EPA-600/9-76-010
MAY 1976
WOhfthY
PROCEDURE
OCSN1
DISPOShL
PERMIT
PROGRN1
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
GULF BREEZE, FLORIDA 32561
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EPA-600/9-76-010
May 1976
BIOASSAY PROCEDURES FOR THE OCEAN DISPOSAL
PERMIT PROGRAM
by
Environmental Research Laboratory
Office of Research and Development
Gulf Breeze-Narragansett-Corvallis
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
ENVIRONMENTAL RESEARCH LABORATORY
GULF BREEZE, FLORIDA 32561
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DISCLAIMER
This report has been reviewed by the Environmental Research Laboratory,
Gulf Breeze, U.S. Environmental Protection Agency, and approved for
publication. Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.
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FOREWORD
Both the Marine Protection, Research, and Sanctuaries Act of 1972 (Sec.
102c) and the Federal Water Pollution Control Act Amendments of 1972 (Sec.
403c) require that applications for permits for the disposal of any
materials into the ocean be evaluated on the basis of their ecological
impact on the marine environment. The disposal of chemical or biological
warfare agents and high-level radioactive wastes is prohibited.
Organohalogen compounds, mercury, cadmium, and petroleum products cannot be
ocean disposed except when present only as trace contaminants of waste
materials. All other materials must meet the limiting permissible
concentration (LPC) of the total pollutant before they may be disposed of in
the marine environment.
The LPC is defined as that concentration of a waste material or chemical
constituent in the receiving water which, after reasonable allowance for
initial mixing in the mixing zone, will not exceed 0.01 of a concentration
shown to be toxic to appropriate, sensitive marine organisms in a bioassay
or otherwise shown to be detrimental to the marine environment. Therefore,
the LPC concept and the bioassay results on which the LPC is calculated
represent the essential technical criteria of the Ocean Disposal Permit
Program.
Bioassay procedures described in this manual were developed for use by
EPA personnel in carrying out the Ocean Disposal Permit Program. They are
not intended as official EPA procedures or standard methods, but as guides
to EPA ocean-dumping personnel when prescribing to permit applicants what
bioassay tests are required. Under the regulations of the Ocean Disposal
Permit Program, an EPA Regional Administrator has the discretionary
authority to require the permit applicant's performance of any of these
enclosed methods, modifications of these methods, or completely different
methods. The Regional Administrator's decision regarding the requirement
for the performance of bioassay tests depends to a great extent on the type
and amount of waste, location of dump site, and proposed method of disposal
as well as other technical considerations.
Dr. Thomas W. Duke
Director
Environmental Research Laboratory
Gulf Breeze, Florida 32561
iii
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ABSTRACT
The bioassay procedures given in this manual were developed to provide
tests for conducting toxicity evaluations of waste materials considered for
ocean disposal under EPA's Ocean Disposal Permit Program.
Nine bioassay procedures are described; three are considered "special"
and are not recommended for routine use. The procedures specify the use of
various organisms representing several trophic levels. Both flow-through
and static tests are included. Methods given vary in their utility and
complexity of performance. The procedures are not intended to be considered
"standard methods," but, depending on the judgment of the EPA Regional
Administrator responsible for the managing of the permit program, are to be
used as reference methods or official methods.
IV
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CONTENTS
Foreword ±±±
Abstract iv
List of Figures vi
List of Tables vii
Acknowledgments viii
I. Introduction 1
II. Bioassay Procedures for Routine Application
A. Background Information For The Performance of Phyto-
plankton Marine Bioassays 2
B. Static Method For Acute Toxicity Tests With
Phytoplankton 19
C. Static Method For Acute Toxicity Tests With
Brine Shrimp (Artemia salina) 26
D. Methods For The Culture And Short-Term Bioassay Of
The Calanoid Copepod Acartia tonsa 31
E. Static Bioassay Procedure Using Grass Shrimp
(Palaemonetes sp.) Larvae 50
F. Static Method For Acute Toxicity Tests Using Fish And
Macroinvertebrates 61
G. Continuous-flow Method For Acute Toxicity Tests Using
Fish And Macroinvertebrates 69
III. Special Bioassay Procedures
A. Flowing Sea Water Toxicity Test Using Oysters
(Crassostrea virginica) 81
B. Chronic Bioassay Using Sheepshead Minnows (Cyprinodon
variegatus) 84
C. Fish Brain Acetylcholinesterase Inhibition Assay 91
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FIGURES
Number
1-A Hypothetical relationship between algal growth
and toxicant concentration 10
2-A Relationship between percent of control growth
rate (0-48 hrs) and copper 13
1-D Algal culture 35
2-D Mass copepod culture (static) 33
3-D Generation cage (after Heinle) 39
4-D Mass copepod culture (flowing) 42
5-D Bioassay protocol 44
1-E Example of a range-finding bioassay 51
2-E Example of a definitive bioassay 52
VI
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TABLES
Number Page
1-A Sea water and sterility enrichments 4
2-A Synthetic sea water formulation for
algal assays 5
3-A Nutrient enrichments for algal bioassay
medium 7
1-B Composition of mixes to be added to
artificial sea water 21
1-D Synthetic sea water formulation 32
2-D Sea water and sterility enrichments 33
3-D Composition of algal diet and recommended
concentration for adult feeding, egg laying,
and naupliar feeding 34
4-D Protocol for mass copepod culture 41
1-F Standard salt water 62
2-F Suggested test temperatures for vertebrates
and invertebrates 65
1-G Artificial sea water 71
2-G Suggested test temperatures for vertebrates
and invertebrates 75
VI1
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ACKNOWLEDGMENTS
The bioassay procedures given in this manual are the result of the Ocean
Dumping Bioassay Committee's deliberations and represent selection of
methodology developed at various EPA laboratories. The laboratories and
investigators involved are as follows:
Continuous-flow Method for Acute Toxicity Tests Using Fish and
Macroinvertebrates
David J. Hansen, Environmental Research Laboratory, Gulf Breeze, Florida
Steven C. Schimmel, Environmental Research Laboratory, Gulf Breeze,
Florida
Del Nimmo, Environmental Research Laboratory, Gulf Breeze, Florida
Jack I. Lowe, Environmental Research Laboratory, Gulf Breeze, Florida
Patrick R. Parrish, (formerly Environmental Research Laboratory, Gulf
Breeze, now Bionomics)
William H. Peltier, EPA, Region IV
Chronic Bioassay Using Sheepshead Minnows (Cyprinodon variegatus)
David J. Hansen, Environmental Research Laboratory, Gulf Breeze, Florida
Steven C. Schimmel, Environmental Research Laboratory, Gulf Breeze,
Florida
Fish Brain Acetylcholinesterase Inhibition Assay
David L. Coppage, Environmental Research Laboratory, Gulf Breeze,
Florida
Flowing Sea Water Toxicity Test Using Oysters (Crassostrea virginica)
Philip A. Butler, Office of Pesticide Programs at Environmental Research
Laboratory, Gulf Breeze, Florida
Jack I. Lowe, Environmental Research Laboratory, Gulf Breeze, Florida
vin
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Static Method for Acute Toxicity Tests With Phytoplankton
Jack Gentile, Environmental Research Laboratory, Narragansett, Rhode
Island
Mimi Johnson, Environmental Research Laboratory, Narragansett, Rhode
Island
Acute Bioassay Using the Copepod (Acartia tonsa)
Jack Gentile, Environmental Research Laboratory, Narragansett, Rhode
Island
Suzanne Sosnowski, Environmental Research Laboratory, Narragansett,
Rhode Island
John Cardin, Environmental Research Laboratory, Narragansett, Rhode
Island
Static Bioassay Procedure Using Grass Shrimp (Palaemonetes sp.) Larvae
Dana Beth Tyler-Schroeder, Environmental Research Laboratory, Gulf
Breeze, Florida
Members of the EPA Ocean Disposal Bioassay Working Group are as follows:
Thomas W. Duke, (Chairman) Office of Research and Development (ORD),
Environmental Research Laboratory, Gulf Breeze, Florida
William P. Davis, ORD, Environmental Research Laboratory, Gulf Breeze,
Florida; Bears Bluff Field Station, South Carolina
Jack Gentile, ORD, Environmental Research Laboratory, Narragansett,
Rhode Island
David J. Hansen, ORD, Environmental Research Laboratory, Gulf Breeze,
Florida
Jack I. Lowe, ORD, Environmental Research Laboratory, Gulf Breeze,
Florida
William E. Miller, ORD, Environmental Research Laboratory, Corvallis,
Oregon
Royal J. Nadeau, Region II, Edison, New Jersey
Carolyn K. Offutt, Office of Water and Hazardous Material (OWHM),
Washington, D.C.
Richard D. Spear, Region II, Edison, New Jersey
ix
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Richard C. Swartz, ORD, Environmental Research Laboratory, Corvallis,
Oregon; Newport Field Station, Oregon
Robert Vickery, Region VI, Dallas, Texas
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SECTION I
INTRODUCTION
The bioassays procedures given in this manual were established to
provide procedures for conducting biological evaluation of waste materials
to be disposed of in the ocean. Tests conducted according to these bioassay
procedures will provide information on the relative toxicity of various
materials to be disposed. However, these bioassay procedures, like all
laboratory bioassay methods, are attempts at simulation of actual conditions
and therefore suffer all the inaccuracies inherent to simulation systems.
Although these bioassay procedures are not "standard" EPA methods, they
are intended as guides for those involved in evaluating ocean dumping
permits. Accordingly, each method differs in detail and style and does not
conform to a standard format. Permit applicants are expected to modify
bioassay procedures according to both the nature of the waste material and
the type of procedure involved.
Three of the bioassay procedures presented in this manual have been
classified as "special" (i.e., not to be used routinely). These procedures
are the oyster shell growth procedure, the chronic fish egg-to-egg
procedure, and the acetylcholinesterase inhibition test. The remaining
bioassays lend themselves to more routine measurement.
The Ocean Dumping Bioassay Committee stated that the minimum number of
species to be used in an evaluation of a dumping permit should be three.
These species should be selected from the different taxonomic groups listed
in the section on the continuous-flow method for acute toxicity tests using
fish and macroinvertebrates (see page 71 ). Note that the brine shrimp
(Artemia salina) is not on this list. The committee felt that the brine
shrimp was not a satisfactory organism for use in marine bioassays.
However, they are readily available, and tests can easily be conducted to
compare their sensitivity for a selected waste material to that of
indigenous species from a particular dump zone. We recommended that
indigenous organisms be used whenever possible in addition to those
organisms recommended in this manual.
The EPA bioassay working group intends to periodically revise these
bioassay procedures as new information becomes available. An annual
revision is anticipated.
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SECTION II
BIOASSAY PROCEDURES FOR ROUTINE APPLICATION
A. BACKGROUND INFORMATION FOR THE PERFORMANCE OF PHYTOPLANKTON MARINE
BIOASSAYS
The primary producer populations of estuaries consist principally of
microscopic phytoplankton. In their role of storing potential energy, via
photosynthesis, these organisms represent the primary energy input into
aquatic ecosystems (Joint Industry/Government, 1969). For this reason, it
is imperative that water quality conditions be favorable to their growth and
reproduction if serious alterations in other components of marine
communities are to be avoided.
Under natural conditions, both the qualitative and quantitative aspects
of phytoplankton population dynamics display a high degree of seasonability,
characterized by well-defined succession patterns. It is essential that not
only the productivity of various systems be maintained, but also the
relative abundance of species according to normal seasonal compositions; as
primary herbivore populations exhibit selectivity in their grazing patterns.
Consequently, while a pollutant may seem to have no apparent adverse effect
on the total primary production, it may have drastically altered community
structure and composition. Such alterations often occur when sensitive
indigenous species are eliminated and ecologically less desirable, but other
equally photosynthetically active species dominate. If the more resistant
species is incompatible with the feeding and/or nutritional requirements of
primary herbivore populations, then energy transfer to higher trophic levels
will be affected and contribute ultimately to significant effects on
naturally occurring desirable populations. To adequately describe and
predict the potential effects of a toxicant upon an estuarine ecosystem
response, data for the phytoplankton is a necessity.
1. Species Selection
In the design of a bioassay program, the selection of test species is
pivotal to the acquisition of realistic and meaningful information. Algal
culture techniques historically have focused upon developing suitable
culture media to sustain complete life cycles. Nutritional levels and
medium composition often bore little resemblance to the actual environmental
conditions the organism encountered. Furthermore, research was often
limited to a few species that were readily maintained in the laboratory.
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Within the last decade, culture techniques have greatly broadened the scope
of species available for investigation.
In choosing species for bioassays, the following criteria are useful
guides:
a. Whenever possible, indigenous species representing a diversity of
phylogenetic types from the major seasonal successions should be studied.
b. Since sensitivities vary among species, when possible, select the
more sensitive species for bioassay.
c. From seasonal and laboratory studies, conditions of greatest
vulnerabilities should also be identified for the species selected.
d. Since a bioassay basically measures the response of an organism to
the product of toxicant concentration and exposure time, the rate of
response of the test species must be considered. Both test species and
culture conditions should permit growth rates of 0.5-1.0 doublings per day
under non-stress conditions.
The above criteria offer maximum flexibility for the experienced
researcher. For workers with limited funds and expertise, two species are
recommended, both to be used if indigenous forms are unavailable. It is
also recommended that these species be used in conjunction with others to
serve as controls on the systems being tested. Skeletonema costatum is an
ecologically important phytoplankter that is common to a wide geographic
range of neretic waters. Thalassiosira pseudonana, while of lesser
ecological significance, is sensitive to heavy metals and has an 8-hour
generation time which offers greater practical value in the establishment of
toxicological responses.
2. Culture Conditions
The culture conditions for the test species generally should reflect
their natural conditions. In order to develop some semblance of uniformity,
two basic regimes are recommended. For temperate species, a temperature of
20° ± 2°C, light intensity of 2500-5000 lux on a 14-hour light and 10-hour
dark cycle (14:10 cycle) is desirable. For cold water forms, a temperature
of 8° ± 2°C, 2500-500 lux on a 10:14 cycle is recomended. Stock cultures of
the test species are to be maintained in enriched natural (Table 1-A), or
synthetic sea waters (Table 2-A).
They should be transferred to the nutritionally dilute culture medium
and allowed to go through two complete growth cycles prior to use in a
bioassay. This is necessary since nutritional history can have marked
effects upon responses. We have found up to five fold differences in
responses of bioassay organisms maintained under high and natural nutrient
levels (Gentile et al. 1973). Stock cultures should be maintained bacteria-
free whenever possible and transferred at 1-2 week intervals.
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TABLE 1-A. SEA WATER AND STERILITY ENRICHMENTS
Sea water enrichments for stock algal culture maintenance (After Guillard and
Ryther, 1962):
NaN03 75 mg/liter
JoO 5 mg/il
Na2Si03.9H20 10 m§/£
Vitamins:
Thiamine HC1 0.10 mg/2,
Biotin 0.50 yg/S,
B12 0.50 yg/£
Trace metals:
CuS04.5H20 0.002 mg/Jl
0.004 mg/il
0.002 mg/il
MnCl2.4H20 0.036 mg/il
NaMo04.2H20 0.001 mg/il
Fe-sequestrine 1-0 mg (0.13 mg
Buffer:
TRIS-500 mg/Jl @ pH 7.8-8.2
Sterility enrichments to be added to enriched sea water medium above before
autoclaving:
Sodium glutamate 25° mS/A
Sodium acetate 25° mS/£
Cycline 25° ln
Nutrient agar 50 mg/A
^crose 250 mg/J
Sodium lactate 250 mg/£
L & D alanine 25° mg/£
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TABLE 2-A. SYNTHETIC SEA WATER FORMULATION FOR ALGAL ASSAYS (After
Kester et al., 1967).
Compound Concentration/liter
NaCl 24.00 g
Na2S04 4.00 g
H3B03 0.03 g
CaCl2 • 2H20 1.47 g
MgCl2 . 6H20 10.78 g
Na2Si:03.9H20 * 30.00 mg
i
KC1 700.00 mg
NaHC03 200.00 mg
*Prepare stock solution in deionized water and adjust to pH 7.8-8.2
3. Sea Water
The choice of sea water is dictated by availability, quality and cost.
Natural sea water can often be used for bioassays even though inherent
variabilities in quality may complicate analysis of results. Clean offshore
water is suitable if proper precautions during collection and processing are
observed. In general, synthetic sea water is preferred for the constancy of
its composition and quality even though trace contaminants must be removed
by additional purification. The cost of the required chemicals and
purification are usually equivalent to the expense of collecting,
transporting and processing natural sea water.
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a. Natural Sea Water
Sea water is collected from 3-10 meters (to avoid surface
contamination) with a non-metallic water sampler, and transported in
autoclavable polyethylene carboys. Glass is also suitable if breakage can
be prevented. Within 24 hours, the water is filtered through acid-washed
membrane filters in a non-metallic filtration system. Filtered sea water is
then stored at 4°C in the dark.
b. Synthetic Sea Water
A modified synthetic sea water formulation (Table 2-A) has been
developed from Kester et al. (1967). This sea water is recommended for
fish, invertebrates and plankton bioassays. This synthetic sea water has
been endorsed by the Environmental Protection Agency, the 14th Edition of
Standard Methods and the A.S.T.M. Committee on Bioassays.
c. Salinity
A salinity of 30 °/oo is recommended for all bioassays. Salinity
adjustments on natural or synthetic sea waters should be made with glass
distilled or deionized water.
d. Sterilization
Sterilization of stock culture maintenance medium can be
satisfactorily achieved by autoclaving since the pH is stabilized by the
presence of TRIS-buffer. Since bioassay medium cannot be_ autoclaved, two
alternative methods are recommended: 1) positive pressure filtration and/or
2) pasteurization (60° ± 2° C for 4 hours). These treatments will not
appreciably alter the physical-chemical properties of the sea water but will
provide effective sterilization.
The medium should, however, be filtered (0.45y) through a previously
acid washed filter 2 N HC1). Removal of residual acid is accomplished by
rinsing the filter with distilled/deionized water and discarding the first
liter of filtered sea water. Medium should be stored in acid stripped
borosilicate glass or linear polyethylene carboys. To these can be
connected a sterile dispensing tower for distributing media.
Sterility checks are made weekly on this test medium by inoculating
2 ml aliquot of sea water into 10 ml of sterile water enriched as in Table
3-A. The tubes are incubated at 20°C in the dark for up to one week.
Contamination is indicated by turbidity and opalescence of the medium.
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TABLE 3-A. NUTRIENT ENRICHMENTS FOR ALGAL BIOASSAY MEDIUM
Nutrient Amount
Na N03 4.42 mg/fc (50 yMN)
K2HP04 0.87 mg/£ 5 yMP)
Thiamine 100.00 yg/&
Biotin 0.50 yg/£
B12 0.50 yg/£
Fe* 25.00 yg/£
Mn 10.00 yg/£
Zn 1.00 yg/X.
Mo 0.50 yg/£
Co 0.10 yg/£
Cu 0.10 yg/£
*Fe as Cl: Dissolve iron sponge or filings in minimum HC1 with warming and
dilute to volume with deionized water.
4. Glassware
All glassware is high grade borosilicate glass (Pyrex/Kimax). The
bioassays are performed in 125 ml Erlenmyer flasks, containing 50 ml of
medium, and foam plugged. Glassware is dry-heat sterilized (170 C for 2
hours) rather than autoclaved, since the stem often carries metal
contaminants which can interfere with bioassays involving metal toxicity.
Rigorous cleaning is necessary for all glassware to insure against
contamination. Glassware is soaked in detergent, hand or mechanically
brushed, rinsed in deionized water, totally immersed in 10% HN03 for 2-b
hours, thoroughly rinsed in double glass distilled or deionized water and
air or oven dried.
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For work involving the toxicity of metals, the glassware should receive
the following post-wash treatment. To eliminate the problems of either
positive or negative contamination, a monolayer of silico-polymer is applied
to all surfaces contacting the sea water. Commercially available SC-87* is
prepared as 5% solution in cyclohexane, poured into and drained, leaving a
film on the surfaces of the glassware that have come in contact with the
solution. The glassware is then air dried and oven cured at 150-175°C for 4
hours. The result is a completely non-wetable surface which, after a double
glass distilled water rinse, is ready for use. One coating often lasts two
or three assays before recoating is necessary. Recoating can be done over
the old coating or a strong alkali (2N NAOH + 10% ETOH) can completely strip
the old coating prior to recoating. In most instances, alcoholic-alkali
stripping can be avoided by using hot detergent each time prior to
recoating.
5. Bioassay Protocol
The bioassay design consists of three major integrated components:
preparation of log-phase inoculum, nutrient enriched bioassay medium and
toxicant solutions.
a. Inoculum
Inoculum for the bioassay is prepared by inoculating 0.5 ml (0.1-1.0
ml) of stock culture into triplicate 125 ml flasks containing nutrient
enriched sea water at bioassay level (Table 3-A). At the point of
inflection of the growth curve, inoculate three new flasks from this series
and follow the second growth curve. Cells from this second or later
transfers are suitable for use in the bioassay. These cells have now become
adapted to the more natural nutrient levels, and their response will more
closely reflect that expected from a natural population of the test species.
b. Bioassay Medium
Filter sterilized and/or pasteurized, enriched sea water is
dispensed into a presterilized 1-2 liter flask that is compatible with a 50
ml Ace-dispenser (Cat. no. 8004, Ace Glass Co., Vineland, N.J.). Nutrients
(Table 3-A) are aseptically added and inoculum (as described above) is added
to give an initial cell density of 2,500 cells/ml to 10,000 cells/ml.
Inoculation of the total medium volume permits the dispensing of a uniform
cell population to all flasks. Initial cell density or biomass is measured.
Fifty milliliters of enriched inoculated medium is dispensed into 125 ml
flasks using a 50 ml Ace-dispenser in a sterile hood.
*SC-87. Product of General Electric: distributed by Pierce Chemical Co.,
Rockford, Illinois.
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The selection of an initial cell density will be dependent upon the
sensitivity of the biomass parameter measuring system. For example, in
clean systems using particle counters, initial cell densities of 2.5 x 10 •*
microscopic counts are employed, initial cell densities of 1 x 10^cells/ml
may be appropriate. For extractive (ATP, Chi "a") or isotope techniques,
the initial cell density can be kept low since the aliquot examined can be
adjusted.
c. Toxicant
Toxicant solutions are prepared in distilled water or suitable
solvent for hydrophobic compounds. Stock solutions or dilutions of a waste
should be prepared to insure that the same volume is added at all test
levels. This addition should not exceed one milliliter/50 ml of test
medium. When working with waste effluents, a maximum of 5 ml addition is
allowed since this will constitute a 10% maximum alteration in salinity.
Toxicant additions are made to the flasks containing inoculated enriched sea
water and placed in an incubator.
d. Design
The bioassay design is in part determined by the type of toxicant
tested. A general format will include a screening of a broad range of
concentrations from which levels are selected for a definitive evaluation.
Generally, preliminary screenings should cover concentrations at four orders
of magnitude with duplicate cultures at each level. The definitive assay
should include one concentration above and two below the calculated 50%
inhibition level using logarithmic bisection of intervals. Triplicate
cultures should be used for the definitive bioassay.
Parameter measurement should be evaluated at least once every 24
hours for the duration of the experiment. This permits calculation of rates
of response which are important in interpreting the behavior of the
toxicant. The duration of the experiment should be adequate for the control
population to complete its logarithmic growth phase and reach a stationary
growth rate. It is also desirable to determine for the inhibited cultures:
the duration of the lag-phase, maximum rate of growth and maximum yield
(Figure 1-A). However, not all this information may be readily available
from a single assay and all concentrations.
e. Modifications
The assay system described above uses small volumes (50 ml/125).
This is not meant to frustrate the expansion of assay volumes. The systems
can be easily scaled up to the following dimensions of 125/250; 250/500;
500/1,000. With larger volume systems media, dispensing can be made
directly into the sterile flasks. Nutrients and test species can also be
added to each flask. This increases the potential for variability and
contamination but, with experience, difficulties may be minimized. The
larger systems require more assay medium and space, however, greater volumes
will permit more frequent analysis of a greater number of parameters. This
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10
6 _
10
5 -
CO
z
UJ
Q
-O
0
96
HOURS
120
CONTROL
IO ugs Cu/l
A A
2O ugs Cu/l
n n
4O ugs Cu/l
80 ugs Cu/l
k A
16 O ugs Cu/l
O O
144
168
Figure 1-A. Hypothetical relationship between algal growth and toxicant
concentration.
10
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allows a more precise characterization of the anomalies resulting from
specific pollutant exposures.
6. Parameters
There are a variety of parameters available that characterize the
response of the algal cultures. These parameters are measures or indices of
biomass at the time of sampling, which, when plotted against time, produce a
growth response curve. This curve can be used to determine log-phase, rate
of log-growth, and a maximum population density for control and exposed
cultures.
a. Population Density
Microscopic measurements of cell density can be made using a
haemocytometer, Palmer-Maloney Chamber, or inverted microscope with settling
chambers. Details of these counting methods are available in the literature
(Schwoerbel, 1970; Weber, 1973).
The microscopic methods present two problems: they are time-
consuming when done properly and their statistical significance decrease
significantly at cell densities below 1 x 10 . Consequently, when large
numbers of assays and replicates are required, it becomes impractical to
count each assay microspically.
The most rapid, practical and statistically accurate measure of
population density is with an electronic particle counter. The initial
cost, while high, is offset by the increased work volume, accuracy and
saving of time.
b. Population Biomass
Biomass values can be calculated from population density data by
using cell dimensions and assuming the cell is a particular geometrical
shape (i.e., sphere, cylinder, etc.). This method, being dependent on cell
counts, is subject to the same limitations mentioned above.
Electronic particle counters can also give volumetric measurements,
but usually such capabilities are obtained at additional cost. Still it is
worth the expense, if large numbers of assays are anticipated.
c. Chlorophyl
Chlorphyl "a" is often used as a measure of algal biomass. Both
spectro-photometric absorbance and fluorescence (in vivo and in vitro)
techniques are available (Strickland and Parsons, 1968). The
spectrophotometric technique lacks the sensitivity particularly at low cell
densities. The fluorescent systems, however, are more sensitive and can be
used at cell densities of less than 1 x 10 cells/ml. The in vivo
fluorescent technique is particularly useful because it does not require
extraction and is very sensitive.
11
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A potential limitation of this measurement is the general
variability of cellular chlorophyl "a" as a function of nutrition and
environmental variables (Odum et al. 1959; Yentsch and Ryther, 1957;
Yentsch and Menzel, 1963).
d. Carbon-14 Assimilation
Productivity measurements, based upon radioactive carbon
assimilation, is a standard technique applicable to both fresh water and
marine algae (Steeman-Neilson, 1952; McAllister, 1961; Jitts, 1963; Jenkins,
1965; Strickland and Parsons, 1968). This is usually used as a short term
measure of photosynthetic activity. Culture aliquots may thus be pulse-
labeled for four hours and to record C-14 label incormay thus be pulse-
labeled for four hours and to record C-14 label incorporated by cells and
use this relative value as a biomass index. This latter approach has shown
a correlation to growth rates as measured by changes in cell number or
biomass. Transient changes in C-14 assimilation, not reflecting long-term
growth responses, have also been noted and warrant cautious interpretation
of this data.
Adequate C-14 counting procedures may be obtained in Brandsom (1970)
and Chase and Rabinowitz (1967).
e. ATP-Concentration
ATP has been suggested as a sensitive and accurate measure of living
biomass due to a constancy of cellular ATP/carbon ratio (Holm-Hansen and
Booth, 1966; Hamilton, Holm-Hansen, 1967; Holm-Hansen, 1969). Studies have
demonstrated excellent correlation between ATP and direct measures of
biomass (particle counting) and pulse labeling with carbon-14 (Gentile et
al. 1973; Cheer et al. 1974). This technique requires instrumentation
(about $5,000) and costs about $1.00 per analysis. Being a measure of
living material, highly contaminated wastes (i.e., sludge) could provide
excessive interference.
The above techniques all offer certain advantages or disadvantages,
depending on the bioassay design, type of effluent tested, facilities and
personnel.
Automated particle counting, while offering the most rapid,
sensitive and statistically valid method, has limitations. The most
restrictive is that related to particle interferences. The test compound or
effluent must have low background in the particle size range of the test
species or inevitable masking and errors will result. This limits the types
of effluents to be evaluated by this technique, unless the particulate
fraction can be removed without jeopardizing the toxic characteristics of
the material.
The other methods work well in systems containing particulate
material, but both chlorophyl "a" and carbon-uptake have potentially
undesirable response patterns that can make data interpretation difficult.
12
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ATP, on the other hand, appears to be an excellent indicator of living
biomass though it is somewhat expensive to measure routinely and may not be
appropriate for biologically contaminated wastes (i.e., sludge).
All data can be converted to percent control for any finite exposure
period and the percent response plotted versus toxicant concentration
(Figure 2-A). From this graph, the relationship between toxicant con-
centration and degree of inhibition can be determined.
100
80 H
40-
20-
10
EC-5O=23ugs Cu/l
0 20 40 60 80 100
PERCENT CONTROL RESPONSE
Figure 2-A. Relationship between percent of control growth rate (0-48 hrs.)
and copper.
13
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7. Data Presentation
The design of the bioassay requires a minimum of one observation every
twenty-four hours for the duration of the experiment. Within this schedule,
various options are available to the researcher. The basic data output
represents a growth curve for all concentration examined. This may provide
rate of growth:
k = £n *5± /AT
No
k: rate of growth
N : population concentration at time zero
N : population concentration at time t
AT: time interval from time zero
and generation time:
G - *I
K
G: generation time
k: rate of growth
AT: time interval from time zero
and comparisons at maximum population density. Slopes of growth curves
representing the logarithmic growth phase of exposed cultures and population
biomass may be compared by standard statistical analysis for difference with
controls.
8. Standard Toxicant
To insure that the technical aspects of the bioassay are properly
performed, an internal standard is recommended (LaRoche et al. 1970). The
compound we use routinely is sodium dedecyl sulfate (SDS), a surfactant and
membrane lytic agent. This compound produces a very sharp response curve
indicating an almost "all or none" effect at concentrations of 1-2 mg/£. In
addition, SDS is both soluble and stable in aqueous solutions.
While the use of an internal standard can serve as a quality assurance
monitor, it does not, in itself, validate an experiment. There can be
situations where the EC50 concentration for the standard toxicants in two
experiments are essentially identical, but the control growth rates differ
by a factor of two. The deviation of control growth from normal is an
indication of a problem and this alone warrants the repetition of the
experiment .
14
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9. Applications
Algal bioassays, with their sensitivty and rapid response, are useful in
many areas of water quality research.
a. The simplest application is for routine screening of potential
toxicants. This represents a well-defined and controlled system where
particle counting is recommended since interferences can usually be mini-
mized. These studies should be designed to produce complete growth curves
with both growth rate and maximum density output.
b. Another application of the algal bioassay is as an evaluation of
water quality. If an impacted area is being investigated, water samples can
be collected along a transect or matrix, depending on hydrographic data.
The water is collected and processed according to techniques described in
Section 4, and then inoculated with the test species that has been cultured
in enriched water from a control station. Growth rate and population
density can then be compared from station to station.
c. The algal assay can also be used to measure the biological impact of
mixed effluents containing suspended solids. In this case, particle
counting may not be practicable due to high levels of interference.
Consequently, the growth of the algal culture can be monitored by obtaining
daily aliquots and evaluating the ATP, chlorophyl "a", or measuring C-14
incorporation after pulse labeling the aliquot (2-4 hours) with NaH CO.,.
The resulting data, when plotted semi-logarithmically with time, will
produce a growth response curve that may be submitted to the interpretation
discussed herein.
d. Mention must be made of in situ applications of phytoplankton
bioassay. Using ATP, C-14 uptake and chlorophyl "a", both in situ living
biomass and productivity of a water mass may be estimated. These studies
can be made at the site, the samples preserved and analyzed at a later date.
Such applications as evaluation of power plant entrainment and point source
pollution monitoring, commonly use this approach.
10. Remarks
It should be stressed that important advances have been made by the
utilization of phytoplankton bioassays in the establishment of realistic
water quality criteria for marine life.
Fundamental biological anomalies in these organisms could impair
survival of high trophic levels and certainly be associated with specific
pollutant exposures. However, it should be noted that problems exist in the
application of laboratory findings to conditions which may be found in the
natural environment. One scientific discipline which has greatly been
neglected in this area is certainly that of phytoplankton systematics. As a
consequence, it is felt that, in many instances of in situ evaluation of
phytoplankton productivity, identification of species will reveal the
importance of knowing the species present.
15
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REFERENCES
The following literature is recommended to the researcher for detailed
discussions of techniques described in the text. It is not an exhaustive
list but is adequate to acquaint the researcher with the analytical
methodologies required to successfully perform the assay.
American Public Health Association. 1970. Standard Methods for the
Examination of Water and Wastewater, 18th Ed. APHA, Washington, D. C.
Bransome, E.D., Jr. (Ed.) 1970. The Current Status of Liquid Scintillation
Counting. Grune and Stratton, Inc., New York, 394 p.
Chase, G.D., and J.L. Rabinowitz. 1967. Principles of Radioisotope
Methodology, 3rd Ed. Burgess Publ. Co., Minneapolis, 633 p.
Cheer, Sue, J.H. Gentile, C.S. Hegre. 1974. Improved Methods for ATP
Analysis. Analytical Biochemistry. 60:102-114.
Davey, E.W., J.H. Gentile, S.J. Erickson and P- Betzer. 1970. Removal of
Trace Metals from Marine Culture Mediu. Limnol. and Oceanogr. 15:486-
488.
Gentile, J.H., S. Cheer, P- Rogerson. 1973. The Effects of Heavy Metal
Stress on Various Biological Parameters in Thalassiosira pseudonana.
Amer. Soc. Limno. and Oceanogr. Abstract 34th Annual Meeting.
Hamilton, R.D., 0. Holm-Hansen. 1967. Adenosine Triphosphate Content of
Marine Bacteria. Limnol. Oceanogr. 12:319-324.
Holm-Hansen, 0. and C.R. Booth. 1966. The Measurement of Adenosine
Triphosphate in the Ocean and its Ecological Significance Limnol.
Oceanogr. 11:510.
Holm-Hansen, 0. 1969. Determination of Microbial Biomass in Ocean
Profiles. Limnol. Oceanogr. 14:740-747.
Holmes, R.W. 1962. The Preparation of Marine Phytoplankton for Microscopic
Examination and Enumeration on Molecular Filters. U.S. Fish and
Wildlife Service, Special Scient. Report. Fisheries No. 433:1-6.
Instruction Manual 760 Luminescene Biometer. 1960. E.I. DuPont De Nemours
and Co., Wilmington, Delaware.
16
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Jackson, H.W. and L.G. Williams. 1962. Calibration and Use of Certain
Plankton Counting Equipment. Trans. Amer. Microscop. Soc. 81:96.
Jenkins, D. 1965. Determination of Primary Productivity of Turbid Waters
With Carbon-14. J. WPCF. 37:1281-1288.
Jitts, H.R. 1963. The Standardization and Comparison of Measurements of
Primary Production by the Carbon-14 Technique. In: Proc. Conf. on
Primary Productivity Measurement, Marine and Fresh Water (M.S. Doty,
ed.) Univ. of Hawaii, Aug-Sept. 1961. U.S. Atomic Energy Comm. Div.
Tech. Inf. T.I.D. 7633:103-113.
Joint Industry/Government Task Force of Eutrophication. 1969. Provisional
Algal Assay Procedure, pp. 16-29.
Kester, E., I. Dredall, D. Connops and R. Pytowicz. 1967. Preparation of
Artificial Sea water. Limnol & Oceanogr. 12:176-178.
Laroche, G, R. Eisler and C.M. Tarzwell. 1970. Bioassay Procedures for
Evaluation of Acute Toxicities of Oil and Oil Dispersants, to Small
Marine Teleosts and Macroinvertebrates. J. Water Pol. Cont. Fed.
42:1982-1989.
Lorenzen, C.J. 1966. A Method for the Continuous Measurement of in vivo
Chlorophyll Concentration. Deep Sea Res. 13:223-227-
Lorenzen, C.J. 1967. Determination of Chlorophyll and Pheopigments:
Spectrophotometric Equations. Limnol. & Oceanogr. 12(2) :343-346.
Lund, J.W., C. Kipling and E.D. Lecren. 1958. The Inverted Microscope
Method of Estimating Algae Numbers and the Statistical Basis of
Estimations by Counting. Hydrobiologia. 11:143-70.
Mackenthun, K.M. 1969. The Practice of Water Pollution Biology. U.S.
Dept. of the Interior, FWPCA. 281 p.
McAllister, C.D. 1961. Decontamination of Filters in the C-14 Method of
Measuring Marine Photosynthesis. Limnol. & Oceanogr. 6:447-450.
McNabb, C.D. 1960. Enumeration of Fresh water Phytoplankton Concentrated
on the Membrane Filter. Limnol. & Oceanogr. 5:57-61.
Moss, B. 1967- A Spectrophotometric Method for the Estimation of
Percentage Degradation of Chlorophylls to Pheo-pigments in Extracts of
Algae. Limnol. & Oceanogr. 12:335-340.
Mullin, M.M., P.R. Sloan and R.W. Eppley. 1966. Relationship Between
Carbon Content, Cell Volume and Area in Phytoplankton Limnol. &
Oceanogr. 11:307-311.
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National Academy of Sciences. 1969. Recommended Procedures for Measuring
the Productivity of Plankton Standing Stock and Related Oceanographic
Properties. NAS, Washington, D.C. 59 p.
Odum, H.T., W. McConnel and W. Abbot. 1959. The Chlorophyl "a" of
Communities. Pub. Texas Inst. Mar. Sci. 5:65-95.
Palmer, C.M. and T.E. Maloney. 1954. A New Counting Slide for
Nannoplanktdn. Amer. Soc. Limnol. Oceanogr. Spec. Publ. No. 21, pp. 1-
6.
Schwoerbel, J. 1970. Methods of Hydrobiology (Fresh water biology).
Pergamon Press, Hungary, pp. 200.
Steeman-Neilson, E. 1952. The Use of Radioactive Carbon (C-14) for
Measuring Organic Production in the Sea. J. Cons. Int. Explor. Mer.
18:117-140.
Strehler, B.L. 1968. Bioluminescence Assay: Methods of Biochemical
Analysis. (Glictz, D., Ed.) Interscience, New York. Vol. 14, 99 p.
Strickland, J.D.H. and T.R. Parsons. 1968. A Practical Handbook of Sea
Water Analysis. Fish. Res. Board of Can., Bulletin No. 167, 311 p.
Tailing, J.R. and G.E. Fogg. 1959. Measurements (in situ) on Isolated
Samples on Natural Communities, Possible Limitations and Artificial
Modifications. In: A Manual of Methods for Measuring Primary Production
in Aquatic Environments (R.A. Vollenweider, Ed.). IBP Handbook, No. 12,
F.A. Davis, Philadelphia, pp. 73-78.
United Nations Educational, Scientific and Cultural Organization (UNESCO)
1966. Monographs on Oceanographic Methodology. In; Determination of
P/hotosynthetic Pigments in Sea water. UNESCO, Paris. 69 p.
Utermohl. H. 1958. Zur Vervollkommung der Quantitativen Photoplankton-
Methodek. Mitl. Intern. Ver. Limnol. 9:1-38.
Weber, C.I. 1968. The Preservation of Phytoplankton Grab Samples. Trans.
Amer. Microscop. Soc. 87:70.
Weber, C.I. 1973. Biological Field and Laboratory Methods for Measuring
the Aulity of Surface Waters and Effluents. Env. Monitoring Series.
EPA. 670:4-73-001.
Yentsch, C.S. and J.H. Ryther. 1957. Short-term Variations in Phyto-
plankton Chlorophyll and Their Significance. Limnol. & Oceanogr.
2:140-142.
Yentsch, C.S. and D.W. Menzel. 1963. A Method for the Determination of
Phytoplankton Chlorophyll and Phaeophytin by Fluorescence. Deep Sea
Res. 10:221-231.
18
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B. STATIC METHOD FOR ACUTE TOXICITY TESTS WITH PHYTOPLANKTON
1. Introduction
The method described here is designed for analysis of effects of ocean-
dumped material on growth of marine unicellular algae. It involves addition
of liquid waste or extracts from sludge to algal growth medium, addition of
algae to the medium, and measurement of growth for 96 hours.
Because the capability of calculating EC50 values from bioassay data is
required by law, dilutions of ocean-dumped material are necessary. As it is
impossible to estimate potential algal toxicity (or stimulatory action of
each batch of ocean-dumped material, the recommended dilutions may not be
sufficient to yield EC50 values in every case). The logistics of algal
bioassay are complicated and time consuming. They must be considered
carefully before definite requirements are imposed upon testing
organizations.
2. Maintenance of Test Organisms
The marine unicellular algal species to be used is Chlorococcum sp.
(Milford "C") when only one species is used. If two or three species are to
be used in the test, Thalassiosira pseudonana (also known as Cyclotella
nana) is the second species of choice, and Porphyridium cruentum the third.
These algae may be obtained from the Department of Botany, Culture
Collection of Algae, Indiana University, Bloomington, Indiana 47401.
The algae may be ordered as:
Number Species
819 Chlorococcum (Milford "C")
1269 Cyclotella sp.
637 Porphyridium sp.
The species are to be maintained in stock culture collections in
artificial sea water medium. The artificial sea water is prepared by
dissolving artificial sea salts (such as Rila Salts, Rila Products, Teaneck,
New Jersey 07666) in glass-distilled water to a salinity of 30 parts per
thousand (30 grams of salt in 1000 ml of artificial sea water). Add 30.0 ml
19
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of metal mix, 2.0 ml of minor salt mix, and 1.0 ml of vitamin mix to each
liter. The compositions of the mixes are given in Table 1-B.
Filter (with suction) the sea water medium through a 0.22p membrane
filter (such as that manufactured by the Millipore Corporation, Bedford,
Massachusetts 01730, Catalog No. GSWP 047 00). Before filtration, pass 1
liter of 0.1 N HC1 and 5 liters of glass-distilled water through the filter.
Dispense 200 ml of medium into 500 ml Erlenmeyer flasks and use polyurethane
foam plugs to seal the flasks. Autoclave at 120°C and 20 Ib pressure for 15
minutes. The flasks must have been cleaned by washing with detergent,
soaking in 10% HC1, and rinsing 10 times with distilled water.
Equilibrate at room temperature for one day, and check the pH of medium
in a flask especially set up, as above, for this purpose. The pH should be
between 7.8 and 8.1. If the pH is not within this range, discard all flasks
and make new medium. The pH should fall within this range before a test is
started.
Add 10 ml of stock algal culture to each flask and incubate without
shaking under 450-500 foot candles illumination at 20° ± 2° C with
alternating periods of light (16 hours) and darkness (8 hours). Use
standard microbiological techniques for flaming the necks of flasks whenever
algae are transferred.
Stock cultures as described above must be renewed every 10 days. They
need not be shaken during incubation.
3. Preparation of Test Medium
a. Liquid Waste
If liquid waste is to be tested, it will not be modified before use.
When liquid samples are taken for analysis, however, they must be taken in
glass containers with Teflon-lined lids. The glassware and liners must be
washed with detergent, soaked overnight in 10% HC1, rinsed 10 times with
glass-distilled water, rinsed once with acetone, and again rinsed 10 times
with glass-distilled water.
Prepare dilutions of liquid waste as follows:
1. Mix 100 ml of liquid waste with 900 ml of artificial sea water
that does not contain trace metal, minor salt, or vitamin mixes. This will
be considered to be undiluted medium.
2. Add 1 part of 1. to 9 parts of artificial sea water. This is a
10% solution of undiluted medium.
3. Add 1 part of 2. to 9 parts of artificial sea water. This is a
1% solution of undiluted medium.
20
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TABLE 1-B. COMPOSITION OF MIXES TO BE ADDED TO ARTIFICIAL SEA WATER
Mix Amount
Metal mix:
Fe C12 • 6 H2 0* 0.480 g
Mn Cl2 - A H2 0* 0.144 g
Zn S04 . 7 H2 G* 0.045 g
Cu S02 . 5 H2 0* 0.157 mg
Co C12 . 6 H2 0* 0.404 mg
H3B03 0.140 g
Na2EDTA 1.000 g
Distilled water 1 £
Vitamin mix:
Thiamin hydrochloride 50.0 mg
Biotinf 0.01 mg
B12"f" 0.10 mg
Distilled water 100 ml
Minor salt mix:
K3P04 3.0 g
Na N03 50.0 g
Na2 SI03 . 9 H2 0 20.0 g
Distilled water 1 £
*Separate aqueous solutions of these metal salts are maintained at such
concentrations that 1 ml of each is added to 1 £ of mix.
Biotin is maintained as 1 mg/100 ml alcoholic stock solution; B^2 in a 10
mg/100 ml aqueous solution.
21
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4. Add 1 part of 3. to 9 parts of artificial sea water. This is a
0.1% solution of undiluted medium.
b. Sludge
When sludge is to be tested, artificial sea water without trace
metals, minor salts, or vitamins will be used as extractant. Salinity of
the extractant is 30 parts per thousand and the procedure is:
1. Place a representative portion of the sludge into a 250 ml
capacity graduated cylinder, filling to the 250 ml mark. Let the sludge
settle overnight (approximately 16 hours). Carefully decant and discard the
supernatant.
2. Add 100 ml of the wet settled sludge to a gallon-capacity wide-
mouthed jar and add 900 ml of artificial sea water at room temperature. If
more growth medium will be required, add more settled sludge and artificial
sea water to the jars, but keep the ratio of 100:900 constant. Cap the jars
tightly and shake on an automatic shaker at about 100 excursions per minute
for 30 min. At the end of the shaking period remove the jar from the
shaker, stand it in an upright position and let settle for 1 hour.
3. Filter the supernatant fluid through glass wool, a membrane
filter of 5.0y porosity, and then through a membrane filter of 0.22y
porosity. When the filters clog, replace them as is needed. The filters
must be washed before use by passing one liter of 0.1 N HC1 and 5 liters of
glassdistilled water through them. All glassware associated with filtration
must be prepared before use by washing with detergent, soaking overnight in
10% HC1, and rinsing with glass-distilled water.
4. The following solutions will be used in the test:
a. Filtered extract. This will be considered to be undiluted
medium.
b. Add 1 part of a. to 9 parts of artificial sea water. This
is a 10% solution of undiluted medium.
c. Add 1 part of b. to 9 parts of artificial sea water. This
is a 1% solution of undiluted medium.
d. Add 1 part of c. to 9 parts of artificial sea water. This
is a 0.1% solution of undiluted medium.
5. After filtration and dilution of liquid or sludge material, add
30.0 ml of metal mix, 2.0 ml of minor salt mix, and 1.0 ml of vitamin mix to
each liter and record the pH.
6. Add 48.0 ml of each solution to sterile 125 ml volume Erlenmeyer
flasks that were washed with detergent, soaked overnight in 10% HC1, rinsed
10 times with glass-distilled water, rinsed once with acetone, and again
22
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rinsed 10 times with glass-distilled water. Prepare three flasks for each
solution and for each algal species used. Use polyurethane foam plugs to
seal the flasks.
7. Suggested apparatus for extraction, or their equivalent are:
a. Laboratory shaker, Eberback 6000 with a 605 Utility Box, or
equivalent, capable of shaking a 1 gallon container at 100 excursions per
minute.
b. Glass jars, wide month, 1 gallon capacity with Teflon lined,
screw top lids. Note: It may be necessary to purchase jars and Teflon
sheets separately, in which case the Teflon lid liners may be prepared by
the laboratory personnel. Jars and lids should be equivalent in quality to
those supplied by the Cincinnati Container Corporation, 2833 Spring Grove
Avenue, Cincinnati, Ohio 45225. Jars, Cat. No. 120-400-F-0-0-4 (128 oz);
Lids, Cat. No. 120-400-White, FTK, PPE. Teflon sheets should be equal in
quality to those supplied by the Cadillac Plastic Co., 3818 Red Bank Road,
Cincinnati, Ohio 45227.
4. Bioassay
a. Preparation of algae
Four days before the bioassay test is performed, add 5 ml of algal
stock culture that is at least 5 days old to 45 ml of sterilized artificial
sea water that contains trace metals, minor salts, and vitamins as described
under 2. "Maintenance of Test Organisms." Do this in 125 ml capacity
Erlenmeyer flasks fitted with polyurethane foam plugs. Incubate these new
cultures under 450-500 foot candles from cool white flourescent tubes at 20
± 2°C. Incubate them on rotary shaker platforms (such as No. G2 shaker
fitted with No. AG2-125 platform from New Brunswick Scientific Co., New
Brunswick, New Jersey 08903, or equivalent) at 140 ± 10 excursions per
minute. The lighting cycle should be 16 hours of light followed by 8 hours
of darkness.
On the first day of testing, add 1.0 ml of algal culture to a
volumetric flask of 25 ml capacity. Bring to approximately half volume with
testing medium, add 2 drops of 10% formalin in growth medium, and bring to
full volume with testing medium. Wait 5 minutes.
Shake each flask to attain a homogeneous suspension of cells.
Quickly, remove a sample of the homogeneous suspension with a small pipette
and fill each side of a Spencer Bright-Line haemocytometer. Be sure that
the suspension does not overflow into the troughs of the haemocytometer. At
100X magnification count all cells within and impinging upon the 4 corner 1
mm squares and the central 1 mm^ of each grid. To find the number of cells
in 1 ml the original suspension, multiply the count from the 10 squares by
25,000.
23
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The object of these counts is to determine the dilution required to
attain a final concentration of 100,000 cells per ml in the original cell
culture. For example, if the number of cells in a ml of culture was
200,000, then the original culture should be diluted 1:1 with test medium to
yield 100,000 cells per ml.
b. Growth of algae
Add, using sterile pipettes 2.0 ml of the algal suspension that
contains 100,000 cells per ml to the flasks that were prepared with 48.0 ml
of test medium.
Place the flasks on rotary shaker platforms and set the platform at
140 ± 10 excursions per minute. Illunination should be from overhead cool
flourescent lights. Intensity of light should be between 450 and 500 foot
candles with a lighting cycle at 16 hours of light followed by 8 hours of
darkness. The temperature should be 20° ± 2°C.
Incubate the shaking cultures for 96 hours. At that time, add two
drops of 10% formalin in artificial sea water to each flask, wait five
minutes, swirl the cultures to resuspend the cells to a homogeneous
suspension and count in a haemocytometer as described above.
c. Untreated controls
Control algal cultures must be grown in untreated medium at the time
bioassays on liquid waste or sludge are being done. In this case, untreated
medium, with its full complement of metal, vitamin, and minor salt mixes is
shaken, filtered and added to flasks in exactly the same manner as when
extracting sludge. The cell suspension used to inoculate the untreated
growth is prepared exactly as described above except untreated growth medium
is used for diluting.
Three flasks are used in growth of controls, and counting is done as
described above.
5. Analysis of Results
Calculate the average values for number of algal cells per mililiter in
control and each dilution of waste-treated flasks.
An EC50 value is the dilution at which waste material causes 50%
reduction in growth. In order to estimate this value, inspect the average
values to learn if numbers of algal cells in the waste-treated flasks were
(1) less than half that in the untreated control flasks, and (2) more than
half that in the untreated control flasks. If not, then an EC50 value
cannot be determined.
If average cell counts among the dilutions fall greater and less than
half of those of the controls, then EC50 values must be estimated. Using
semilogarithmic coordinate paper, plot the average cell count for a dilution
24
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that yielded more than half, and the average cell count in a dilution that
yielded less than half of the average cell count of control flasks. The
dilution should be plotted on the logarithmic axis and the percentage of
growth in relation to the control on the arithmetic axis. Draw a straight
line between the two points. The concentration at which this line crosses
the 50% growth line is the EC50 value.
25
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C. STATIC METHOD FOR ACUTE TOXICITY TESTS WITH BRINE SHRIMP, Artemia Salina
1. Introduction
The test organism is the brine shrimp, Artemia salina. Artemia are
ecologically insignificant and may be more resistant to many toxicants than
are natural zooplankton. However, the lack of culture and confinement
procedures for natural zooplankton species limit their use even during
periods of peak abundance.
Twenty-four hour nauplii larvae are exposed to 6 concentrations of the
test material for 48 hours. Each concentration and control is replicated;
one is aerated and one is unaerated. The concentration effective against
50% of the animals (EC50) can then be determined by inspection of a graph of
the data. The toxicity of waste to brine shrimp should be compared to that
with appropriate sensitive marine organisms which include, when possible,
indigenous species at the dumpsite. Brine shrimp should not be used alone.
2. Selection and Preparation of Test Organisms
Artemia was selected as the test organism due to its universal and
nonseasonal availability and relative low cost of the assay. The eggs of
Artemia can be purchased from local pet stores and stored in a dessicated
but viable state for long periods of time, and the required number of
organisms can readily be obtained at any time through the use of proper
hatching procedures. Eggs from the San Francisco Bay area are preferable.
A rectangular tray (glass or enamel) having approximately 1300 square
centimeters (cm) of bottom surface is suitable for hatching Artemia eggs.
Divide this tray into two parts by a partition that extends from the top
down to about 2.0 to 1.5 cm from the bottom. This partition may be of any
opaque, biologically inert material (a pasteboard strip, sealed with
paraffin wrapping is satisfactory). Raise one end of the tray about 1.5 cm
and add 3 liters of sea water formulation. Spread approximately 0.5 g of
brine shrimp eggs in the shallow end of the tray. Cover this end of the
tray with a piece of cardboard to keep the eggs in darkness until hatching
is complete. After 20-23 hours incubation, direct a narrow beam of light
across the uncovered portion of the tray. Brine shrimp are phototactic and
will swim beneath the partition into the illuminated end of the chamber.
The Artemia concentrated in the beam of light can be easily collected with
the use of collecting pipette or siphon connected to a 30.5 cm (12 inches)
rubber tube and mouthpiece and transferred to a beaker or shallow dish
containing a small amount of salt water.
26
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3. Salt Water
For acute toxicity tests with Artemia, a practical criterion for an
acceptable salt water is that healthy test animals will survive in it for
the duration of hatching and testing without showing signs of stress. Salt
water should be prepared from commercially available formulations or from
ingredients listed on page 5, using deionized or glass-distilled water.
Deionized or distilled water should be used to dilute the salt water to a
salinity of 30 parts per thousand (°/oo).
4. Recommended Procedure for Testing Material
a. Experimental Design
The recommended test procedure consists of two separate 48-hour
bioassays with a control and six concentrations of the material to be
tested. One 48-hour bioassay will be without aeration and the second will
be with aeration. In the latter, containers will be aerated with clean air
at the rate of 100 ± 15 bubbles per minute delivered from a glass tube 1
millimeter (mm) inside diameter.
There must be at least 20 control animals and at least 20 animals
must be exposed to each concentration or dilution of the material to be
tested, but they may be divided between two or more test containers. The
use of more animals and replication of treatments is desirable. If
replicates are used, they should be true replicates with no water connection
between the replicate test containers. Stratified randomization of the
treatments (random assingment of one test container for each treatment in a
row followed by random assignment of a second test container for each
treatment in another or an extension of the same row) or total randomization
of the treatments is recommended. A representative sample of the test
animals should be impartially distributed to the test containers, either by
adding one (if there are to be less than 11 animals per container) or two
(if there are to be more than 11 animals per container) test animal to each
container, and then adding one or two more to each test container, and
repeating the process until each test container has the desired number of
test animals in it. Alternatively, the animals can be assigned either by
total randomization or by stratified randomization (random assignment of one
animal to each test container, random assignment of a second animal to each
test container, etc.).
Every test requires a control which consists of the same salt water,
conditions and animals as are used in containers with test material. A test
is not acceptable if more than 10% of the control animals die.
b. Temperature
Test water temperature must be maintained within 1°C of the water
temperature listed on page 65.
27
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c. Salinity
Test water salinity should be 30 °/oo before the material to be
tested is added.
d. Test Containers
Use Carolina culture dishes (or their equivalent) having dimensions
approximately 9.0 by 6.5 centimeters.
Test containers must be cleaned before use. New containers must be
washed with detergent and rinsed with 10% hydrochloric acid, acetone, and
tap or other clean water. At the end of every test, if the test containers
are to be used again, they should be (1) emptied, (2) rinsed with water, (3)
cleaned by a procedure appropriate for removing the toxicant, e.g., acid to
remove metals and bases; detergent, organic solvent, or activated charcoal
to remove organic compounds; and (4) rinsed with water. Acid is useful for
removing scale and hypochlorite (bleach) is useful for removing organic
matter and for disinfecting. All test containers must be rinsed with salt
water just before use.
e. Preparation of Material to be Tested (See other section of manual on
this subject.)
f. Concentrations
Dilutions of samples, by volume, of 10% (100,000 ppm, 100 ml/a), 1%
(10,000 ppm, 10 ml/a), 0.1% (1,000 ppm, 1 ml/£), 0.01% (100 ppm, 0.1 ml/a ),
0.001% (10 ppm, .01 ml/a), and 0.0001% (1 ppm, 0.001 ml/£) are recommended
as initial test concentrations.
The highest concentration (dilution) will be prepared as follows: 9
volumes of salt water will be added to 1 volume of the stirred sample.
(Adequate space should be reserved in the test container for stirring and
addition of animals.)
Each succeeding concentration will be prepared by a similar 1-in 10
serial dilution from the previous test container. Adequate stirring of the
contents of the test container is essential before each dilution.
g. Aeration of One Bioassay
Following diolution, aeration of one set of concentrations should
begin using 100 ± 15 bubbles per minute delivered through a 1 mm ID glass
tube.
h. Transfer of Animals
Animals must be added to the test containers within 1 hour after the
proper dilutions of the material to be tested have been made and aeration of
one set of test containers begun.
28
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i. Feeding
The organisms must not be fed while in the test containers.
j. Measurements
The dissolved oxygen concentration, pH, and temperature must be
measured (1) before adding animals and (2) at 24-hour intervals thereafter
in the highest and lowest concentration and the control of both aerated and
unaerated bioassays. Additional measurements are required in containers in
which animals die. Water samples should be taken midway between the top,
bottom, and sides of the test containers and should not include any surface
scum of material stirred up from the bottom or sides.
k. Observations
At a minimum, the number of dead or affected animals must be
recorded at 24-hour intervals throughout the test. More observations are
often desirable, especially near the beginning of the test. Dead animals
must be removed as soon as they are observed.
To count the dead animals accurately and with relative ease, place
the test dishes on a black surface and hold a narrow beam of light parallel
to the bottom of the dish. By searching the surface, bottom, and
intermediate areas, account for the live animals. The use of a dropping
pipette or medicine dropper to remove the live animals facilitates the
counting and decreases the confusion of trying to count moving larvae. Due
to the turbidity that may accompany the higher concentrations of sludge
materials, a 5 minute settling period may be helpful. Pouring the contents
of the test container into a clean container with a larger surface area may
make the animals more visible. Various magnifying devices may also be of
use.
The adverse effect most often used to study acute toxicity with
aquatic animals is death. However, death may not be easily determined for
some Artemia, and so an EC50 (effective concentration to 50% of test
animals) is often measured rather than an LC50 (lethal concentration to 50%
of test animals.) The effect generally used for determining an EC50 is
immobilization, which is defined as the inability to move except for minor
activity of appendages, or loss of equilibrium.
5. Calculations and Reporting
At the end of the test period, the bioassays are terminated and the LC50
or EC50 values are determined.
a. Calculations
An LC50 is a concentration at which 50% of the experimental animals
died and an EC50 is a concentration at which 50% of the experimental animals
were affected. Either may be an interpolated value based on percentages of
29
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animals dying or affected at two or more concentrations. Estimating the
LC50 or EC50 by interpolation involves plotting the data on semilogarithmic
coordinate paper with concentrations on the logarithmic axis and percentage
of dead or affected animals on the arithmetic axis. A straight line is
drawn between two points representing death or effect in concentrations that
were lethal to or effective against more than half and to less than half of
the organisms. The concentration at which the line crosses the 50%
mortality or effect line is the LC50 or EC50 value. If 50% of the test
animals are not affected by the highest concentration, the percent affected
should be reported.
b. Reports
Any deviation from this method must be noted in all reports of
results. A report of the results of both aerated and unaerated tests must
include:
1. name of method, author, laboratory, and date tests were
conducted;
2. a detailed description of the material tested, including its
source date and time of collection, composition, known physical and chemical
properties, and variability of the material tested;
3. the source of the salt water, date prepared and method of
preparation;
4. detailed information about the test animals, including name and
source;
5. a description of the experimental design, the test containers,
the volume of test solution, the way the test was begun, the number of
organisms per concentration, and the loading;
6. definitions of the criteria used to determine the effect and a
summary of general observations on other effects or symptoms;
7. percent of control organisms that died or were affected in each
test container during the test;
8. the 24- and 48-hour LC50's or EC50's;
9. methods used for and the results of all dissolved oxygen, pH,
and temperature measurements; and
10. any other relevant information.
30
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D. METHODS FOR THE CULTURE AND SHORT TERM BIOASSAY OF THE CALANOID COPEPOD
Acartia tonsa
1. Introduction
The methodology described in this section is designed to provide
bioassay data on the effects of a toxicant on a marine copepod. The
techniques described have been used for several years by EPA and represent
the synthesis of many researchers' efforts both from government and
universities.
The bioassay format recommended is not new and has been employed in
aquatic toxicology for many years. Basically, dose response curves are
constructed from mortality rate data collected from 24, 48, 72, and 96-hour
exposure observations. While these observation intervals should be
considered a requirement, more frequent observations or exposures longer
than 96 hours should be included when the research design dictates. From
the above observations, estimates of LC10, LC50, LC100 and confidence limits
can be determined (Litchfield and Wilcoxon, 1949; Finney, 1964, 1971;
Standard Methods, 1971, 13th Edition).
2. Collection and Preparation of Sea Water
There are two distinct aspects to the sea water requirements for this
bioassay. The sea water for these functions should, if possible, be
collected from the study area. First, the sea water when adjusted to 30 °
/oo salinity and 20° C must support survival of the adult copepod Acartia
tonsa for the 96-hour bioassay period.
A second and more demanding requirement of the sea water is that, with
the proper enrichments, it supports growth of the food algae and the
complete life cycle of the test species. If no visible suitable natural sea
water is available that satisfies these requirements a synthetic sea water
formulation may be employed, (Table 1-D).
Niskin or Van Dorn samplers can be used to collect sea water from 3 to
10 meters depth to avoid surface contamination. Collected sea water can be
transported to the laboratory in glass or polyethylene carboys that have
been aged in sea water. Upon return to the laboratory the water is filtered
through a l.Oy acid washed filter (glass fiber, cellulose, acetate, nylon or
polycarbonate) to remove particulate matter and the water stored at 4° C in
the above containers.
31
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Measurements of salinity, dissolved oxygen and pH should be recorded at
the time of collection.
TABLE 1-D. SYNTHETIC SEA WATER FORMULATION*
Chemical
NaCl 24.00
Na0SO. 4.00
/ 4
CaCl .2H20 1.47
MgCl2.6H20 10.78
KC1 0.70
H BO 0.03
NaHC03 0.20
*Medium is modified from Kester et al. (1967). Medium has salinity of 34
loo and pH 8.0 and must be adjusted to 30 °/oo with distilled or deionized
water. Trace metal contaminants from major salts must be eliminated by ion-
exchange stripping (Davey et al. 1970). Na_EDJA (300 mgs/£) may be used
for holding and culture but must be omittea in bioassay studies with trace
metals.
3. Algal Food Cultures
Although a variety of algal diets have been used for copepod cultures
(Zillioux & Wilson 1966; Heinle 1969; Katona 1970; Nassogne 1970) the
following modification of Wilson & Parrish (1970) has been used in our
laboratory quite successfully (Table 3-D). We have added Skeletonema
costatum because it is a naturally occurring food for Acartia tonsa.
32
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TABLE 2-D. SEA WATER AND STERILITY ENRICHMENTS
(A) Sea water enrichments for stock algal culture maintenance (After Guillard
and Ryther, 1962):
Item
Amount
NaN0
Na2Si03.9H20
Vitamins :
Thiamine HC1
Biotin
B12
75 mg/liter
5 mg/a
10 mg/£
0.10 mg/fl,
0.50 yg/£
0.50
Trace Metals:
01804.5H20
ZnS04-5H20
CoCl2.6H20
MnCl2.4H20
NaMo04.2H20
Fe-sequestrine
0.002 mg/£
0.004 mg/l
0.002 mg/Ji
0.036 mg/H
0.001 mg/£
1.0 mg
(0.13 mg Fe)/£
Buffer:
TRIS-500 mg/£ @ pH 7.8-8.2
(B) Sterility enrichments to be added to enriched sea water medium above before
autoclaving:
Sodium Glutamate
Sodium Acetare
Cycline
Nutrient Agar
Sucrose
Sodium
L & D Alanine
250 mg/S,
250 mg/fc
250 mg/fc
50 mg/SL
250 mg/a
250 mg/£
250 mg/H
33
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TABLE 3-D. COMPOSITION OF ALGAL DIET AND RECOMMENDED CONCENTRATION FOR
FEEDING, EGG LAYING, AND NAUPLIAR FEEDING
Adult &
Item Copepodite Naupliar Egg Laying
Skeletonema costatum
5.0 X 106 5.0 X 105 1.5 X 107
Thalassiosira psuedonana 7.0 X 106 7.0 X 10 2.1 X 10
Isochrysis galbana 5.0 X 106 5.0 X 105 1.5 X 107
Rhodomonas baltica 3.0 X 106 3.0 X 105 9.0 X 106
Total cells/liter 2.0 X 107 2.0 X 106 6.0 X 107
These algae are grown axenically in filtered natural or synthetic sea
water at 30 ° /oo salinity and 20° C with 2500-5000 lux continuous
illumination or 14L:10D. The nutrient enrichments are modifications of
those of Guillard and Ryther (1962), (Table 2-D).
Algal cultures may be grown either in standard test tube or flash
cultures if desired; or in the fill and draw semi-continuous system
described below.
Enriched sea water is dispensed into either screw-capped test tubes (50
ml) or erlenmeyer flasks fitted with Teflon lined caps. After standard
autoclaving (15 min. @ 15 psi & 250°F) the medium is allowed to cool and
equilibrate with atmospheric gases for 48 hours. Sterility checks are made
on each set of autoclaved medium by randomly selecting a representative
number of tubes or flasks and inoculating 1 ml of their contents into tubes
of sterility check medium (Table 2-D)- Caps are tightened and the
inoculated tubes stored in the dark for up to 2 weeks. The appearance of
turbulence or opalescence in the test medium indicates the presence of
contamination.
Tubes or flasks are inoculated with each alga on a regular basis to
continually provide a log-phase, high density food source, the frequency of
which will be determined from interpretation of algal growth curves. The
cultures should be harvested at their maximum log-growth phase cell density.
While the above system does work, it requires many manipulations which can
result in culture contamination. It is very time consuming since it
requires frequent cell counts and a large turnover of glassware.
The recommended algal culture system is of a fill-draw type where
cultures are easily maintained near their maximum log-phase cell density and
growth rate, (Figure 1-D). It is then a simple matter to draw off and
34
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OJ
Ln
MEDIA
40 L
STOPPER
MEDIA
12 L
O
121
40 WATT FLUORESCENT UGHTS
COOL WHITE
PINCH CLAMP
COTTON PLUG
TO AIR SUPPLY
O
70% ETON
AIR VENT —
COTTON PLUG
ALUMINUM CLAMP
MEDIA TUBE
TUBING CONNECTOR
PINCH CLAMP
VENT
ALUMINUM CLAMP
ALGAL CULTURE
AIR STONE
SPIN BAR
MAGNETIC MIXER
PINCH CLAMP
STERILE DISPENSING
TUBE
Figure 1-D. Algal culture.
-------
replace a constant volume with fresh medium so that within 24 hours the
culture will have reached the same cell density. When longer than 24 hour
intervals occur between harvests, proportionally greater amounts of culture
are drawn off and replaced. This system can be scaled up or down depending
on food needs. But most important, this system produces algal food that is
physiologically and nutritionally consistent. Thus the nutritional history
of the test species is better controlled. If this system is used, a series
of tube cultures of each of the four algal foods must be kept concurrently
in case of contamination of the large cultures.
Algal cell densities may be determined in a variety of ways. Direct
microscopic counts can be made using a haemocytometer, Palmer-Maloney
chamber, or Utermohl chamber (inverted scope) (Schwoerbel, J. 1970)
(Standard Methods, 1971). In addition, an electronic particle counter is an
accurate and rapid method for determining unialgal densities. Finally, if
manual counts are necessary, it is useful to relate these to chlorophyl
absorbance at 440 my or 665 my using a spectrometer. A curve that compares
cells/ml with absorbancy should be prepared from serial dilutions of each
algal culture. Then a rapid and simple measure of absorbancy can be used to
replace the cell count.
4. Zooplankton Culture
a. Collection
Zooplankton (including Acartia tonsa) are collected by slowly (5. 4
km/hr) towing a plankton net (aperature 150-250 microns) at a depth of 1 to
3 meters. Captured animals are carefully transferred to insulated
containers three-fourths filled with ambient sea water. The density should
not exceed ca.250/& to assure that the dissolved oxygen concentrations
remains adequate if the organisms aren't returned to the laboratory within 1
to 2 hours. It is imperative to measure and record the temperature,
salinity, dissolved oxygen and pH at the time of the collection since these
parameters must be maintained during the initial stages of laboratory
culture.
b. Holding
Immediately upon return to the laboratory the samples are trans-
ferred to 2.3/H (190 x 100 mm) borosilicate crystallizing dishes. Volume is
adjusted to 2000 ml with filtered sea water at ambient temperature and
salinity and each dish is then fed the adult algal diet, (Table 3-D). The
cultures are then incubated at ambient temperature and 14L:10D cool white
illumination at 1000 lux. After 24 hours, acclimation of cultures to 20° C
and 30 °/oo salinity should commence. Salinity and temperature increments
of 5°C and 5 °/oo per day are satisfactory. Organisms can remain in the
original vessel and culturing volumes changed by alternately siphoning and
then adding sea water of a different salinity. Organism transfers are made
by either pipetting or siphoning to new vessels. During acclimation, a
daily feeding scheduled is maintained.
36
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Holding and acclimating can also be accomplished by adding the tow
collections to large aspirator bottles equipped with low rpm stirring
motors. Organism density should be adjusted to 1:10 ml of culture volume.
c. Sorting and Identification
The Plankton tow contains a mixture of species from which Acartia
tonsa must be isolated. For basic information on the taxonomy and biology
of the genus Acartia and other coastal calanoids the following papers are
recommended (Conover, 1956; Heinle, 1966, 1969; Wilson, 1932; Rose, M.,
1933; Fraser, J.H. & Hansen, V. Kr., Ed., Series Fiches D'Identification Du
Zooplancton).
To facilitate capture, the culture volume is reduced from 2000 ml to
500 ml by slowly siphoning sea water using 150 micron plankton netting over
the siphon intake. Individual adult organisms are carefully drawn up into a
wide bore (>_ 2 mm) transfer pipette and individual animals placed in
depression slides, identified, and transferred to food enriched filtered sea
water at 30 °/oo and 20°C. Care should be taken to exclude all nauplii and
juvenile forms in order to eliminate contaminant species.
d. Mass Culture
The objective of this system is to provide large quantities of
Acartia tonsa of standard age for short-term bioassays.
The mass culture unit is derived from culture systems used by Mullin
and Brooks (1967) and Frost (1972). The culture vessel is a pyrex aspirator
bottle whose size can range from 3.5 to 12.0 gallons depending on the number
of copepods needed. The contents are gently mixed by a low rpm motor (<_25
rpm) mounted above the culture vessel. The slow mixing maintains algal food
in suspension where these planktonic copepods normally feed. It must be
emphasized that water movement is gentle and free of vorticies such as
produced by magnetic stirrers (Figure 2-D) . Cool white flourescent lights
provide 2000 lux illumination incident to the culture surface on a 14L:10D
cycle.
Acartia tonsa females are capable of producing in excess of 30 eggs
per female per day when fed the food ration recommended in Table 3-D (Wilson
& Parrish, 1971). Thus if 250 or more gravid females are brooded,
theoretically over five thousand eggs will be produced within 24 hours. For
this potential number of adults, a 40/& culture vessel would be desirable.
Generally, the relationship between culture volume (mis) and organism
density is 10ml:1.
Fifty to one hundred gravid females are placed in each of three to
five generation cages (Figure 3-D) immersed in 2.3/Jl crystallizing dishes
containing ca.2000 ml of sea water and fed at 3 times the usual
concentration (Table 3-D). The generation cage allows the eggs to pass
through the net and hatch eliminating the possibility of cannibalism by
adults. After 24 hours the adults are removed by gently lifting each
37
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OJ
00
LOW RPM MOTORS
1/4" DRILL CHUCK
-PLEXIGLASS RODS -
-ASPIRATOR BOTTLES
COOL WHITE FLUORESCENT
LAMPS
-SILASTIC TUBING
-HOFFMAN CLAMPS
Figure 2-D. Mass copepod culture (static).
-------
125 X 90mm PLEXIGLASS CYLINDER
2000ml FILTERED SEAWATER
PLANKTON NETTING, 250 MICRONS
APERATURE - 25 mm FROM BOTTOM
2.3 LITER, 190 X 100mm PYREX CRYSTALIZING DISH
Figure 3-D. Generation cage (after Heinle).
-------
generation cage out of the dish and quickly immersing it in another dish
with 3 times the usual food density. The remaining sea water from all
dishes containing eggs and nauplii is carefully siphoned into a glass
aspirator bottle containing filtered sea water. The final volume is
adjusted and the naupliar culture is fed as in Table 3-D. If a second mass
culture is desired the procedure is repeated after 24 hours.
The average length of each developmental stage in the life cycle of
Acartia tonsa at 20°C and 20 °/oo is as follows:
Stage Length in Days
Egg (newly oviposited) 1
Nauplius (6 instars) 7
Copepodite (6 instars) 6
Adult (until gravid) 3
Total life cycle 17
During the first 6 days of mass culture only naupliar stages are
present. Daily feeding should be at 2 x 106 cells/a (Table 3-D) and 50% of
the culture medium should be siphoned off and replaced with clean medium on
the 3rd and 7th days. The intake end of the siphon should be covered with
60 micron netting to prevent loss of nauplii.
After the 7th day copepodites should be present and from this point
on, feeding should be 2 x 10 cells/£/day with 50% replacement of the
culture volume with filtered sea water every third day as described above.
Within 16 to 17 days the population will have reached maturity and can be
bioassayed or used to start new cultures. Average adult life span at 20° C
is <30 days.
We have also found it useful to maintain a non-age-standardized mass
culture in reserve. Gravid females from the original generation cages are
used to start a 12 liter (3 1/2 gallon) system, fed the adult food ration
and 50% of their culture water replaced every 3rd day. In addition,
approximately 1/3 of the culture (including organisms) is harvested
periodically (10-14 days) to keep the population at ca. 50 adults and
copepodites/£. This precaution is worth the effort since the high density
cultures have occasionally "crashed" for no apparent reason. A protocol for
this system is given in Table 4-D.
If a constant source of filtered sea water is available, a flowing
water mass culture system can be used (Figure 4-D) . This sytem consists of
a constant head tank which feeds two large cylindrical reaction vessels.
Dilution water flow is controlled by capillarly restriction or clamps. The
four specie algal food is proportionally metered by peristatic pump to
40
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TABLE 4-D. PROTOCOL FOR MASS COPEPOD CULTURE
Step
Day of culture
Age-standardized
Non-age-standardized
11
12
1-3
4
3
4
5
6
7
8
9
10
5-6
7
8-9
10
11-12
13
14-15
16
17-18
19
Naupliar diet daily
(Table 3)
Replace 50% culture
medium with filtered
s.w. retaining organ-
isms. Feed as in 1
As in 1
Repeat step 2
As in 1
Repeat step 2
Adult diet
Adult diet daily
Repeat step 6
As in 7
Repeat step 6
As in 7
Harvest for
Bioassays
Adult diet daily
(Table 3)
As in step 2. Feed
as adults.
As in 1
Repeat step 2
As in 1
Repeat step 2
As in 1
Repeat step 2
As in 1
Harvest 33% of culture
including organisms.
Transfer remaining 67%
to a clean carboy by
siphon* & add filtered
s.w. to volume.
Repeat steps 1-10
*Rate of siphoning is controlled by difference in "head pressure". Do not
constrict the siphon tube or animals will be damaged.
41
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t-FILTERED SEAWATER
CONSTANT HEAD TANK
I--STANDPIPE
ALGAL
FOOD
PUMP
lol
20 LITER CYLINDRICAL
— PLEXIGLASS CULTURE -
VESSELS
-VALVE DRAINS
LOW RPM MOTOR
STIRRING ROD
-150 MICRON
NET
COLLAR
STANDPIPE —
DRAIN—
Figure 4-D. Mass copepod culture (flowing).
-------
provide a constant cell density of 2-5 x 10 cells/Jl. This cell density can
sustain culture densities in excess of 100 adults/copepodites/Jl though
harvesting is recommended to keep the density at 50/Ji.
The reaction vessels are 30 cm high with 30 cm diameter and a 25 cm
standpipe. This provides approximately 18£ culture volume. The standpipe
has a collar of 150 micron nitex net which effectively retains both eggs and
nauplii even though they are considerably smaller. Too fine a net produced
excessive clogging. It is likely that bacterial and algal growths reduce
the effective mesh size to occlude particles as small as 50 microns. This
net collar requires brushing periodically to maintain effective drainage.
The reaction vessels are illuminated as in the static system and are
equipped with low rpm (<25) motors to maintain the population in suspension.
The dilution rate is approximately 10 ml/min which results in a effective
replacement of 50% of the culture volume every 24 hours although the total
volume pumped is 80% of the reaction volume. Flow rates >10 ml/min can be
used, but with caution, as one doesn't want to wash out eggs and/or nauplii.
This basic culture design has been scaled down to 4-liter reaction
vessels and is presently being used for long-term bioassay studies on
population structure and reproduction.
e. Harvesting
Mass cultures of copepods that have reached the adult stage are
harvested for use in bioassays as follows: the culture volume is reduced by
75% using a slow siphon whose intake is covered with 60 micron plankton
netting. The remaining 25% of the culture including organisms is carefully
transferred to 2.3£ pyrex crystallizing dishes (ca. 2000 ml/dish). This
transfer is critical and is best performed as follows. Because of the
fragility of the organism, it is not advisable to constrict the discharge
tube to reduce flow. The discharge flow through the ventral tubulation on
the aspirator is controlled by minimizing the head pressure between the
culture vessel and the crystallizing dish. A slow flow minimizes turbulence
and the opportunity for organisms to collide with vessel walls.
Harvested animals can be further concentrated in the crystallizing
dishes by siphoning the culture medium. Capture is facilitated by using
positive phototactive response of the test species.
5. Short-Term Bioassays
Using adult Acartia tonsa (Dana) and culture conditions from previously
described culture methods, the following short-term bioassays are performed.
(Also See Figure 5-D.)
Range Finding Bioassays
1) Ten adult Acartia are tested per replicate with 3 replicates
required per test concentration and control. Feeding is omitted for the
duration of the assay. A solvent control must be included when appropriate.
43
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Exploratory Bioassay
Harvested Adults (ca. 50)
I I I I I
Control 0.1 1.0 10.0 100.0
10 Adults 10 Adults etc. etc. etc.
Evaluate Mortality and Moribundity after 24
and 48 hours
Ex. 0% 0% 25% 100% 100%
Range Finding Bioassay
Harvested Adults (ca. 180)
1
Control
1 1 1
) 10 10
\
0.1
! 1 1
etc.
0.33
1 1 1
etc.
1.0
1 1 1
etc.
3.3
1 1 1
etc.
I
10
1 1 !
etc.
1
Adults
Evaluate Mortality and Moribundity at 24-hour
intervals for a 96-hour exposure
Calculate LC-50 for 96-hour data
Definitive Bioassays
Harvested Adults (ca. 360)
1 ' <^'
Control etc. etc. etc. etc. etc.
I 1 1 I
15 15 15 15
Adults
Evaluate Mortality and Moribundity at 24-hour
intervals for 96-hour exposure
Figure 5-D. Bioassay protocol.
44
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2) Test container will consist of a suitable flat bottom borosilicate
glass dish containing 100 ml sea water. The depth of medium must be > 2.0
cm.
3) Toxicant Concentration Selection
Generally a broad range of concentrations covering at least four
orders of magnitude is chosen initially. This is followed by a progressive
bisection of intervals on a logarithmic scale (see Standard Methods, 1971).
4) Toxicant Administration
a. Water miscible toxicants immediately prior to the addition of
the test species.
b. Water immiscible toxicants will be dissolved in a suitable
solvent prior to addition to the test medium. Solvent evaluation must be
performed to insure solvent concentrations used are not toxic.
5) Ten adult Acartia are captured from stock cultures with a wide bore
transfer pipette and transferred to a 20 ml beaker containing undosed
filtered sea water (ca. 5 ml). Adjust the final volume of this beaker to 15
ml. The animals and the 15 ml of medium are added to 85 ml of toxicant-
dosed medium by immersing the beaker and gently rinsing.
6) Exposure period will be 96 hours. The number of dead and moribund,
copepods will be observed and recorded at 24, 48, 72, and 96 hours of
exposure. To ascertain if a motionless animal is dead it is gently touched
with a sealed glass capillary probe. Dead animals are removed at each
observation point. Control mortalities in excess of 15% invalidate the
experiment.
7) At each observation period measures of dissolved oxygen and pH
should be made particularly if wastes contain high amounts of organic
matter. Since the test species is very sensitive to agitation, these
measurements must be made on a series of concurrently prepared uninoculated
samples at all test concentrations.
Definitive Short-Term Bioassay
1) General culture conditions and handling will follow previous
discussion. The specifications for this assay are as follows.
2) Fifteen adults are to be tested per each of 4 replicates per
toxicant concentration and control.
3) Test vessels will be as described above.
4) Concentration ranges for toxicant will be chosen so as to include at
least two levels above and below the 96-hour LC50 determined from range
finding bioassays.
45
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5) Exposure and data collection will be as above.
6) Calculations, data presentation are as described in Standard Methods
(14th Ed.) pp. 565-577. Alternate methods of data presentation are
desirable, particularly the application of confidence limits. See
Litchfield and Wilcoxon, 1949; and Finney, 1964, 1971.
The bioassay methodology is at best a general framework which is subject
to modifications as determined by the type of toxicant and the experimental
design.
For example, in assays with toxicants that readily adsorb to container
walls and fail to remain in solution, transfer of organisms to freshly dosed
media is required. The frequency of transfer being determined after rates
of solubility and adsorption have been determined.
6. Standard Toxicant
In order to assure that the technical aspects of the bioassay are
performed properly, and internal standard toxicant is recommended. The
compound we use is sodium dodecyl sulfate (SDS), a surfactant and membrane
lytic agent. This compound produces a sharp response curve indicating an
almost "all or none" effect. While the use of an internal standard can
serve as a quality assurance monitor, it does not, in itself, validate an
experiment. Adequate control survival >85% is the primary criteria for the
success or failure of a bioassay.
7. Conclusion
The above culture system, while designed for Acartia tonsa, has worked
equally well for Eurytemora affinis and Psuedodiaptimus coronatus. The
generation cages, however, were only suitable for ^. tonsa because it
releases eggs individually. Both J5. af finis and P_. coronatus produce egg
sacs.
In the event that natural sea water is not suitable quality to allow
survival, growth, and reproduction of the test species, the following
synthetic formulations are recommended. The formulation in Table 1-D has
been used for both whole life history culture and numerous bioassay studies
at this laboratory. Heinle (1969) found the commercial sea water Instant
Ocean suitable for the culture of both A. tonsa and _£._ af finis. No data is
available on the use of Instant Ocean in bioassays and comparisons to
natural sea water. In lieu of this, I would only recommend it for culture
and not bioassays until suitable comparative data is available.
While it is desirable to work with an age standardized culture this is
not always possible. The mass culture system described can be used as a
holding and acclimation system for indigenous populations. For example, in
many geographical areas A. tonsa is replaced by A. claiisi during the winter
months. Using the above system we have held A. clausi at 10°C for several
weeks. These organisms were used in bioassays with excellent results. Thus
46
-------
we feel that this system, with appropriate modifications, can be used to
hold and culture a variety of zooplankters.
47
-------
REFERENCES
American Public Health Service. 1971. Standard Method for the Examination
of Water and Wastewater. 13th Ed. New York. 874 p.
Conover, R.J. 1956. Oceanography of Long Island Sound, 1952-1954. VI
Biology of Acartia clausi and A. tonsa. Bull. Bingham Oceanogr. Coll.
15:156-233.
Davey, E.W., J.H. Gentile, S.J. Erickson and P. Betzer. 1970. Removal of
Trace Metals from Marine Culture Medium. Limnol. & Oceanogr. 15:486-
488.
Finney, D.J. 1964. Statistical Method in Biological Assay. 2nd Ed. Hafner
Publishing Co., New York. 668 p.
. 1971. Probit Analysis. 3rd Ed. Cambridge Univ. Press. London. 333
P-
Fraser, J.H. and V.Kr. Hansen (Ed.) Series Fiches D*Identification Du
Zooplancton.
Frost, B.W. 1972. Effects of Size and Concentration of Food Particles on
the Feeding Behavior of the Marine Planktonic Copepod Calanus pacificus.
Limnol. & Oceanogr. 17 (6):805-815.
Gentile, J.H., J. Cardin, M. Johnson, S. Sosnowski. 1974. Power Plants,
Chlorine & Estuaries. Amer. Fish Society, 36th An. Meeting Honolulu,
Sept. 9-11.
Guillard, R.R. and J.H. Ryther. 1962. Studies of Marine Planktonic
Diatoms. I Cyclotella nana Hustedt, and Detonula confervacia (Cleve)
Grant. Canadian Journ. Microbiol. 8:299-339.
Heinle, D.R. 1966. Production of a Calanoid Copepod Acartia tonsa, in the
Patuxent River Estuary. Chesapeake Sci. 7:59-74.
. 1969a. Effects of Temperature on the Population Dynamics of
Estuarine Copepods. Ph.D. Thesis, Univ. of Maryland, College Park. 132
P-
. 1969b. Culture of Calanoid Copepods in Synthetic Sea Water. J.
Fish Res. Bd. Canada. 26(1):150-153.
48
-------
Katona, S.K. 1970. Growth Characteristics of the Copepods Eurytemora
affinis and E. herdmani in Laboratory Cultures. Helgolander wiss.
Meeresunters. 20:373-384.
Kester, E., I. Dredall, D. Connors and R. Pytowicz. 1967. Preparation of
Artificial Sea Water. Limnol. & Oceanogr. 12(1):176-178.
Litchfield, J.T. and F. Wilcoxon. 1949. A Simplified Method of Evaluation
Dose-Effect Experiments. J. Pharm. Exper. Ther. 96 (2):99-115.
Mullin, M.M. and E.R. Brooks. 1967. Laboratory Culture, Growth Rate, and
Feeding Behavior of a Planktonic Marine Copepod. Limnol. & Oceanogr.
12:657-666.
Nassogne, A. 1970. Influence of Food Organisms on the Development and
Culture of Pelagic Copepods. Helgolander wiss. Meeresunters. 20:333-
345.
Rose, M. 1933. Faune de France. No. 26. Copepodes Pelagiques. Librairie
de la Faculte des Sciences. Reprinted 1970 by Kraus Reprint, Nendein
Luchtenstein.
Schwoerbel, J. 1970. Methods of Hydrobiology. Pergamon Press, New York.
Wilson, C.B. 1932. The Copepods of the Woods Hole Region, Massachusetts.
Smithsonian Institute, United States National Museum. Bulletin 158.
Wilson, D.F. and K.K. Parrish. 1971. Remating in a Planktonic Marine
Calanoid Copepod. Marine Biology. 9:202-204.
Zillioux, E.J. and D.F. Wilson. 1966. Culture of a Planktonic Calanoid
Copepod Through Multiple Generations. Science. 151:996-998.
49
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E. STATIC BIOASSAY PROCEDURE USING GRASS SHRIMP (Palaemonetes sp.) LARVAE
1. Introduction
Procedures for static 96-hour bioassays utilizing grass shrimp larvae,
Palaemonetes sp., are outlined here. The grass shrimp is an obvious
bioassay choice for several reasons. Three species of the genus, P_. pugio,
vulgaris, and intermedius, are common inhabitants of estuaries along the
Gulf and Atlantic coasts of the United States (Holthuis, 1949, 1952). They
are easy to collect and maintain in the laboratory. Field populations are
usually quite large, allowing greater numbers to be brought into the
laboratory for testing. By manipulating environmental conditions of
temperature and photoperiod, it has been possible to induce spawning in the
laboratory (Little, 1967), opening the way to laboratory cultures of genetic
uniformity. Developing larvae are also available throughout the year for
testing with these methods.
Larval stages of the three species are hardy and easy to culture in the
laboratory. Developmental stages have been described for all species
(Broad, 1957a, b; Broad and Hubschman, 1962; Hubschman and Broad, 1974), and
salinity-temperature optima are known for the larval development of P_.
vulgaris (Sandifer, 1973). Developing larvae have demonstrated a
susceptibility to polychlorinated hydrocarbons greater than adults or
juveniles (Tyler-Schroeder, unpublished manuscript).
2. Culture Methods
Palaemonetes sp. are easily collected from the field using dip nets or
seines in grassy, shallow estuarine areas. They can also be reared in
enclosed holding ponds.
(Ri
To obtain larvae, 8" glass culture bowls, such as the Carolina culture
dish, containing 1H of filtered sea water are stocked with 3 ovigerous
female shrimp per bowl. In order to produce enough shrimp larvae for a 96-
hour test series (210 per replicate, 630 per test series; see Figures 1-E
and 2-E) at least 17-25 bowls of ovigerous females (51-75 shrimp) must be
maintained continuously in the laboratory. The species of each female is
® CarolinaBiological Supply Company, Burlington, North Carolina 27215.
Mention of commercial products or trade names does not constitute
endorsement by The Environmental Protection Agency.
50
-------
Concentration(s)
(rag/liter - ppm)
Control
0.01
0.1
1.0
10
Number larvae per
test container
Total _ 150 larvae
Replicate
30
30
30
30
30
1st Day larvae
3 replicates
Larval age and
Number of replicates
18 Day larvae
3 replicates
Total number of
larvae
(150 larvae/replicates) X (3 replicates/test) - 450 larvae
(450 larvae/test) X (2 test ages) = 900 larvae
Total = 900 larvae
Test Series
Example mortality:
%
ppm
0
Control
3
0.01
10
0.1
80
1.0
97
10.0
Estimated LC50 between 0.1 and 1.0 ppm
Figure 1-E. Example of a range-finding bioassay.
-------
Concentrations (ppm)
(chosen from
Range-Finding
Tests, Figure 1-E)
Control
0.1
0.159
0.252
0.399
0.631
1.0
Number of
larvae per
test concentration
30
30
30
30
30
30
30
Ln
ro
Larval age
and
number of
replicates
1st Day larvae
3 replicates
18th Day postlarvae
3 replicates
Total number
Larvae
(210 larvae/replicate) X (3 replicates/test) = 630 larvae
(630 larvae) X (3 test ages) = 1890 larvae
Total = 1890 larvae
Test Series
Figure 2-E. Example of a definitive bioassay.
-------
confirmed and the chelipeds removed with fine surgical scissors to prevent
removal of the eggs by the females. Shrimp in culture bowls are fed Artemia
nauplii daily and water is changed if a slight cloudiness appears. Since
eggs are carried for 2-3 weeks before hatching; it is advisable to select
females with eggs in the more advanced stages of development.
Larvae are removed from bowls containing ovigerous females each morning
and mixed together to insure uniformity of test animals. They are randomly
dispensed into 8" culture bowls containing U of filtered sea water (200
larvae/5, ), fed Artemia nauplii and reared to the desired test age. Food is
added daily and water changed when a slight cloudiness appears. There
should always be sufficient live food in rearing and test chambers, since
insufficient food accentuates developmental variability (Broad, 1957b) and
produces undesirable variation in test results.
When rearing larvae to a particular age, a 10-15% mortality should be
expected when figuring the number of larvae needed for that test. Ideally,
the larvae to be used in a series of 96-hour acute tests should be hatched
at one time and reared in mass culture. Samples of larvae would be removed
from this culture at designated times for testing. This technique would
minimize or circumvent problems due to possible seasonal variation in larval
susceptibility to waste material.
Salinity-temperature optima for P^ vulgaris larvae indicate a broad
range of tolerance to environmental conditions, which is most likely true
for P^. pugio and P^. internedius. Survival of P^ pugio is approximately the
same when reared in the laboratory at a temperature of 25°C and salinities
of from 15-25 °/oo (A.N. Sastry, personal communicationl). Bioassays should
be performed within this range, preferably closer to 15 °/oo salinity, as P_.
pugio taken from the field are most commonly found in water of this
salinity, or lower.
3. Preparation of Test Media, Selection of Test Containers
The nature of the material being tested dictates choice of test
container size and shape, preparation of test concentrations and frequency
of test media replacements. Problems posed by various wastes include
insolubility in sea water, adsorption to exposed surfaces, decomposition by
hydrolysis, photolysis, etc., loss by volatilization, high BOD and bacterial
growth. Such problems can affect results by causing variation from the
calculated concentration of waste being tested, changing pH of test medium,
releasing breakdown products which may be more or less toxic than parent
compounds, and causing mortality of test animals not related to direct
effect of toxicants.
J-A.N. Sastry, Graduate School of Oceanography, University of Rhode Island,
Kingston, Rhode Island 02881
53
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When choosing container size, it is important to choose a small vessel
surface area to volume ratio because of possibility of adsorption. A larger
volume is also important because of stocking density requirements. The 8"
diameter Carolina culture dish, containing l£ of media, has been found to be
a satisfactory test container for bioassay of Palaemonetes larvae, allowing
maximum volume per vessel surface area and an acceptable stocking density of
30 larvae/I.
Test media should be prepared fresh at the time of replacement, so that
decomposition of toxicant, adsorption to preparation containers, depletion
of oxygen and bacterial growth is minimal. Likewise, it is necessary to
change solutions in test containers at least every 24 hours, preferably
every 12 hours.
All sea water to be used should be of natural origin, preferably from
the dumping site. It should be filtered through a filter of ly porosity.
To adjust salinity the addition of either distilled water or a high-salinity
brine is necessary. The high-salinity brine may be of natural or artificial
origin. If natural origin is desired, place a closed container one-half to
three-quarters full of filtered sea water (x30 °/oo salinity) in a freezer
until solid throughout, usually 2-3 days. Subsequent to removal from the
freezer, the supernatant is drained after the first 2-3 hours thaw.
Supernatant should be 80-110 °/oo salinity or above and can be stored
indefinitely.
An artificial brine may be made using any of the commercial artificial
sea salts and distilled water, but should be used with caution as several of
these preparations contain one or more chelator substances, e.g., EDTA,
which would bias test results with waste material containing heavy metals.
The use of artificial sea water in place of natural sea water totally, is
not recommended at this time. In addition to various chelators in
commercial preparation, the presence of high levels of contaminant heavy
metals in artificial or laboratory prepared sea salt mixes should be
checked. Several shelf chemicals are known to have background levels of Cu,
for example, as high as 5-10 ppb (yg/&) (Erickson et al. 1970; J.H. Gentile,
personal communication^). Unwanted trace metals can be removed by passing
the sea water through a column containing a deionizing resin (Davey et al.
1970), but this method may not be practical for large volumes of water.
Many effluents to be tested are complex mixtures having both solid and
liquid components. There may also be gaseous components. The following
guidelines should be followed when preparing test media:
2j.H. Gentile, National Marine Water Quality Laboratory, South Ferry Road,
Narragansett, Rhode Island 02882
54
-------
A. If Liquid Only
Waste material should be stirred or shaken thoroughly before use.
Waste material may be used directly as a stock, or a stock prepared by
dilution with filtered sea water to a desired concentration. All stocks and
test concentrations should be prepared on a weight to volume basis (gm/£ ,
mg/£ , y g/£). If volume/volume basis is used, a correction should be made
for specific gravity of the material being tested, i.e., (weight/volume) *
(specific gravity) = volume/volume.
B. If Solid and Liquid
1. Agitate the entire material, solid and liquid, until thoroughly
mixed. Remove a volume and use as a stock, or dilute with filtered sea
water to a desired stock concentration. It will be necessary to provide
agitation, e.g., aeration or stirring, in test containers to maintain solid
materials in suspension.
2. a) Centrifuge the material to separate solid and liquid
fractions. Decant liquid-do not filter. Use as stock or dilute to desired
stock concentration as above.
b) It may be desirable to test the effects of solids on
Palaemonetes larvae. Solids can be added to the test containers by weight,
agitated to keep them in suspension and combined toxic-mechanical effects
determined. Alternately, one volume of solid material may be diluted with
four volumes of sea water to prepare a standard elutriate. The "standard
elutriate" is the supernatant resulting from the vigorous 30 minute shaking
of one part bottom sediment with four parts water from the proposed disposal
site followed by 1 hour of letting the mixture settle and appropriate
filtration or centrifugation (Federal Register 38(94):12874, Wednesday, May
16, 1973). Use the standard elutriate as a stock and assay.
Complex wastes often contain substances insoluble in sea water,
necessitating the use of a water miscible solvent to introduce them into the
test media. Only the minimum amount of solvent necessary to dissolve the
toxicant should be used, preferably no more than 100 yl solvent/^ of test
media and only 10 yl/£, if possible. Test concentrations should be prepared
so that the amount of solvent added to each test container is constant. A
solvent control must be run with each test to show the solvent by itself
exerts no adverse effects on test organisms. Suggested solvent carriers are
polyethylene glycol (M.W. 200), triethylene glycol, acetone and ethanol.
All test glassware should be thoroughly washed using the following
procedure:
1) Empty old test solution and rinse with cold water.
2) Rinse with acetone, followed by a warm water rinse.
55
-------
3) Wash with laboratory soap and a brush. Rinse thoroughly with warm
water 4-5 times.
4) A rinse of 10% HC1 or HNO-, is required also, if the toxicant contains
heavy metals.
5) Rinse with distilled water and allow to dry. If an acid rinse is
used, it should be followed by 4-5 thorough rinses with deionized water.
4. Bioassay Procedures
Because Palaemonetes normally exhibits variability in molting and
developmental rates during larval life, it is not feasible to produce suffi-
cient larvae of individual stages for testing. Therefore, tests are begun
using larvae of specified ages (e.g., ages 1, and 18 days). Most larvae
will metamorphose to postlarvae (PL) on approximately day 18-21. Hence,
one bioassay is performed on 18 day old larvae and one on postlarvae, to
determine if the biochemical and physiological changes accompanying
metamorphosis alter the response to the toxicant. For the same reasons, a
bioassay using day 30 postlarvae may be required.
Palaemonetes larvae are added to test containers using a method of
random selection (total randomization, stratified randomization, etc.).
Larvae are removed from culture dishes using a rectangular piece of fine
mesh Nitex nylon net and stocked in test dishes at a density of 30
larvae/liter of test media/culture dish. Larvae are fed an excess supply of
Artemia nauplii throughout the test. Artemia are added with each change of
test media. Mortalities of Palaemonetes are recorded at the time of each
test media change (every 12 or 24 hours), and all dead animals removed at
this time. All test and control culture dishes should be maintained at 25°C
in a culture cabinet, BOD incubator, or water table. Tests may be run in
total darkness or on a 12 hr light - 12 hr dark regime. All tests should
include 4-6 concentrations and a sea water control. Control mortality
exceeding 10% invalidates test results. Because of the inherent variability
of each age group of larvae, 2 to 3 replicates must be run simultaneously
for each test concentration in each experiment. These basic test conditions
are to be followed for both range-finding and definitive bioassays as
discussed below (See Figures 1-E and 2-E).
Initially, a series of range-finding 96-hour assays are performed using
1 and 18 day old larvae to determine the range of toxicity of the material
being examined, and to determine the best test conditions. A broad range of
concentrations covering at least four orders of magnitude should be tested,
e.g., 0.01, 0.1, 1.0 and 10 mg/& (ppm), or gm/& (%). Temperature, pH and
dissolved oxygen (DO) levels should be monitored throughout these tests to
help determine need for aeration and frequency of test solution change.
After the range-finding test is completed and an LC50, concentration
lethal to 50% of the shrimp, approximated, a series of definitive bioassays
are performed. The purpose of the definitive bioassay is to more clearly
determine the limits of toxicity of a waste and better estimate the LC50.
56
-------
Concentrations chosen for definitive bioassays are determined by results
from the range-finding tests, i.e., the lowest definitive test concentration
should equal or be greater than the greatest concentration in range-finding
tests that killed none or few test organisms. Likewise, the greatest
definitive test concentration should be equal to or less than the least
concentration in range-finding tests that killed all or almost all of the
test organisms (See Figures 1-E and 2-E). Once upper and lower definitive
test concentrations are chosen, intermediate concentrations are calculated
using progressive bisection of intervals on a logarithmic scale (Standard
Methods, 1965). At least five, and preferably more, test concentrations are
to be used in order to yield mortality data that lies on either side of a
50% kill, a condition necessary for statistical treatment of data using
Probit Analysis.
Growth is often a more sensitive indication of effect than mortality and
is useful in choosing concentrations to be used for chronic tests.
Therefore, at the end of each test the rostrum-telson length of surviving
larvae from each test concentration and controls should be measured using an
ocular micrometer. A sample of untreated larvae should be measured at the
beginning of the test for comparative purposes. Additional observations,
such as loss of equilibrium, cessation of feeding, irregular movements, and
other behavioral aberrations should be noted at the time of each test media
change.
5. Analysis of Data
Data from 96-hour acute bioassays should be analyzed using Probit
Analysis (Finney, 1964a, b). This method estimates a value for LC30, 70 and
90, as well as the LC50. Because Probit Analysis is generally performed by
computer, it is wise to check the computer output, by plotting percent kill
in probits against logarithm of concentration and comparing computer and
graphed LCSO's. The line thus plotted should closely resemble that
determined by the computer. The Litchfield-Wilcoxon method of LC50
estimation or graphical interpolation using Probit graph paper (Standard
Methods, 1965) can be used when data does not meet the more rigorous
specifications required by Probit Analysis (Litchfield, 1949; Litchfield and
Wilcoxon, 1949, 1953). The 95% confidence limits should be indicated for
all data.
6. Reports
At the completion of testing and data analysis, a report is usually
required. Such reports should include the following information:
1. Name of method, investigator, laboratory, and date tests were
conducted.
2. Detailed description of material tested, source, date and time of
collection, composition, known physical and chemical properties.
3. Source of sea water, date and method or preparation.
57
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4. Detailed information about test animals, including scientific name,
life stage, age, source, history and acclimation procedure for larvae, if
necessary.
5. A description of experimental design, test containers, volume of test
solution, way test was begun, number of organisms at each concentration,
number of organisms in each control and types of controls run.
6. Definitions of response used to determine the effect and a summary of
general observations of other effects or symptoms.
7. Percent of control organisms that died or were affected during the
test.
8. LC50, with confidence limits. LC30, 70 and 90, if pertinent.
9. Methods used for and results of all DO, pH, and temperature
measurements.
10. Any deviations and reasons.
11. Other relevant information.
58
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REFERENCES
Broad, A. Carter. 1955. Reproduction, Larval Development and Metamorphosis
of Some Natantia from Beaufort, N.C. Ph.D. Thesis. Duke University.
87 p.
. 1957a. Larval Development of Palaemonetes pugio Holthuis. Biol.
Bull. 112:144-161.
. 1957b. The Relationship Between Diet and Larval Development of
Palaemonetes. Biol. Bull. 112:162-170.
and Jerry H. Hubschman. 1962. A Comparison of Larvae and Larval
Development of Species of Eastern U.S. Palaemonetes With Special
Reference to the Development of Palaemonetes intermedius Holthuis.
Amer. Zool. 2(3): 172 (Abstr.).
Davey, E.W., J.H. Gentile, S.J. Erickson and P. Betzer. 1970. Removal of
Trace Metals from Marine Culture Medium. Limnol. Oceanog. 15(3):333-
490.
Erickson, S.J., N. Lackie and T.E. Maloney. 1970. A Screening Technique
for Estimating Copper Toxicity to Estuarine Phytoplankton. J. Water
Poll. Cont. Fed. 42:R270-R278.
Federal Register, Part II. Environmental Protection Agency - Ocean Dumping
Criteria, Wednesday, May 16, 1973. 38(94):12874.
Finney. D.J. 1964a. Probit Analysis: A Statistical Treatment of the
Sigmoid Response Curve. Cambridge at the University Press, Cambridge.
318 p.
. 1964b. Statistical Method in Biological Assay. 2nd Ed. Hafner,
N.Y. 668 p.
Holthuis, L.B. 1949. Notes On the Species of Palaemonetes (Crustacea,
Decapoda) Found in the United States of America. Konin, Neder. Akad. v.
Weten. 52:87-95.
. 1952. A General Revision of the Palaemonidae (Crustacea, Decapoda,
Natantia) of the Americas. II. The Subfamily of Palaemoninae. Occ.
Pap. Allen Hancock Fdn. 12:1-369.
59
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Hubschman, J.H. and A.C. Broad. 1974. The Larval Development of
Palaemonetes intermedius Holthuis, 1949 (Decapoda, Palaemonidae) reared
in the laboratory. Crustaceana 26(1):89-103.
Litchfield, J.T., Jr. 1949. A Method For Rapid Graphic Solution of Time-
percent Effect Curves. J. Pharmacol. Exp. Therapeutics. 97:399-408.
and F. Wilcoxon. 1949. A Simplified Method of Evaluating Dose-effect
Experiments. J. Pharmacol. Exp. Therapeutics. 96:99-113.
. 1953. The Reliability of Graphic Estimates of Relative Potency from
Dose-percent Effect Curves. J. Pharmacol. Exp. Therapeutics. 108:18-
25.
Little, Georgiandra. 1968. Induced Winter Breeding and Larval Development
in the Shrimp, Palaemonetes pugio Holthuis (Caridea, Palaemonidae).
Crustaceana, Supplement 2: Studies on Decapod Larval Development. 19-
26.
Sandifer, Paul A. 1973. Effects of Temperature and Salinity on Larval
Development of Grass Shrimp, Palaemonetes vulgaris (Decapoda, Caridea).
Fish. Bull. 71(1):115-123.
Standard Methods for the Examination of Water and Wastewater. 12th Ed.
1965. American Public Health Association, Inc. New York, N.Y. 769 p.
60
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F. STATIC METHOD FOR ACUTE TOXICITY TESTS USING FISH AND MACROINVERTEBRATES
1. Equipment
a. Facilities
For maximum convenience and versatility, the facilities should
include tanks or live cars for holding and acclimating test animals, a tank
for salt water, and a temperature-controlled recirculating water bath or
controlled-environment room for the test containers. The holding and
acclimation tanks should be equipped for temperature control and the holding
tank should be equipped for aeration. Because air used for aeration must
not contain oil or fumes, it must be taken from a well-ventilated, fume-free
area and powered by a surface aerator or an oil-less rotary or piston-type
air compressor. During holding, acclimation, and testing, test animals
should be shielded from disturbances.
b. Construction Materials
Construction materials and commerically purchased equipment that
may contact any water into which test animals are to be placed, should not
contain any substances that can be leached or dissolved by the water. In
addition, materials and equipment should be chosen to minimize sorption of
toxicants from water. It is suggested that glass, #316 stainless steel, or
perfluorocarbon plastics be used whenever possible.
c. Test Containers
1. Type: For fish and invertebrates, the test solution should be
between 15 and 20 centimeters (cm) deep. These animals can be tested in
19.6£ (5-gallon) wide-mouthed soft glass bottles containing 15£ of
solution. Alternatively, test containers can be made by welding, not
soldering, stainless steel or by gluing double-strength window glass with
clear silicon adhesive. As little adhesive as possible should be in contact
with the water; extra beads of adhesive should be on the outside of the
containers rather than on the inside. Some invertebrates can be tested in
3.9£ (1 gallon) wide-mouthed soft glass bottles or battery jars.
2. Cleaning: Test containers must be cleaned before use. New
containers must be washed with detergent and rinsed with 10% hydrochloric
acid, acetone, and tap or other clean water. At the end of every test, if
the test containers are to be used again, they should be (1) emptied; (2)
rinsed with water; (3) cleaned by a procedure appropriate for removing the
toxicant test, e.g., acid to remove metals and bases; detergent, organic
61
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solvent, or activated charcoal to remove organic compounds; and (4) rinsed
with water. Acid is useful for removing scale and hypochlorite (bleach) is
useful for removing organic matter and for disinfecting. All test
containers must be rinsed with salt water just before use.
2. Salt Water
For acute toxicity tests, a practical criterion for an acceptable salt
water is that healthy test animals will survive in it for the duration of
acclimation and testing without showing signs of stress, such as unusual
behavior or coloration. Salt water should be prepared from commercially
available formulations or from ingredients listed in Table 1-F using
deionized or glass-distilled water. Deionized or distilled water should be
used to dilute the salt water to a salinity of 30 parts per thousand (°/oo).
Natural salt water may also be used if it satisfies the acclimation
requirement.
TABLE 1-F. STANDARD SALT WATER*
Ingredient
SrCl2.6H20
H3B03
KBr
CaCl2.2H20
Na SO,
2 4
Amount (g)
0.02
0.03
0.10
1.10
4.00
Ingredient
MgCl2.6H20
NaCl
Na0SiO,.9H00
232
EDTAt
Amount (g)
10.0
23.50
0.02
0.003
tEthylene diamine tetracetate.
*To formulate this water, mix technical grade salts with 900 m£ of distilled
or demineralized water in the order and quantities listed. Then add enough
distilled or demineralized water to make the final volume 1H . Dilute the
water with distilled or demineralized water to achieve a salinity of
30 °/oo. if necessary, add NaHCO to adjust final pH of water to between
8.0 and 8.2. Before the water is used, filter it through a 0.22-micron
membrane filter.
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3. Test Organisms
a. Species
Recommended test animals are as follows (specific names must be
verified and reported):
Invertebrates:
White sea urchin, Tripneustes esculentus
White shrimp, Penaeus setiferus
Pink shrimp, J?. duorarum
Brown shrimp, £. aztecus
Grass shrimp, Palaemonetes sp.
Shrimp, Crangon sp.
Oceanic shrimp, Pandalus jordani
Blue crab, Callinectes sapidus
Dungeness crab, Cancer magister
Vertebrates:
Sheepshead minnow, Cyprinodon variegatus
Mummichog, Fundulus heteroclitus
Silverside, Menidia sp.
Threespine stickleback, Gasterosteus aculeatus
Pinfish, Lagodon rhomboides
Spot, Leiostomus xanthurus
Shiner perch, Cymatogaster aggregata
Buffalo sculpin, Enophrys bison
Pacific staghorn sculpin, Leptocottus armatus
English sole, Parophrys vetulus
Other species indigenous to the dumping area may be used if
approved by EPA. The specific name of the animals must be verified and
reported. Samples of the test animals may be requested by EPA. Tests on
other animals under other experimental conditions may be required by EPA.
b. Source
Test animals are usually collected from wild populations in
relatively unpolluted areas. (Collecting permits may be required by local
or state agencies.) Some animals may be purchased from commercial
suppliers. All animals should be healthy and as uniform in size and age as
possible.
c. Size
1. Fish: Fish that weigh between 0.5 and 5.0 grams each are
usually desirable. In any single test, the standard length (tip of snout to
end of caudal peduncle) of the longest fish should be no more than two times
the standard length of the shortest fish.
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2. Invertebrates: Maximum size should be:
shrimp - less than 10 cm rostrum-telson length
crabs - less than 10 cm carapace width
Since cannibalism occurs in many species of arthropods, the claws of crabs
should be banded, or the individuals should be physically isolated.
d. Care and handling
If the animals are to be tested at a temperature or salinity other
than that at which they are collected, they should not be subjected to more
than a 3°C change in water temperature in any one hour period or to more
than a 5 °/oo change in salinity in any 24 hour period. To maintain animals
in good condition during holding and acclimation, crowding should be
avoided. Animals should be fed at least once a day if held for an extended
period and tanks should be cleaned after feeding.
Animals should be handled as little as possible. When they must be
handled, it should be done as gently, carefully and quickly as possible.
A group of organisms must not be used for a test if they appear 'to
be diseased or otherwise stressed or if more than 3% of the individuals die
during the 48 hours immediately prior to transferral to test containers.
4. Recommended Procedure for Testing Material
a. Experimental Design
The recommended test procedure consists of two separate 96-hour
bioassays with a control and six concentrations of the material to be
tested. One 96-hour bioassay will be without aeration and the second will
be with aeration. In the latter, containers will be aerated with clean air
at the rate of 100 ± 15 bubbles/minute delivered from a glass tube 1 mm
inside diameter. (Use of several disposal pipetts connected to a side-arm
vacuum bottle provides for easier control of air flow.)
There must be at least 10 control animals and at least 10 animals
must be exposed to each concentration or dilution of the material to be
tested, but they may be divided between two or more containers. The use of
more animals and replication of treatments is desirable. If replicates are
used, they should be true replicates with no water connection between the
replicate test containers. Stratified randomization of the treatments
(random assignment of one test container for each treatment in a row
followed by random assignment of a second test container for each treatment
in another or an extension of the same row) or total randomization of the
treatments is recommended. A representative sample of the test animals
should be impartially distributed to the test containers, either by adding
one (if there are to be less than 11 animals per container) or two (if there
are to be more than 11 animals per container) test animals to each
container, and then adding one or two more to each test container, and
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repeating the process until each test container has the desired number of
test animals in it. Alternatively, the animals can be assigned either by
total randomization or by stratified randomization (random assignment of one
animal to each test container, random assignment of a second animal to each
test container, etc.).
Every test requires a control which consists of the same salt
water, conditions and animals as are used in containers with test material.
A test is not acceptable if more than 10% of the control animals die.
b. Temperature
Test water temperature must be maintained within 1°C of the water
temperature listed in Table 2-F.
TABLE 2-F. SUGGESTED TEST TEMPERATURES FOR VERTEBRATES AND INVERTEBRATES*
Region Temperature
I 20°C
IIfand III 25°C
IV, VI and IX 30°C
X 15°C
*Temperatures in this table should be revised to the highest average monthly
temperature of oceanic surface waters at dump sites in each region.
tPuerto Rico and Virgin Islands are in Region II but should use temperatures
suggested for Region IV.
c. Salinity Test
Test water salinity should be 30 °/oo before the material to be
tested is added.
d. Loading
The mass of animals in each test container must be limited so that
the animal's oxygen requirements alone do not influence the test results.
For the recommended test animals, the grams of animals per liter of test
solution in the test containers should not exceed 1.0. Tests at high
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temperature may require reduced loading. Proper loading can be confirmed by
measuring dissolved oxygen concentration in the water of the unaerated
control containers. It must not be less than 4 mg/£ (ppm).
e. Preparation of Material to be Tested (See other section of manual
on this subject)
Samples, whether liquid waste or sludge, will be stirred to a
uniform consistency before dilutions are made.
f. Concentrations
Dilutions of samples, by volume, of 10% (100,000 ppm, 100 ml/£), 1%
(10,000 ppm, 10 ml/A), 0.1% (1,000 ppm, 1 ml/A), 0.01% (100 ppm, 0.1 ml/A ),
0.001% (10 ppm, .01 ml/a), and 0.0001% (1 ppm, 0.001 ml/£) are recommended
as initial test concentrations.
The highest concentration (dilution) will be prepared as follows: 9
volumes of salt water will be added to 1 volume of the stirred sample.
(Adequate space should be reserved in the test container for stirring and
addition of animals.)
Each succeeding concentration will be prepared by a similar l-in-10
serial dilution from the previous test container. Adequate stirring of the
contents of the test container is essential before each dilution.
g. Aeration of One Bioassay
Following dilution, aeration of one set of concentrations should
begin using 100 ± 15 bubbles per minute delivered through a 1 mm ID glass
tube.
h. Transfer of Animals
Animals must be added to the test containers within 1 hour after
the proper dilutions of the material to be tested have been made and
aeration of one set of test containers begun.
i. Feeding
The organisms must not be fed while in the test containers.
j. Measurements
The dissolved oxygen concentration, pH, and temperature must be
measured (1) before adding animals and (2) at 24-hour intervals thereafter
in the highest and lowest concentration and the control of both aerated and
unaerated bioassays. Additional measurements are required in containers in
which animals die. Water samples should be taken midway between the top,
bottom, and sides of the test containers and should not include any surface
scum of material stirred up from the bottom or sides.
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k. Observations
At a minimum, the number of dead or affected animals must be
recorded at 24-hour intervals throughout the test. More observations are
often desirable, especially near the beginning of the test. Dead animals
must be removed as soon as they are observed.
The adverse effects most often used to study acute toxicity with
aquatic animals is death. Criteria for death are no movements, especially
no opercular movement in fish, and no reaction to gentle prodding. However,
death is not easily determined for some invertebrates, and so an EC50
(effective concentration to 50% of test animals) is often measured rather
than an LC50 (lethal concentration to 50% of test animals). The effect
generally used for determining an EC50 with invertebrates is immobilization
which is defined as the inability to move except for minor activity of
appendages, or loss of equilibrium. Other effects can be used for
determining an EC50, but the effect and its definition must always be
reported. General observations on such things as erratic swimming, loss of
reflex, discoloration, changes in behavior, excessive mucous production,
hyperventilation, opaque eyes, curved spine, hemorrhaging, molting, and
cannibalism should be reported.
5. Calculation and Reporting
At the end of the test period, the bioassays are terminated and the LC50
or EC50 values are determined.
a. Calculations
An LC50 is a concentration at which 50% of the experimental animals
died and an EC50 is a concentration at which 50% of the experimental animals
were affected. Either may be an interpolated value based on percentages of
animals dying or affected at two or more concentrations. Estimating the
LC50 or EC50 by interpolation involves plotting the data on semilogarithmic
coordinate paper with concentrations on the logarithmic axis and percentages
of dead or affected animals on the arithmetic axis. A straight line is
drawn between two points representing death or effect in concentrations that
were lethal to or effective against more than half and less than half of the
organisms. The concentration at which the line crosses the 50% mortality or
effect line is the LC50 or EC50 value. If 50% of the test animals are not
affected by the highest concentration, the percent affected should be
reported.
b. Reports
Any deviation from this method must be noted in all reports of
results. A report of the results of both aerated and unaerated tests must
include:
1. name of method, author, laboratory, and date tests were
conducted;
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2. a detailed description of the material tested, including its
source, date and time of collection, composition, known physical and
chemical properties, and variability of the material tested;
3. the source of the salt water, date prepared and method of
preparation;
4. detailed information about the test animals, including name,
standard length, weight, source, history, and acclimation procedure used;
5. a description of the experimental design, the test containers,
the volume of test solution, the way the test was begun, the number of
organisms per concentration, and the loading;
6. definitions of the criteria used to determine the effect and a
summary of general observations on other effects or symptoms;
7. percent of control organisms that died or were affected in each
test container;
8. the 24-, 48- and 96-hour LC50, or EC50;
9. methods used for and the results of all dissolved oxygen, pH and
temperature measurements; and
10. any other relevant information.
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G. CONTINUOUS - FLOW METHOD FOR ACUTE TOXICITY TESTS USING FISH AND
MACROINVERTEBRATES
1. Introduction
Continuous-flow (often referred to as "flow-through") bioassays have
definite advantages over static tests in evaluating certain types of wastes
to be disposed of at sea. They are desirable in testing waste chemicals
that have high biochemical oxygen demands, and are unstable or volatile.
Many test species of fish and macroinvertebrates have high rates of
metabolism and are difficult to maintain in jars or tanks of standing sea
water. Continuous-flow bioassays, conducted under proper conditions,
provide for well-oxygenated test solutions, nonfluctuating concentrations of
the toxicant, and continual removal of metabolic wastes of the test
organisms. (Standard Methods, 13th Edition, 1971).
This method provides general procedures for conducting a 96-hour, flow-
through bioassay on marine fish and macroinvertebrates such as shrimp and
crabs. Evaluation of different types of waste will, no doubt, require some
modification of these procedures.
2. Equipment
a. Facilities
For maximum convenience and versatility, the facilities should
include tanks or live cars for holding and acclimating test animals, a tank
for sea water, and a temperature-controlled recirculating water bath or
controlled-environment room for the test containers. The holding and
acclimation tanks should be equipped for temperature control and the holding
tank should be equipped for aeration for emergency use. During holding,
acclimation, and testing, test animals should be shielded from unnecessary
disturbances.
b. Construction Materials
Construction materials and commercially purchased equipment that
may contact any water into which test animals are to be placed should not
contain any toxic substances that can be leached or dissolved by the water.
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In addition, materials and equipment should be chosen to minimize sorption
of toxicants from water. It is suggested that glass, #316 stainless steel,
or perfluorocarbon plastics be used whenever possible.
c. Test Containers
1. Type: For fish and invertebrates, the test solution usually
needs to be between 15 and 20 cm deep. Test containers can be made by
welding, not soldering, stainless steel or by gluing double-strength window
glass with clear silicon adhesive. As little adhesive as possible should be
in contact with the water; extra beads of adhesive should be on the outside
of the containers rather than on the inside. Plywood tanks coated with
fiberglass resin are also acceptable.
2. Cleaning: Test containers must be cleaned before use. New
containers must be washed with detergent and rinsed with 10% hydrochloric
acid, acetone, and tap or other clean water. At the end of every test, if
the test containers are to be used again, they should be (1) emptied; (2)
rinsed with water; (3) cleaned by a procedure appropriate for removing the
toxicant tested, e.g., acid to remove metals and bases; detergent, organic
solvent, or activated charcoal to remove organic compounds; and (4) rinsed
with water. Acid is also useful for removing scale and hypochlorite
(bleach) is useful for removing organic matter and for disinfecting. All
test containers must be rinsed with uncontaminated sea water just before
use.
3. Sea Water
For acute toxicity tests, a practical criterion for an acceptable sea
water is that healthy test animals will survive in it for the duration of
acclimation and testing without showing signs of stress, such as unusual
behavior or coloration. Natural sea water (particularly from the dump site)
is preferable to artificial sea water; however, at times due to logistical
or economical problems, artificial sea water s more prctical. Artificial
sea water may be prepared from commercially available formulations or from
ingredients listed in Table 1-G using deionized or glass-distilled water.
Salinity of test water should ideally be that of the dump site; however,
requirements of the individual species to be tested must be considered.
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TABLE 1-G. ARTIFICIAL SEA WATER*
INGREDIENT
SrCl .6H 0
H3BO
KBr
CaCl2.2H20
Na0SO,
2 4
AMOUNT (g)
0.
0.
0.
1.
4.
02
03
10
10
00
INGREDIENT
M8C12.6H20
NaCl
Na2SiO .OH20
EDTAt
AMOUNT (g)
10.00
23.50
0.02
0.003
tEthylene diamine tetracetate (EDTA)
*To formulate this water, mix technical grade salts with 900 milliliters of
distilled or demineralized water in the order and quantities listed. Then
add enough distilled or demineralized water to make the final volume one
liter. Dilute the water with distilled or demineralized water to achieve a
salinity of 30 °/oo. If necessary, add NaHCO to adjust final pH of water to
between 8.0 and 8.2. Before the water is used, filter it through a 0.22
micron membrane filter.
4. Test Organisms
a. Species
Recommended species are as follows (specific name must be verified
and reported):
Invertebrates:
Copepods, Acartia sp.
White sea urchin, Tripneustes esculentus
White shrimp, Penaeus setiferus
Pink shrimp, ]?. duorarum
Brown shrimp, P^. aztecus
Grass shrimp, Palaemonetes sp.
Shrimp, Crangon sp.
Oceanic shrimp, Pandalus jordani
Blue crab, Callinectes sapidus
Dungeness crab, Cancer magister
Vertebrates:
Sheepshead minnow, Cyprinodon variegatus
Mummichog, Fundulus heteroclitus
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Longnose killifish, F_. similis
Silverside, Menidia sp.
Threespine stickleback, Gasterosteus aculeatus
Pinfish, Lagodon rhomboides
Spot, Leiostomus xanthurus
Shiner perch, Cymatogaster aggregata
Buffalo sculpin, Enophrys bison
Pacific staghorn sculpin, Leptocottus armatus
English sole, Parophrys vetulus
Other species indigenous to the dumping area may be used if
approved by EPA and the specific name of the organism is verified and
reported. Samples of the test animals may be requested by EPA. Tests on
other organisms under other experimental conditions may be required by EPA.
b. Source
Test animals are usually collected from wild populations in
relatively unpolluted areas. (Collecting permits may be required by local
or state agencies.) Some animals may be purchased from commercial
suppliers. All animals should be healthy and as uniform in size and age as
possible. Juvenile stages are preferable.
c. Size
1. Fish: Fish that weigh between 0.5 and 5.0 g each are usually
desirable. In any single test, the standard length (tip of snout to end of
caudal peduncle) of the longest fish should be no more than two times the
standard length of the shortest fish.
2. Invertebrates: Maximum size should be:
shrimp - 5 to 10 cm rostrum-telson length (5-8 g live
weight)
crabs - less than 7 cm carapace width
d. Acclimation
Depending on the desired test conditions, different periods of
acclimation may be necessary. Acclimation to ambient laboratory conditions
should be the most severe stress the animals will encounter (before the
actual test) i.e., handling in the field and transportation to the
laboratory with the inevitable shock of placing the animals in water
different than that of the transporting media. Initial transfer of the
animals in the lab should be made to water with the temperature and salinity
adjusted very closely to those conditions found in the transporting media.
Acclimation to ambient laboratory conditions should be considered successful
if less than 10% of the animals die during 4-7 days of holding. If the
exposure test is to be run at controlled conditions of temperature and
salinity other than ambient, the test animals should be proportioned into
the exposure tanks containing water at ambient conditions. The water
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conditions can then be changed gradually (8-24 hours) to the test conditions
if temperature changes less than +3°C or -5°C and salinity changes less than
5 °/oo. Under these conditions acclimation without handling should continue
for 4-10 days with adequate water flow.
e. Care and Handling
Animals should be handled as little as possible. When they must be
handled, it should be done with a dip net as gently, carefully, and quickly
as possible. Animals should be fed daily during acclimation but should not
be fed for a period of 48 hours before or during the actual test. It may be
necessary, however, to feed certain invertebrates during the actual test.
Crowding should be avoided. Cannibalism occurs in many species of
arthropods; therefore, in some cases it may be necessary to isolate
individuals in compartmented aquaria. Banding the claws of crabs, and
placing a 2-3 cm (about 1 in.) layer of sand in the bottom of the aquaria
for shrimp are useful techniques.
5. Recommended Procedure for Testing Material
a. Experimental Design
The recommended test procedure consists of a 96-hour bioassay with
a control and at least five concentrations of the material to be tested.
Acute static tests are useful in determining range of toxicity of the
material and selecting concentrations for the flow-through tests (See
Section 5-f. Range finding and definitive tests).
For the definitive test a minimum of 20 organisms is required for
the control and each concentration or dilution of the material to be tested,
but they may be divided between two or more test containers. The use of
more organisms and replication of treatments is desirable but consideration
of "loading" must be made. If replicates are used, they should be true
replicates with no water connection between the replicate test containers.
Stratified randomization of the treatments (random assignment of one test
container for each treatment in a row followed by random assignment of a
second test container for each treatment in another or an extension of the
same row) or total randomization of the treatments is recommended.
The test animals should be impartially distributed to the test
containers by adding no more than 10% to each container, repeating the
process until each test container has the desired number of test animals in
it. Alternatively, the animals can be assigned either by total
randomization or by stratified randomization (random assignment of one
animal to each test container, random assignment of a second animal to each
test container, etc.).
Every test requires a control which consists of the same salt
water, conditions and animals of same species and size as are used in
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containers with test material. A test is not acceptable if more than 10% of
the control animals die.
b. Toxicant Delivery System
Flowing sea water tests are preferable to static tests because test
solutions are renewed continually, assuring a steady concentration of the
toxicant, however, they require metering pumps or other devices for accurate
delivery of the toxicant or test material into the sea water flowing through
the test aquaria.
Most toxicant delivery systems have been designed for introducing
solutions of toxicants and solvents into fresh water, and may not be
applicable to all wastes. Many materials proposed for disposal at sea are
not homogenous mixtures and innovative toxicant delivery systems will be
required to introduce representative samples of the materials into the test
containers. Stirring may be required to maintain suspended solids in non-
homogenous dump material.
Many toxicant delivery systems have been described and used in
various types of bioassay (Sprague, 1969; Freeman, 1971; Bengtsson, 1972;
Cline and Post, 1972; Granmo and Kollberg, 1972; Lowe et al. 1971 and 1972;
Lichatowich et al. 1973; Abram, 1973), but the proportional diluter (Mount
and Brungs, 1967) has probably been used routinely (in fresh water) more
than any other system. A small chamber to promote mixing of toxicant-
bearing water and dilution water should be used between the diluter and the
test containers for each concentration. If duplicate test containers are
used, separate delivery tubes can be run from this mixing chamber to each
duplicate. Alterations in the design of the proportional diluter, have been
useful in some situations (Esvelt and Conners, 1971; McAllister, Mauch and
Mayer, 1972; Benoit and Pulglisi, 1973; Schimmel, Hansen and Forester,
1974).
The flow rates through the test containers must be at least 5 tank
water volumes per 24 hours, and in many cases it is desirable to construct
the toxicant delivery system so that it can provide 10 or more tank water
volumes per 24 hours. Some systems may provide for a continuous flow of sea
water. The flow rates through the test containers should not vary by more
than 10% from any one test container to any other or from one time to
another within a given test.
The calibration of the toxicant delivery system should be checked
carefully before, during and after each test. This should include
determining the volume of stock solution and dilution water used in each
portion of the toxicant delivery system and the flow rate through each test
container. The general operation of the toxicant delivery systems should be
checked daily during the test.
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c. Temperature
Test water temperature should be maintained within 1°C of the water
temperature listed in Table 2-G (unless seasonal bioassays are performed).
This may be accomplished by preheating the sea water before it enters the
test containers, by immersing the test containers in a constant temperature
water bath, or by a combination of these methods.
TABLE 2-G. SUGGESTED TEST TEMPERATURES FOR VERTEBRATES AND INVERTEBRATES*
Region Temperature
I 20°C
IIf and III 25°C
IV, VI and IX 30°C
X 15°C
*Temperature in this table should be revised to the highest average monthly
temperature of oceanic surface waters at dump sites in each region.
1"Puerto Rico and Virgin Islands are in Region II but should use temperatures
suggested for Region IV.
d. Salinity
The salinity of test water should be that of the dump site if: (a)
dump site water is used or (b) artificial sea water is prepared. The
salinity of any other natural sea water used should be -15 °/oo.
e. Loading
Excessive weight (grams/liter) of organisms in a test container
may adversely affect results of test. Therefore, the loading must be
limited so that:
1. the concentration of dissolved oxygen in the control container
does not fall below 60% saturation;
2. the concentration of metabolic products does not become too
high, specifically, the concentration of non-ionized ammonia does not exceed
20
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3. the concentration of toxicant is not lowered by more than 20%
because of uptake by the test organisms; and
4. the organisms are not stressed because of crowding - for the
species listed under 4. (Test Organisms) the loading in the test containers
must not exceed 2 g/£/day. Lower loadings must be used if necessary to meet
the four criteria listed above.
f. Range-Finding and Definitive Tests
Much time and effort may be saved by conducting a series of "range-
finding", static tests with a few animals and a wide range of
concentrations, before setting up the "definitive" flow-through tests which
will be used to calculate the final LC50 or EC50 (See Standard Methods, 13th
Edition, 1971 for details). For example, waste concentrations of 10, 1,
0.1, and 0.01% by volume and two or three animals in each concentration
might be tested first for a period of 24 hours. Definitive test
concentrations should then fall between the highest concentration at which
all animals survive and the lowest concentration at which all or most
animals die.
g. Observations
At a minimum, the number of dead or affected animals must be
recorded at 24 hour intervals throughout the test. More observations are
often desirable, especially near the beginning of the test. Dead animals
must be removed as soon as they are observed.
The adverse effect most often used to study acute toxicity with
aquatic animals is death. Criteria for death are no movements, especially
no opercular movement in fish, and no reaction to gentle prodding. However,
death is not easily determined for some invertebrates, and so an EC50
(effective concentration to 50% of test animals) is often measured rather
than an LC50 (lethal concentration to 50% of test animals). The effect
generally used for determining an EC50 with invertebrates is immobilization,
which is defined as the inability to move except for minor activity of
appendages, or loss of equilibrium. Other effects can be used for
determining an EC50, but the effect and its definition must always be
reported. General observations on such things as erratic swimming, loss of
reflex, discoloration, changes in behavior, excessive mucous production,
hyperventilation, opaque eyes, curved spine, hemorrhaging, molting, and
cannibalism should be reported.
6. Calculations and Reporting
At the end of the test period, the bioassays are terminated and the LC50
or EC50 values are determined.
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a. Calculations
An LC50 is a concentration at which 50% of the experimental animals
would be expected to die and an EC50 is a concentration at which 50% of the
experimental animals would be expected to be affected. Either may be an
interpolated value based on percentages of animals dying or affected at two
or more concentrations. Estimating the LC50 or EC50 by interpolation
involves plotting the data on logarithmic - probability graph paper with
concentrations on the logarithmic axis and percentage of dead or affected
animals on the probability axis. A line is drawn between all data points.
The concentration which the line crosses the 50% mortality or effect line is
the LC50 or EC50 value. In fitting, the line points nearest the 50% effect
level should be given more weight. Ideally data should consist of enough
intermediate (between 0 and 100%) effects to conduct statistical tests such
as probit analysis to determine confidence limits.
If 50% of the test animals are not affected by the highest
concentration, the percent affected at each concentration must be reported.
b. Reports
The final report should include:
1. name of method, author, laboratory, and date tests were
conducted;
2. a detailed description of the material tested, including its
source, date and time of collection, composition, known physical and
chemical properties, and variability of the material tested;
3. the source of the salt water, date prepared and method of
preparation;
4. detailed information about the test animals, including name,
standard length of fishes, carapace width of crabs, total length of shrimp,
weight, source, history, and acclimation procedure used;
5. a description of the experimental design, the test containers,
the volume of test solution, the number of organisms per concentration, and
the loading (water flow to each tank);
6. definitions of the criteria used to determine the effect and a
summary of general observations on other effects or symptoms;
7. percent of control organisms that died or were affected in each
test container;
8. the 24-, 48-, and 96-hour LCSO's or ECSO's;
9. methods used for the results of all dissolved oxygen, pH, and
temperature measurements; and
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10. any other relevant information.
78
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REFERENCES
Abram, F.S.H. 1973. Apparatus for Control of Poison Concentration in
Toxicity Studies With Fish. Water Res. (Oxford) 7 (12):1875-1879.
American Public Health Association. 1971. Standard Methods for the
Examination £f_ Water and Wastewater. 13th Edition, Amer. Pub. Health
Ass., Wash., D.C. 874 p.
Bengtsson, B.E. 1972. A Simple Principle for Dosing Apparatus in Aquatic
Systems. Arch Hydrobio. (Stuttgart) 70 (3):413-415.
Benoit, D.A. and F.A. Puglisi. 1973. A Simplified Flow-splitting Chamber
and Siphon for Proportional Diluters. Water Res. (Oxford) 7(12):1915-
1916.
Cline, T.F. and G. Post. 1972. Therapy for Trout Eggs Infected With
Saprolegnia. Progr. Fish-Cult. 34 (3):148-151.
Esvelt, L.A. and J.D. Conners. 1971. Continuous-flow Fish Bioassay
Apparatus for Municipal and Industrial Effluents. In: L.A. Esvelt, W.J.
Kaufman and R.E. Selleck. Toxicity Removal from Municipal Wastewaters.
Volume IV of "A Study of Toxicity and Biostimulation In San Francisco
Bay-Delta Waters." Sanitary Engineering Research Laboratory, Univ.
California. Berkeley, pp. 155-182.
Freeman, R.A. 1971. A Constant Flow Delivery Device for Chronic Bioassay.
Trans. Amer. Fish Soc. 100(1):135-136.
Granmo, A. and S.C. Kollberg. 1972. A New Simple Water Flow System for
Accurate Continuous Flow Tests. Water Res. 6(9):1597-1599.
Lichatowich, J.A., P.W. O'Keefe, J.A. Strand and W.L. Templeton. 1973.
Development of Methodology and Apparatus for the Bioassay of Oil. In:
Proceedings of Joint Conference on Prevention and Control of Oil Spills.
American Petroleum Institute, Environmental Protection Agency, and U.S.
Coast Guard. Washington, D.C. pp. 659-666.
Lowe, J.I., P.O. Wilson, A.J. Rick and A.J. Wilson, Jr. 1971. Chronic
Exposure of Oysters to DDT, Toxaphene and Parathion. Proc. Natl.
Shellfish. Assoc. 61:71-79
Lowe, J.I., P.R. Parrish, J.M. Patrick, Jr. and J. Forester. 1972. Effects
of the Polychlorinated Biphenyl Aroclor^ 1254 on the American Oyster,
Crassostrea virginica. Mar. Biol. (Berl.) 17:209-214.
McAllister, W.A., Jr. W.L. Mauch and F.L. Mayer, Jr. 1972. A Simplified
Device for Metering Chemicals in Intermittent-flow Bioassays. Trans.
Amer. Fish Soc. 101(3):555-557.
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Mount, D.I. and W.A. Brungs. 1967- A Simplified Dosing Apparatus for Fish
Toxicological Studies. Water Res. (Oxford) 1(1):21-29.
Schimmel, S.C., D.J. Hansen and J. Forester. 1974. Effects of Aroclor 1254
on Laboratory-reared Embryos and Fry of Sheepshead Minnows (Cyprinodon
variegatus). Trans. Am. Fish. Soc. 103(3):582-586.
Sprague, J.B. 1969. Review Paper: Measurement of Pollution Toxicity to
Fish. 1. bioassay methods for acute toxicity. Water Research
3(11):793-821.
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SECTION III
SPECIAL BIOASSAY PROCEDURES
A. FLOWING SEA WATER TOXICITY TEST USING OYSTERS (Crassostrea virginica)
The following test procedure is included as a "special bioassay" for use
in evaluating short-term effects of specific wastes on marine molluscs. It
is recommended only for use with the commercial Eastern oyster, Crassostrea
virginica, and requires flowing unfiltered, natural sea water. This test
should be used only with materials which can be dissolved in water or other
solvents and then metered into test aquaria. The test has proved to be a
valuable bioassay procedure at the Gulf Breeze Environmental Research
Laboratory (EPA) where it has been used for several years to evaluate the
effects of insecticides, herbicides, and other toxic organics on oysters
(Butler, 1965).
This procedure, as described below, was taken almost verbatim from a
report by the Subcommittee on Mollusks of the Standard Bioassay Committee
for the 14th Edition of Standard Methods for the Analysis of_ Water and Waste
Water. It is included in this manual by permission of Dr. Philip A. Butler,
Chairman of the Subcommittee.
1. Shell Deposition Test
The deposition of new shell in juvenile oysters is directly affected by
changes in ambient water quality. The degree of inhibition in shell
deposition is quantitatively related to the amount of environmental stress.
This 96-hour test demonstrates the comparative toxicity of pollutants to
young oysters. The test is conducted with flowing unfiltered sea water in
the temperature range between 15° and 30°C. Actively feeding oysters extend
their mantle edges to the periphery of the shell or valves. The body can
contract, however, to occupy a much smaller area. If the peripheral value
edges are mechanically ground away, the oysters respond by depositing new
shell to replace this loss.
The growth of new shell is primarily linear during the first week and
the rate of deposition is an index of the animal's reaction to ambient water
quality. With acceptable water conditions, 25 mm and larger oysters deposit
as much as 1.0 mm of peripheral new shell per day. Small oysters (less than
50 mm) are more suitable than large ones because typically they form new
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shell deposits at temperatures ranging from about 10 to 30°C in contrast to
mature oysters, which tend to become less active at temperature extremes.
Test data are independent of minor fluctuations in temperature and
salinity during the 96-hour exposure, since the simultaneous shell
deposition in control oysters is considered to be the norm or 100%.
a. Procurement and Preparation of Oysters
Oysters, about 25 to 50 mm in height, i.e., the long axis, with
reasonably flat, rounded shape are culled to singles, cleaned and maintained
in trays in the natural environment. At the time of the test, oysters are
re-cleaned and about 3-5 mm of the shell periphery are removed, leaving a
smoothly rounded blunt profile. This is conveniently done by hand-holding
the oysters against an electric disc grinder. Removal of too wide a rim of
shell will make an opening into the shell cavity and such damaged oysters
should be discarded.
Test aquaria may be fabricated of glass or fiberglassed wood, and
should measure about 64 x 38 x 10 cm deep (25 x 15 x 4 inches) to provide
adequate space for 20 oysters. Such containers permit adequate circulation
of the water while avoiding physical agitation of the oysters by the water
current.
The unfiltered water supply in a constant head reservoir is
delivered by calibrated siphons to the aquaria via a mixing trough into
which the toxicant in an appropriate solvent is also metered. Stock
solutions of the toxicant are prepared so that a delivery of 1 or 2 ml per
minute by means of a calibrated pump will result in the desired
concentration. Baffles in the trough ensure adequate mixing and aeration
before the water enters the test aquaria.
The aquaria contain about 18H at 75% capacity and with a flow rate
of 100& hour~l will provide 5H of water hour~l oysters.~l Small oysters
feed and grow readily under these conditions.
b. Bioassay Procedure
Oysters are prepared and randomly distributed so that each control
and test aquarium contains 20 individuals. Oysters are placed with the
left, cupped value down and the anterior hinged ends all oriented in one
direction. One control aquarium receiving toxicant solvent alone and one
aquarium for each desired concentration of the toxicant are established.
At the end of 96 hours, all oysters are removed from the water and
the shell increments are measured. Shell deposition is not uniform on the
periphery and so the length of the longest "finger" of new shell on each
oyster, measured to the nearest 0.5 mm, is recorded.
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c. Calculation
The ratio of the mean growth of a group of test oysters to the mean
growth of the control oysters provides a percentage index of the tolerance
of the oysters to a specified toxicant concentration. A 96-hour EC50
(concentration inhibiting shell deposition by 50%) may be calculated from an
appropriate exposure series for the indicated test conditions. These values
are relative and may differ significantly under different salinity or
temperature regimes. Appropriate statistical techniques should be used to
determine confidence limits when possible.
A preliminary exposure series is helpful in establishing a suitable
range of toxicant concentrations. In general, three or four oysters exposed
for 48 hours to appropriate concentrations of the test material will bracket
the range of toxicant concentrations required to determine 96-hour EC50
data.
REFERENCE
Butler, Philip A. 1965. Reaction of Some Estuarine Mollusks to
Environmental Factors. In: Biological Problems In Water Pollution -
Third Seminar - 1962. U.S. Department of Health, Education, and
Welfare, Public Health Service Publication No. 999-WP-25 June, 1965.
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B. CHRONIC BIOASSAY USING SHEEPSHEAD MINNOWS (Cypfinodon variegatus)
1. Purpose and Limitations
The purpose of this procedure is to provide a method of determining the
effect of a material on survival of sheepshead minnow embryos and fry, their
growth to adulthood, and spawning success. Spawning success is measured by
observing the ability of pairs of fish to spawn naturally, their fecundity,
fertilization success and survival of embryos and fry.
This test has several limitations and should not be considered valid for
assessing toxicity of all types of materials. Sheepshead minnows are
tolerant of low oxygen and a wide range of temperature and salinity and
bioassays using this fish may underestimate the toxicity of materials that
are toxic because they alter these environmental conditions. Materials
tested should be ones that mix well with water. Insoluble or highly turbid
materials mix poorly, and their toxicity may be under or overestimated.
2. Physical Systems
a. Water Source
1. The source of test water should be (1) from the dump site or (2)
a natural source of sea water with salinity >15 °/oo.
2. Sea water must be filtered to remove particles 15y and larger,
but filtration should not affect the chemical composition of the natural sea
water. Filtration must remove planktonic larvae which could prey upon eggs,
fry and juvenile fish in the chronic bioassay.
3. Any source proposed must be analyzed for possible pollutants
(e.g., pesticides, PCB's and heavy metals). Special determinations should
be made for those toxicants being investigated.
b. Dosing Apparatus
A number of apparatus would be acceptable for this bioassay
including those of Mount and Brungs, 1967; Hansen et al. 1971; Hansen et al.
1974 or Schimmel et al. 1974.
c. Toxicant Mixing
A mixing chamber is necessary to assure adequate mixing of the test
material. Aeration should not be used for mixing. Mixing is extremely
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important because if materials are not adequately mixed with water, toxicity
can not be properly assessed. Improper mixing can either expose the animal
to too much or too little of the material, and toxicity would be over or
underestimated.
d. Duplicates
True duplicates should be used for each concentration in all tests
(no water connection between aquaria). Aquaria location should be by random
selection.
e. Aquaria
Each duplicate glass aquarium should measure a minimum of 30 x 90 x
30 cm high and have a water depth of 15 cm.
f. Embryo and Fry Chambers
1. Embryo and fry chambers should be constructed to allow for
adequate exchange of water and to insure that the proper quantity of
material is entering the chambers. Embryo chambers (incubation cups) can be
constructed from J.13 g, 5 cm, OD round glass or beakers with bottoms cut
off. The bottoms are replaced with 40 mesh stainless steel or nylon
screening. Chambers are hung on an oscillating rocker arm apparatus hat is
driven by a 1-5 rpm electric motor (Mount, 1968). These chambers must be
brushed daily to prevent clogging. Chambers may also be constructed from
Petri dishes to which nylon or stainless steel screen is glued (Schimmel et
al. 1974).
2. Embryo and fry chambers should be designed so that water can be
drained down to 2.5 cm (1 inch) in order to facilitate growth measurements
of fry.
3. Embryo and fry chambers may be supplied test water by (1)
separate delivery tubes from the mixing chamber, (2) splitting the flow from
the aquaria or (3) egg cups on a "rocker" arm. Care must be taken that each
embryo and fry chamber receives an equal amount of the toxicant solution.
g. Flow Rate
1. Flow rates to each duplicate aquaria should be great enough to
(1) provide 90% replacement in 8-12 hours (Sprague, 1969); (2) maintain
dissolved oxygen, 60% saturation; and (3) maintain the toxicant
concentration.
2. The test system should be equipped with an alarm system to
insure continuation of water and toxicant flow.
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h. Photoperiod
A 16-hour light/8-hour dark cycle should be maintained throughout
the test. It may be desirable to control lights by a timing switch
(Drummond and Dawson, 1970).
i. Temperature
Test temperature should be maintained at 30°C (±1°C) . Temperatures
can be maintained by (1) preheating the diluted water to the prescribed
temperature, and/or (2) placing the test aquaria in a temperature controlled
water bath.
j . Cleaning
All aquaria should be cleaned whenever organic material builds up.
Aquaria should be brushed down and siphoned to remove accumulated material.
To reduce stress, fish can be left in the aquaria but the end of the siphon
should be covered with screen. Care should be exercised in cleaning to
prevent loss or damage to the fry, juveniles or adults. Embryo and fry
chambers may need to be cleaned frequently or replaced when screens clog or
organic material collects. Frequency of cleaning will vary but it may be
daily. Special care is required to prevent injury to fry.
k. Spawning Chambers
Chambers should be constructed of either glass or #316 stainless
steel. Chambers should be at least 13 x 30 x 15 cm high, with a large mesh
screen attached 1 cm above the bottom to enable eggs to pass through. This
reduces cannibalism by the parents.
1. Disturbance
All test chambers containing fish should be shielded from excessive
outside disturbances. Tanks should be shielded from all outside light
sources that would interfere with the photoperiod.
m. Concentrations
1. A minimum of 5 concentrations of toxicant and a control, all
duplicated, should be utilized in all acute and chronic tests.
2. Concentrations selected for chronic toxicity experiments should
be based on results of acute flow-through bioassays. Concentrations should
be selected so that at least one will adversely affect some life stage of
the sheepshead minnow and one will not affect any stage.
3. We believe that chemical analyses are justified because of the
complexity of this bioassay and the need for interpreting the results.
Analyses should be made of the material itself, of the water during this
test and of the adult fish at the conclusion of the test. At a minimum,
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water from each aquarium at the beginning and end of the test, and fish from
each aquarium (10 or more fish each) at the end of the test should be
analyzed. It is highly desirable to chemically analyze additional samples
of water and of fish including, each life stage, muscle tissue and gametes.
n. Acute Tests
Acute flow-through bioassays should be conducted prior to
initiation of any chronic test. It is desirable for these tests to be on at
least two different age classes (e.g., fry, juveniles or adults). Consult
section on suggested acute flow-through bioassay methods.
3. Biological Systems
a. Source of Adult Fish
Adult fish should be obtained from the same source, either from
wild population or suitable culture laboratories. They should be held in
flowing 30°C sea water of >15 °/oo salinity for a minimum of two weeks
before obtaining eggs.
b. Obtaining Eggs from Adult Fish
To obtain a sufficient number of eggs to begin a chronic exposure,
two methods may be employed: (1) natural spawning from laboratory stocks;
and (2) artificial inducement, where egg production is stimulated by
injection of human gonadotrophic hormone and fertilized with sperm excised
from males. (Schimmel, et al. 1974). The former may be preferable.
c. Beginning the Test
The test begins when fifty fertilized eggs are placed in two or
more separate embryo and fry chambers in each duplicate aquarium. Survival
of embryos and fry (which constitute the parental stock F^ ) are to be
checked and recorded daily. After 4 weeks fish are reduced to 25 per
duplicate aquarium. This should provide enough fish so that at least five
pairs of adults will be available in each replicate aquarium for spawning.
d. Food
1. Fry should be fed live brine shrimp nauplii two or more times
daily for about 2 weeks. (Do not use frozen nauplii.) After 2 weeks
supplement with dry trout pellets (e.g., Oregon Moist) or dry mollie flakes
(e.g., Biorell) for 2 additional weeks.
2. Juveniles and adults can be fed twice daily on dry food
supplemented with frozen adult brine shrimp. Each batch of food should be
checked for pesticides (DDT, dieldrin, endrin, etc.) and metals. In
addition, chemical analysis should also include chemicals in the material to
be tested.
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e. Disease
Disease outbreaks should be handled according to their nature with
each aquarium being treated similarly even though disease is not evident in
all aquaria. All treatments should be kept to the minimum and recorded as
to type, amount and frequency.
f. Measuring Fish
A sample of fish of the FI generation should be measured at one day
post-hatch, 4 weeks, 8 weeks and at adult termination. Larval ^2) fish are
to be measured at one day post-hatch and weighed and measured at week 4
(termination). Suggested techniques for measuring fish include the
photographic method outlined by McKim, J.M. and D.A. Benoit, (1971) or
placing fish in a beaker or petri dish containing .63 cm (1/4 inch) of water
on a Xerox machine or other photocopier.
g. Thinning
At 28 days post-hatch, the F, larvae should be randomly reduced to
25 fish per duplicate aquaria.
h. Spawning Chambers
As mature adults (F, ) begin courtship (indicated by sexual
dimorphism, territoriality, aggressive behavior and courtship by the male),
separate pairs should be placed in individual spawning chambers in the
aquaria. Pairs should be left in the chambers until a sufficient number of
eggs have been collected to insure statistical comparisons of fecundity,
fertility, and survival of embryos and fry can be made. A minimum of five
pairs of fish should be used in each aquarium for spawning studies.
i. Removing Eggs
1. All eggs should be removed at a fixed time each day so the fish
are not overly disturbed and that disruption of spawning activity will not
occur.
2. Daily records of spawnings, egg numbers, and egg fertility must
be kept. All eggs must be removed daily, examined for fertility, and then
used for survival studies or residue analyses, or discarded. Each pair
should be observed daily for a minimum of 2 weeks.
j. Egg Incubation
1. Fifty fertile eggs should be collected and incubated from adults
in each aquarium. Preferably they should be from each of the 5 spawning
pairs. If necessary because of small spawns, eggs can be collected over an
extended period in order to obtain 50 eggs.
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2. If no spawning occurs in the highest concentration, eggs should
be transferred from control spawns and incubated in the highest
concentration to gain additional information.
3. Groups of 50 eggs are placed in two or more egg cups. Survival
of embryos, time required to hatch, hatching success, and survival of fry
for 4 weeks will be determined and recorded.
4. Additional groups of 50 eggs from fish from contaminated aquaria
should be placed in control aquaria to determine if they contain chemicals
toxic to embryos or fry.
k. 1?2 Embryos and Fry
Survival of embryos and fry will be recorded daily for 4 weeks.
Fry are to be measured on one day post hatch and weighed and measured on
week 4 (termination). Daily records should be kept on mortalities and
development of abnormalities.
1. Termination of Adults
1. With many chronic procedures utilizing other fishes, termination
of the test is considered the time when no spawning activity has occurred
over a 2 week interval. For the sheepshead minnow, however, termination
should occur after 2 or more weeks of spawning. Enough spawns have to occur
to statistically predict the effect of the material that is tested. It is
our experience that most fish will spawn readily and almost daily unless
they are affected by a pollutant or are immature.
2. Adult fish should be weighed, measured, sexed and retained for
residue analysis.
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REFERENCES
American Public Health Association, American Water Works Association and
Water Pollution Control Federation, 1971. Standard Methods for the
Examination of Water and Wastewater, 13th Ed. Am. Publ. Health Assoc.,
Inc. New York, N.Y. pp. 879.
Committee on Methods for Toxicity Tests with Aquatic Organisms, 1974.
Methods for Toxicity Tests with Aquatic Organisms. EPA Ecological
Research Series. (In Press).
Drummond, Robert A. and Walter F. Dawson. 1970. An Inexpensive Method for
Simulating Diel Patterns of Lighting in the Laboratory. Trans. Amer.
Fish Soc. 99(2):434-435.
Hansen, D.J., P.R. Parrish, J.I. Lowe, A.J. Wilson, Jr. and P.D. Wilson.
1971. Chronic Toxicity, Uptake and Retention of Arocloi®1254 in Two
Estuarine Fishes. Bull. Environ. Contain. Toxicol. 6:113-119.
f&
Hansen, D.J., S.C. Schimmel and J. Forester. 1974. Aroclor5' 1254 in Eggs
of Sheepshead Minnows. Effect of fertilization success and survival of
embryos and fry. Proc. S. E. Assoc. Game Fish Comm. (In Press).
McKim, J.M. and DA.A. Benoit. 1971. Effect of Long-term Exposures to
Copper on Survival, Growth and Reproduction of Book Trout (Salvelinus
fontinalis). J. Fish. Res. Board Canada 28(5):655-662.
Mount, Donald I. 1968. Chronic Toxicity of Copper to Fathead Minnows
(Pimephales promelus, Rafinesque). Water Research 2:21-29.
Mount, Donald I. and William Brungs. 1967. A Simplified Dosing Apparatus
for Fish Toxicology Studies. Water Research 2:21-29.
Schimmel, S.C. and D.J. Hansen. 1974. Effects of Aroclor^ 1254 on the
Embryo and Fry of Sheepshead Minnows. Trans. Amer. Fish Soc.
103(3):522-586.
Sprague, J.B. 1969. Review Paper: Measurement of Pollution Toxicity to
Fish. 1. Bioassay methods for acute toxicity. Water Research
3(11):793-821.
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C. FISH BRAIN ACETYLCHOLINESTERASE INHIBITION ASSAY
1. Introduction
The purpose of this procedure is to provide a method for determining the
effect of materials to be dumped in the ocean on acetylcholinesterase (AChE)
in fish brains,. This is a test for nerve poisons which disrupt nerve
impulse transmission by inhibiting AChE, the enzyme that modulates levels of
the neurotransmitter .acetylcholine (Koelle, 1963; Karczmar, 1970). This
procedure is not necessary for materials that contain no AChE inhibiting
poisons.
It has been shown that brain-AChE of fishes is inhibited by ±n vivo
exposure to organophosphate and carbamate pesticides under laboratory
conditions (Weiss, 1958, 1961; Carter, 1971; Coppage, 1972). Furthermore,
environmental water pollution by these pesticides has been monitored by
measuring AChE activity in fish brains (Williams and Sova, 1966; Holland et
al. 1967; Coppage and Duke, 1971). Coppage (1971) defined the conditions
necessary for obtaining reliable and reproducible data in the laboratory
AChE assays and reported in vitro effects of four pesticides on AChE
activity in brains of sheepshead minnows (Cyprinodon variegatus). Coppage
and Matthews (1974) further refined assay techniques and reported acute
effects of in vivo exposure to organophosphate pesticides on cholinesterases
of four estuarine fishes and a shrimp.
2. Recommended Procedure for Exposing Animals
Fish should be exposed to the material as recommended in the definitive
test of the continuous-flow method for acute toxicity using fish and
macroinvertebrates as described in this manual. Fish to be assayed for AChE
should be from control aquaria and, if possible, from three contaminated
aquaria in which some fish have died. Live control fish should be divided
into three groups of three to six fish each for assay. Three to six fish
from the contaminated aquaria should also be assayed using the following
method.
3. Recommended Procedure for AChE Assay
a. Preparation of Fish Brains (3 to 6 brains are pooled for each
sample).
1. Weigh 5 cm square of aluminum foil in following manner: pick up
and place foil on balance pan with forceps (fingers can leave enough
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moisture to cause weight error at this low weight). Weigh, then leave on
pan at full rest.
2. Place another larger piece of foil in dissecting area.
3. Kill fish by placing them in a clean beaker containing acetone
for about 3 minutes.
4. Pour fish into clean sink or pan and scale heads under running
water with scalpel. (Check all dissecting equipment used. Make sure it has
been cleaned and rinsed with acetone to avoid pesticide residues. As heads
are scaled, place fish in another beaker containing acetone.)
5. Pour fish into clean sink or pan and then place them on paper
and blot dry.
6. With scissors, clip away skull from above the brain.
7. After all skulls have been clipped, remove brains by pulling off
bone flap with forceps and digging bone and flesh away from spinal cord with
probe if necessary. Cut spinal cord about 2 mm behind brain.
8. Strip brain from optic nerves after lifting from behind, and
place on larger piece of foil.
9. After all brains are removed, transfer them with forceps to the
preweighed foil on the balance pan and determine weight in milligrams.
Divide weight by five.
10. Transfer weighed brains to nylon cup (See next section) and add
about 4 ml of distilled water.
11. Homogenize for 1 minute then pour into graduated cylinder.
Rinse cup several times with distilled water and pour into cylinder.
12. Add distilled water to cylinder until total volume (in ml)
equals the number found by dividing the brain weight by five. Pour this
into beaker to gently mix. Assay within 30 minutes after preparation.
b. Assay for AChE
AChE activity should be determined by using an automated recording
pH stat to measure normal and in vivo-inhibited brain AChE. The following
procedure applies: mix 2 ml of diluted brain homogenate with 2 ml of 0.03 M
acetylcholine iodide in distilled water; titrate the liberated acetic acid
with carbonate-free 0.01 N NaOH; carry out the reaction at pH 7 and 22°C
while passing nitrogen over the liquid to prevent adsorption of atmospheric
carbon dioxide. Calculate the micromoles of substrate hydrolyzed per unit
of time from the number of micromoles of NaOH required to neutralize the
liberated acetic acid per unit of time, and express AChE activity as
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micromoles of ACh hydrolyzed per hour per mg brain tissue in reaction
vessel.
4. Calculations and Reporting
Assay results of exposed and control fishes are compared, and
percentages of normal brain AChE activity of exposed fish are reported.
Results should be subjected to statistical analysis (student's t-test, for
example) to determine statistical validity. Original control fish may be
divided into groups of five and brains pooled for each group of five to
obtain samples for normal AChE and statistical comparisons to exposed fish
replicates.
5. Reports
Any deviation from this method must be noted in all reports of results.
A report of the results of a test must include:
1. name of method, author, laboratory, and date tests were conducted;
2. a detailed description of the material tested, including its source,
date and time of collection, composition, known physical and chemical
properties, and variability of the material tested;
3. the source of the salt water, its date and method of preparation;
4. detailed information about the test animals, including name, standard
length, weight, age, source, history, and acclimation procedure used;
5. a description of the experimental design, the test containers, the
volume of test solution, the number of organisms per concentration, and the
loading;
6. period of exposure and number of animals dead at end of exposure;
7. percent of control organisms that died or were affected during the
test;
8. methods used for and the results of all test material, dissolved
oxygen, pH, and temperature measurements; and
9. any other relevant information.
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REFERENCES
Carter, F.L. 1971. _In vivo Studies of Brain Acetylcholinesterase
Inhibition by Organophosphate and Carbamate Insecticides in Fish.
Unpublished Ph.D. dissertation, Louisiana State Univ., Baton Rouge,
Louisiana.
Coppage, D.L. 1971. Characterization of Fish Brain Acetylcholinesterase
With an Automated pH Stat for Inhibition Studies. Bull. Environ.
Contam. Toxicol. 6(4):304-310.
Coppage, D.L. 1972. Organophosphate Pesticides: Specific Level of Brain
AChE Inhibition Related to Death in Sheepshead Minnows. Trans. Am.
Fish. Soc. 101(3):534-536.
Coppage, D.L. and T.W. Duke. 1971. Effects of Pesticides in Estuaries
Along the Gulf and Southeast Atlantic Coasts. In: Proceedings of the
2nd Gulf Coast Conference on Mosquito Suppression and Wildlife
Management, pp. 24-31. (C.H. Schmidt, Ed.) National Mosquito Control-
Fish and Wildlife Management Coordinating Committee, Washington, D.C.
Goppage, D.L. and E. Matthews. 1974. Short-term Effects of Organophosphate
Pesticides on Cholinesterase of Estuarine Fishes and Pink Shrimp. Bull.
Environ. Contam. Toxicol. 11(5):483-488.
Holland, H.T., D.L. Coppage and P.A. Butler. 1967. Use of Fish Brain
Acetylcholinesterase to Monitor Pollution by Organophosphorus
Pesticides. Bull. Environ. Contam. Toxicol. 2(3):156-162.
Karczmar, A.G. (Ed.). 1970. Anticholinesterase Agents. Pergamon Press,
New York.
Koelle, G.B. (Ed.). 1963. Cholinesterases and Anticholinesterase Agents.
Springer-Verlag, Berlin.
Weiss, C.M. 1958. The Determination of Cholinesterase in the Brain Tissue
of Three Species of Fresh Water Fish and Its Inactivation in vivo.
Ecology 39:194-199.
Weiss, C.M. 1961. Physiological Effect of Organic Phosphorus Insecticides
On Several Species of Fish. Trans. Am. Fish. Soc. 90:143-152.
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Williams, A.K. and R.C. Sova. 1966. Acetylcholinesterase Levels in Brains
of Fishes From Polluted Waters. Bull. Environ. Contam. Toxicol.
1:198-204.
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/9-76-010
3. RECIPIENT'S ACCESSI Ot* NO.
4. TITLE AND SUBTITLE
Bioassay Procedures for the Ocean Disposal
Permit Program
5. REPORT DATE
May 1976 (Issuing Date)
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
10. PROGRAM ELEMENT NO.
Environmental Research Laboratory, Gulf Breeze, Florida
Environmental Research Laboratory, Narragansett, R.I.
Environmental Research Laboratory, Corvallis, Oregon
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Gulf Breeze, Florida 32561
13. TYPE OF REPORT AND PERIOD COVERED
In-house
14. SPONSORING AGENCY CODE
EPA-ORD
15. SUPPLEMENTARY NOTES
16. ABSTRACT
The bioassay procedures given in this manual were developed to provide tests
for conducting toxicity evaluations of waste materials considered for ocean disposal
under EPA's Ocean Disposal Permit Program.
Nine bioassay procedures are described; three of which are considered "special"
and are not recommended for routine use. The procedures specify the use of various
organisms representing several trophic levels. Both flow-through and static tests
are included. Methods given vary in their utility and complexity of performance.
The procedures are not intended to be considered "standard methods", but are to be
used as reference methods or official methods dependent on the judgement of the EPA
Regional Administrator responsible for the management of the permit program.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Bioassay, Oysters, Marine fishes,
Algae, Crustacea
Bioassay procedures,
Ocean Disposal Permit
Program, Marine organisms,
Marine phytoplankton,
Brine shrimp, Calanoid
copepods, Macroinverte-
brates, Fish brain
acetylcholinesterase
6F
6T
13. DISTRIBUTION STATEMENT
Unlimited
19. SECURITY CLASS (ThisReport)
Unclassified
21. NO. OF PAGES
106
20. SECURITY CLASS (Thispage)
Unclassified
22. PRICE
EPA Form 2220-1 (9-73)
96
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