = Observed mortality % - control mortality %
100 - control mortality %
The LCc0 values should be given as percent of sample soil (W/W).
A-66
-------
SOIL RESPIRATION
Introduction and Rationale
Soil/litter microcosms can be used to define the impact of pollutants
upon primary productivity in terrestrial ecosystems (Lighthart, 1980). The
measurement of evolved C02 from microbial respiration in these microcosms
can indicate the degree of pollutant stress within the system. Low CX^
levels indicate high stress whereas no significant change or an increase in
C0£ can identify low pollutant stress or even stimulation of microbial
activity.
A simple soil/litter microcosm toxicity test can be conducted within 14
days. Soil micro-organisms are exposed to various concentrations of the
test materials (soil and/or soil extracts) at standard moisture and
temperature conditions. Evolution of C0« is measured at predetermined time
intervals throughout the test. Results are expressed as percent inhibition
(EC,-Q) or stimulation (SC^n) between CO^ evolved in control and amended
microcosms at specified time intervals.
Materials and Methods
Glassware includes one quart regular Mason (one liter) jars with air
tight lids and one ounce (30 ml)- glass bottles with air tight lids. These
should be washed according to GENERAL INSTRUCTIONS for the aquatic tests.
A-67
-------
Triplicate microcosms for each treatment are prepared by placing 100 g
of air-dry artificial soil, or combinations with test soil (Table A-ll)
sieved to pass a 2mm screen into each of three Mason jars. Then, 20 ml of
deionized water is added for appropriate moisture conditions in the
microcosm. Finally, a one ounce glass bottle with C02 trapping solution is
added and the air-tight Mason jar lid is sealed securely. A special blank
must be used to correct for atmospheric C02 during titration. This consists
of 3 clean Mason jars without soil and C02 trap but which are run
concurrently with controls and test jars.
Equipment should include a constant-temperature room or incubator
capable of providing temperature control of 20 ^2°C. A ten liter capacity
ball mill with 25 3.2 x 3.2cm (1-1/4") burundum cylinders and a 2mm mesh
sieve. Standard laboratory equipment such as balances, pH meters,
pipettors, magnetic mixers and bars, drying ovens, and appropriate glassware
necessary to prepare reagents and perform the titration of C02 are also
needed.
Test Procedure
The test material is introduced into the microcosm either as a soil or
soil extract. The soil sample may be added directly (lOOg) or by weight
\ *
percentages as in the earthworm test and added to the artificial soil. The
aqueous test extracts are introduced into the artificial microcosms on a
percent basis (V/W), i.e. 100, 50, 25, 12.5, 6.25, 3.125, where 100 percent
represents 25 ml of soil extract and further dilutions of extract are made
with deionized water (Table A-8). The extracts and acid traps are added
A-68
-------
after two days of incubation at 20°C in the dark if extract is to be
studied.
Respiratory carbon dioxide is measured in the alkali traps twice weekly
for the duration of the test which is typically 2 weeks.
Three replicate microcosms are required for each control and test (soil
and/or soil extract) concentration. Each microcosm is incubated at 20 _+2°C
in the dark for 14 days. The evolution of C02 is measured twice weekly by
titration. Reagents and titration procedures are outlined in Table A-12.
Results and Data Interpretation
The total C02 produced during the 14 day test is obtained by summing
individual C02 measurements for each interval. Percent inhibition (I), or
stimulation (S), is calculated after 14 days for each test concentration
according to the following formulas:
*I = ^-^- X 100
«S = ^-£ X 100
where C is the mean C02 evolution in the control and T is the mean growth in
the treated microcosm. Three endpoints are calculated from the percent
response vs. concentration data. For samples which are inhibitory, an EC5Q
(defined as the lowest test concentration causing growth inhibition of 50
A-69
-------
Table A-12
PROCEDURES OF TITRATING CO, IN TRAPS AND
METHODS FOR PREPARING REAGENTS
Taken from Lighthart, 1980; Unpublished Procedure)
A. C02 Titration Procedure
a. Replace the C02 traps at the designated intervals by openina the
microcosm and removing the exposed C0? trap and replacing it with
an unexposed one. (At the same time tnis step is being performed,
insert an open vacuum line to aid in properly replenishing the air
in the microcosm. Remove at least 3 times the volume of the air
space.)
b. As quickly as practical, place an air tight cap on the exposed C0«
trap; return the microcosms to the 20 C dark incubator.
c. Add five ml of 1.3N of BaCl? and a stir bar to each exposed C0?
trap immediately prior to tltration.
d. Titrate excess 0.6N NaOH remaining in the trap to pH 9.0 with a
buret and pH meter (or autotitrator) using Trizma standardized
0.6N HC1 to measure milligrams of C0« produced.
Formula for the Calculation of C0? Production:
mg of C0? = (Blank ml - Sample ml) x 22 mg of CO?/ml/N x Normality of Acid
e.g., mg^of C0? = (10.40 ml - 6.93 ml) x 22 mg of C0?/ml/n x 0.6013 N
« 45.90 mg of C02 produced
B. Preparation of Reagents
1. 0.6 NaOH
a. Rinse 20 liter glass carboy with distilled H?0.
b. Place on a large magnetic stir plate; add degassed distilled H90
to the 18.9 liter mark. *
c. Add 454 grams (1 Ib) of NaOH pellets.
d. Stopper and stir overnight before use. (Maintain the NaOH stock
solution in a C02 free atmosphere by using ascarite traps.
2. 0.6N HC1
a. Rinse 20 liter glass carboy with distilled H?0.
b. Add 1.0 liter of concentrated HC1.
c. Add distilled KLO until the 20 liter mark.
d. Stopper and stir overnight.
e. Titrate 5 "Tris" samples (0.5 to 0.9 grams of "tris" in 10.0 ml of
. .. distilled H?0 and 5 ml of 1.2N BaCl9) to pH 5.0 with ca. 0.7N
HC1; calcolate mean and standard deviation ("s"). (If "s" is
larger than 0.0015, do 5 more samples and combine results.)
ffl.1211 g/meq) (ml of HC1 usedV
Normality of HC1 = (Weight of Tris in grams)
(0.1211 q/meq) (9.69 ml)
e.g., Normality of HC1 »0.7089 gramsm 0i6041N
A-70
-------
Table A-12 (Continued)
3. 1.3N Bad-
a. Weign 317.56 grams BaCl2:2H20.
b. Dissolved in degassed distilled H90 in a 1 liter volumetric
flask. *
4. Tris
Aminonrethane(hydroxymethyl)tris--Trizma Base (Sigma Chemical Company,
St. Louis, Missouri).
A-71
-------
percent relative to control) is calculated. For samples which are
stimulatory, and SCgQ (defined as the lowest concentration causing growth
stimulation of 20 percent relative to control) is calculated. Also, the
measurements of C(L made a other times can be used to evaluate anomalous
results and to observe time trends of C02 production.
A-72
-------
REFERENCES CITED
American Public Health Association. 1981. "Standard Methods for the
Examination of Water and Wastewater," 15th ed. New York.
Asplund, R.D. 1969. "Some Quantitative Aspects of the Phytotoxicity of
Monoterpenes." Weed Sci., pp 454-455.
ASTM. 1980. Standard Practice for conducting acute toxicity tests with
fishes, macroinvertebrates, and amphibians. E 729-80. Amer. Soc.
for Testing and Materials. Philadelphia, Pennsylvania. 25 p.
Brusick, D.J. and R.R. Young. 1982. "IERL-RTP Procedures Manual: Level
1 Environmental Assessment Biological Tests." USEPA Report. PB
82-228/966. NTIS, 5285 Port Royal Road., Springfield, Virginia 22161.
Committee on Methods for Toxicity Tests with Aquatic Organisms. 1975.
"Methods for Acute Toxicity Tests with Fish, Macroinvertebrates and
Amphibians." EPA-660/3-75-009, PB 242105. NTIS, 5285 Port Royal Road,
Springfield, Virginia 22161
Davis, H.G. 1953. "Culture of Disease of Game Fishes." University of
California Press, Berkeley, California.
Durrant, M.J., Draycott, A.P. and Payne, P.A. 1974. "Some Effect of NaCl
on Germination and Seedling Growth of Sugar Beet," Ann. Bot.,
38:1045-1051.
Edwards, C.A. and J. Lofty. 1972. Biology of earthworms. Chappman and
Hall, Ltd. London.
Hikino, H. 1978. "Study on the Development of the Test Methods for
Evaluation of the Effects of Chemicals on Plants," Chemical Research
Report No. 4, Office of Health Studies, Environmental Agency, Japan.
Hoffman, G.L. and Meyer, F.L. 1974. "Parasites of Freshwater Fishes,"
TFH Publications, Inc. Neptune City, New Jersey.
Horowlitz, M. 1976. "Application of Bioassay Techniques to Herbicide
Investigations," Weed Research, 15: 209-215.
Imai, I. and Siege!, S.M. 1973. "A Specific Response to Toxic Cadmium
Levels in Red Kidney Bean Embryos" Physiol. Plant., 29:118-120.
Kenega, E. 1981. Comparative Toxicity of 131496 Chemicals to Plant Seeds.
Ecotoxicology and Environmental Safety, 5:469-475.
Lighthart, B. and Bond, H. 1976. "Design and Preliminary Results from
Soil/Litter Microcosms." Int. J. Environ. Stud., 10:51-58.
Lighthart, B. 1980. Effects of Certain Cadmium Species on Pure and Litter
Populations of Microorganisms. Antonie van Leeuwenhoek, 46:161-167.
A-73
-------
Miller, W.E., Greene, J.C. and Shiroyama, T. 1978. "The Selenastrum
capricornutum Printz Algal Assay Bottle Test," EPA-600/9-78-Q18.
USEPA. Corvallis, Oregon.
Mount, D.I. and Brungs, W.A. 1967. "A Simplified Dosing Apparatus for
Fish Toxicological Studies," Water Res., 21-29.
Muller, W.H. 1965. "Volatile Materials Produced by Salvia leucophylla:
Effects on Seedling Growth and Soil Bacteria," Bot. Gaz., 126:195-200.
Neiman, R.H. and Poulsen, L.L. 1971. "Plant Growth Suppression on Saline
Media: Interactions with Light," Bot. Gaz., 132:13-19.
OECD. 1981. Test Guidelines for the Assessment of Toxicity to Earthworms
(Eisenia foetida Sav.). Laboratory Test. OECD, Chemical Testing
Programme, Ecotoxicology Group. GGA AP - 3000 bu. 2600. Paris.
Porcella, D.B. and Cleave, M.L. 1981. The Use of Bioassay Approaches for
Assessing Phytoplankton Growth in Lakes and Reservoirs.
Phytoplankton - Environmental Interactions in Reservoirs. Volume I.
(M.W. Lorenzen, Ed.) Tech Rept. 9-81-13. U.S. Army Engr. Waterways
Exp.Station. Vicksburg, Mississippi, pp 276-314.
Reichenback-Klinke, H. and Elkan, E. 1965. "The Principal Diseases of
Lower Vertebrates," Academic Press, New York.
Rubinstein, R. et.aJL,1975. "Test Methods for Assessing the Effect of
Chemicals on Plants," EPA-560/5-75-008 (NTIS PB 248198), Franklin
Institute Research Laboratories, Philadelphia, Pennsylvania.
Santelman, P.W. 1972. "Herbicide Bioassay," Research Methods in Weed
Science, Weed Sci. Soc., USA, pp 91-101.
Snieszko, S.F. (Ed.). 1970. "A Symposium on Diseases of Fishes and
Shell-fishes," Spec. Publ. 5, American Fisheries Society, Washington,
D.C.
Stephan, C.E. 1977. "Methods for Calculating an LC,-n," Aquatic Toxicology
and Hazard Evaluation, ASTM STP 634 (F.L. Mayir and J.L. Hamelink,
Eds.), American Society for Testing Materials. Philadelphia,
Pennsylvania.
USDA. 1952. "Manual for Testing Agricultural Horticultural Seeds,"
Agriculture Handbook No. 30. Washington, D.C.
USEPA; 1979.NationalEnvironmental Research Center, Methods Development and
Quality Assurance Research Lab. "Methods for Chemical Analysis of
Water and Wastes," USEPA-600/4-79-020, EPA, Office of Technology
Transfer, Washington, D.C. -
USEPA, 1980 (Draft). "Guidelines and Specifications for Implementing
Quality Assurance Requirements: Demonstration Grants and Cooperative
Agreements Involving Environmental Measurements." QAMS-003/80/01.
USEPA. ORD. Washington, D.C.
A-74
-------
Van Duijn, C. 1973. "Diseases of Fishes," (3rd ed.) Charles C. Thomas.
Springfield, Illinois.
Veith, G.D., D.L. Defoe, and B.V. Bergstedt. 1979. Measuring and
estimating the bioconcentration factor of chemicals in fish. J. Fish.
Res. Board Can., 36:1040-1048.
Walley, K., Kahn, M.S.I, and Bradshaw, A.D. 1974. "The Potential for
Evolution of Heavy Metal Tolerance in Plants. I. Copper and Zinc
Tolerance in Aqrostis tenuis." Heredity, 32:309-319.
Wellington, P.S. 1961. "Handbook for Seedling Evaluation," National
Institute of Agricultural Botany. Cambridge, United Kingdom.
A-75
-------
APPENDIX B
GUIDANCE ON SAFETY PROCEDURES
FOR WORKING WITH
SAMPLES FROM HAZARDOUS WASTE SITES
USING THE
BIOASSESSMENT PROTOCOL
-------
GUIDELINES AND CONCEPTS OF SAFE PROCEDURES
The objective of these guidelines is to protect workers, the public,
and the environment, and to insure that contamination does not occur and
interfere with valid laboratory results. The major factor in providing this
protection is the common sense of the staff performing the bioassessments.
The guidance presented herein is designed to complement this common sense.
For example, it is extremely important that all staff follow good
housekeeping procedures and maintain personal grooming and cleanliness
within the confines of the laboratory area. There are safety courses that
are available through OSHA and these should be taken wherever possible.
Also there is access to experienced personnel within states, regions, or
local communities. These should be drawn on prior to starting and whenever
any possible hazards might occur that were not considered.
Whichever safety procedures are utilized, they must be commensurate
with the hazard, and hazard depends on the concentration and types of
materials that will cause exposure. Hopefully, explosive, ignitable,
corrosive, or otherwise highly reactive samples will not be evaluated in the
bioassessment protocol. The intent of the protocol is to assess the acute
toxicity of soil and water samples. Sample size should be adequate for
needs and not in great excess as disposal then becomes more of a problem.
A key factor is the safety committee to oversee procedures written in
advance of the setup of the laboratory facilities. This safety committee
should include at least a chemist and a biologist and, whenever possible,
these persons should have experience in hazardous wastes. In the absence of
such experience, a state or other local expert should be utilized.
B-l
-------
The facilities are a key part in maintaining the integrity of the
protective plans. Wherever possible, separate self-contained facilities
should be utilized. For example, field equipment should be labeled for
hazardous waste site use only. Disposable sample containers and protective
gear and equipment should be used where possible. Solid and liquid waste
handling procedures should be specified and materials should be placed in
unbreakable containers that are easily transported. A separate storage area
a separate preparation room, and a separate experimental area should be
required. All of these facilities should be lockable and maintained under
safe and secure conditions.
Air should be supplied using forced air fans and complete exchange of
the air supply in the preparation area should occur on an average of once
every five minutes at the minimum. Air supply in general should be
commensurate with potential hazard and the cost of providing air exchange.
Chemical fume hoods are an excellent means of providing this kind of safety
for sample preparation.
Personnel are protected with respirators, gloves, and laboratory
clothing which are disposable or, in the absence of severe exposures,
washable. Laboratory services should be isolated with appropriate check
valves and/or supply services. This can include air supply, water supply
and gas supplies. Vacuum services should be entirely separate; small
vacuum pumps can provide adequately for most cases.
Finally, personnel involved should be clearly identified and their
utilization of the laboratory facilities involved with hazardous waste
materials should be controlled and recorded. Medical surveillance should be
B-2
-------
implemented where needed. In any case, a complete physical examination with
chemical measurements of blood and urine samples should be implemented prior
to any work in the laboratory area with known hazardous materials. One
should avoid the perception as well as the reality of risk. Specific
procedures are identified in the following paragraphs.
1. Work schedule and procedures. All work to be performed should
be detailed in advance and written out for all personnel
involved and for review by the safety committee. A
responsible person should be designated the hazardous waste
material disposal officer. Detailed procedures on handling of
soil and water samples, dilution procedures in water and
synthetic soils, disposable and unbreakable containers,
storage access, analytical measurements, and pertinent
information should be written out in advance of any
experiment. Standard procedures should be followed to
minimize the paper work involved. However, all personnel
involved must sign a form stating that they have read and
understood the instructions. A simple test designed to
determine their understanding of the procedures can be
maintained in the personnel file.
.. 2«. Sampling, handling and storage. Sampling should be done using
careful procedures since actual sites will have more hazardous
exposure than will the laboratory facilities. In some cases,
it will be wise to subcontract to a firm specializing in
hazardous waste handling in order to collect samples.
Designated, separate field sampling equipment should be
B-3
-------
utilized for collecting water and soil samples.
Samples should be stored in disposable, non-breakable
sample containers. Metals should be placed in polyethylene
containers. Organics should be collected and stored in
disposable glass bottles and bottles packed in absorbent
material that can account for the entire liquid in the sample.
Labels should be affixed to all samples.
3. Personnel. Personnel who are allowed to have access to the
sampling and laboratory facilities should be identified
clearly. Personnel testing or monitoring should be performed
and recorded. For each type of waste, the need for medical
surveillance should be evaluated. Personnel should be
medically tested whenever especially hazardous conditions
occur.
5. Chemical information form. All available information on the
chemicals that are potentially present at a site should be
accumulated. This information will be invaluable in terms of
analyzing potential environmental hazards that exist at a site
for the protocol as well as protecting the personnel.
6. Identification of the potentially most hazardous operations.
It is important that all -operations be written down as 1n item
1 above. The most potentially hazardous operations should be
identified in this section and described in detail. After
identifying these operations it is important to explain what
B-4
-------
procedures should be followed during potential accidents
and/or routine safety procedures. Each operation that may be
included in this section should be carefully identified and
discussed with appropriate clean-up and disposal procedures.
7. Accidental exposure and emergency treatment requirements and
procedures. Based on the chemical list and the potentially
most hazardous operations, appropriate procedures should be
spelled out. Monitoring for potential health problems that
might occur should be detailed in this section.
8. Accidental release information. Accidental releases from
bioassessment procedures are probably not a critical factor.
However, a chain of custody form (see Section 16) must be used
for all sample handling so that storage, utilization,
dilution, and ultimate disposal by the laboratory disposal
officer will be recorded for future use.
9. Waste disposal procedures. All toxic materials, original
samples and high dilutions of samples, must be packaged in
unbreakable containers and deliverable to the disposal
officer. For safety and public relations reasons it is
important to dispose of all contaminated materials in a safe
manner to a hazardous waste site. Segregation of protective
clothing and samples that- are in the low response levels into
one category and intermediate to high response dilutions and
actual samples into a high level category will aid disposal
operations. It is the responsibility of the disposal officer
B-5
-------
to dispose of these using approved state procedures or other
applicable regulations.
10. Personnel protection. Protective clothing including
laboratory coats, and shoes, covers, eye protection, gloves,
and face protection should be specified where necessary. In
addition, respirators and dust filters generally should be
utilized in the preparation room and also where hazards
suggest that it is necessary in the experimental area. Each
individual should carefully wash in a secure area that is
outside of both the preparation room and the laboratory
experimental area. Soap and water are usually adequate for
washing. However, it is important that the protective
clothing be utilized in the preparation room and discarded at
the door in safe containers before exiting to the wash area.
11. Signs. The laboratory facility should be isolated from
general public contact. Authorized personnel should be the
only persons allowed in the experimental area. Warning signs
should be posted and controlled access should be maintained at
all times.
12. Work area identification and access control. Although signs
are necessary for information reasons, it is important that
all areas be locked and public access kept under surveillance
and minimized. The storage area in particular should have
double locking procedures with a signature form and chain of
custody form for samples.
B-6
-------
13. Work surface protection. Preparation rooms and laboratory
facility areas should be covered with disposable plastic
backed absorbent paper.
14. Contaminant devices. All samples should be stored in sealed
containers in the locked storage area. Subsamples can be
prepared in the preparation room and samples returned to the
storage area. After an assay, all samples should be delivered
in disposable containers to the disposal officer.
15. Storage. The storage of samples should be minimized where
possible. Only enough sample to meet the needs of the
bioassessment should be collected plus a minimal safety
margin. After bioassessment and analysis and review of the
results, the samples should be disposed of to prevent
accumulation of old and unusable samples. The storage area
should be double locked and only authorized personnel be
permitted to utilize the locked storage area. Chain of
custody forms should be used to follow all samples.
16. Laboratory transport. A chain of custody form detailing
sources and dates of sampling, descriptions of sample
materials, and potential hazards should follow all samples.
This form should be easy to use and cross referenced to a
permanent record of the sample. As the sample is transported
from individual to individual, a signoff should occur. The
chain of custody form is signed off finally by the disposal
officer after final disposal and then the form returned to the
B-7
-------
file for permanent storage. All laboratory transport should
follow a prescribed procedure from field, to transport, to
laboratory storage, to preparation room where subsampling
occurs and is recorded. The primary sample is returned to
storage and the subsample is analyzed using bioassessment
procedures. At successful termination of the bioassessment
the waste material from the experimental assay and the primary
sample should be transported to the disposal officer and then
disposed of safely at a hazardous waste site.
17. Housekeeping. Good laboratory practices are the best
guarantee of safety of personnel. Detailed procedures
specifying handling, treatment, and disposal of samples and
bioassay organisms should eliminate most potential problems.
Prompt cleanup of all problems should occur to prevent more
serious problems.
18. Laboratory facilities. Separate vacuum lines, water plumbing,
and waste drainage must be provided. Careful labeling and
isolation of facilities and maintenance equipment will
minimize problems.
19. Emergency personnel. Potential problems that might occur and
require emergency personnel should be carefully reviewed.
Samples that do not require emergency personnel should be
handled separately from those that might. If emergency
personnel might be required, it is important to check with
them in advance of such requirements and it is the
B-8
-------
responsibility of the safety committee to insure that this
process is followed.
B-9
-------
TECHNICAL REPORT DATA
(Please read IiiOntctions on the reverse before complenng)
i. REPORT NO.
EPA-600/2-83-054
3. RECIPIENT'S ACCESSION-NO.
241737
J. TITLE AND SUBTITLE
Protocol for Bioassessment of Hazardous Haste Sites
5. REPORT DATE
July 1983
6. PERFORMING ORGANIZATION CODE
7. AUTHOR1S)
D.B. Porcella
8. PERFORMING ORGANIZATION RLPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
10. PROGRAM ELEMENT NO.
Tetra Tech, Inc.
3746 Mt. Diablo Blvd.
Lafayette, CE 94549
11. CONTRACT/GRANT NO.
TC-3547-1 PO 2B0177NALX
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Corvallis, Oregon 97333
13. TYPE OF REPORT AND PERIOD COVERED
project report- final
14. SPONSORING AGENCY CODE
EPA/600/02
15. SUPPLEMENTARY NOTES
Project Officer: '.-I.E. Miller, FTS 420-4669 / S.A. Peterson, FTS 420-4794
16. ABSTRACT
The bioassessment protocol is one of several tools, including chem.ical analysis and
field study, tnat can be used to characterize the potential environmental risk associ-
ated with hazardous waste sites. The protocol can be applied to priority ranking for
deciding the need for cleanup of a site compared to other sites, and to assess cleanup
effectiveness by testinn for potential hazards at the site boundaries or along a samp-
ling transect. Bioassessment involves using defined biological tests to determine the
biological response to concentrations of the biologically active components of soil anc
water samples from a hazardous waste site. The tests are described in the report App-
endix and include aquatic and terrestrial tests. The key to defining site priority or
cleanup effectiveness is in the experimental sampling design. Careful definition of
general and site-snecific issues is necessary. The design should be evaluated in terms
of cost-benefit so that costly errors in environment-risk and economic risk are mini-
mized. Important points about how these concepts relate to sampling design are dis-
cussed. The bioassessment protocol is designed to be a set of tools that are applied
as appropriate to a specific site. Necessary samples are collected to address the
specific issues that occur at the site. Data from chemical analyses and field studies
may be available or may be required based on the results obtained from bioassessment.
7.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b IDENTIFIERS/OPEN ENDED TERMS
C. COSATI i iclii.'Gioup
13. DISTRIBUTION STATEMENT
Release to public
19. SECURITY CLASS (Tins Report/
unclassified
21.
OF PAGES
142
20. SECURITY CLASS (Thispage/
unclassified
22. PRICE
EPA Form 2220-1 19-73)
------- |