EPA-560/5-77-002
A CONTINUOUS FLOW SYSTEM USING FISH AND
AMPHIBIAN EGGS FOR BIOASSAY DETERMINATIONS
ON EMBRYONIC MORTALITY AND TERATOGENESIS
FINAL TECHNICAL REPORT
APRIL 1977
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF TOXIC SUBSTANCES
WASHINGTON.D.C. 20460
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NOTICE
This report has been reviewed by the Office of Toxic
Substances, U. S. Environmental Protection Agency, and
approved for publication. Approval does not signify that
the contents necessarily reflect the views and policies
of the U. S. Environmental Protection Agency. Mention of
tradenames or commercial products is for purposes of
clarity only and does not constitute endorsement or
recommendation for use.
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EPA-560/5-77-002
A CONTINUOUS FLOW SYSTEM USING FISH AND AMPHIBIAN EGGS
FOR BIOASSAY DETERMINATIONS ON
EMBRYONIC MORTALITY AND TERATOGENESIS
BY
Wesley J. Birge
Jeffrey A. Black
EPA Contract No. 68-01-4321
EPA Project Officer
I. Eugene Wallen, Ph.D.
For
U. S. Environmental Protection Agency
Office of Toxic Substances
4th and M Streets, S. W.
Washington, D. C. 20460
April 1977
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TABLE OF CONTENTS
SUBJECT
PAGE
INTRODUCTION
ADVANTAGES OF FISH AND AMPHIBIAN EMBRYOS AS BIOASSAY TEST ORGANISMS
BIOASSAY PROCEDURES
Bioassay System
Modifications of Present Bioassay System
Culture Water
Test Conditions and Monitoring Procedures
Monitoring of Toxicant
Test Concentrations and Exposure Time
Sample Size and Test Responses
Selection of Animal Test Species
FIGURES
Figure 1. Bioassay Screening of Environmental Pollutants
Figure 2. Continuous Flow Bioassay System
TABLES
Table 1.
Table 2.
Table 3.
APPENDIX I:
APPENDIX II:
APPENDIX III:
BIBLIOGRAPHY
Performance of the Continuous Flow System
Syringe Pump and Peristaltic Pump Flow Rates and
Dilution Ratios
Synthetic Culture Water
SUPPORTING DATA (Figures 3-6; Tables 4-19)
SYNOPSIS OF BIOASSAY PROCEDURES
NORMAL AND TERATOGENIC STAGES OF RAINBOW TROUT
ALEVINS AND FRY (Plates I-VI)
6
6
11
11
14
15
17
17
18
5
7
9
10
13
20
42
49
56
ii
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INTRODUCTION
It is becoming increasingly apparent that, concerning the toxic effects
of many environmental trace contaminants, embryonic and early juvenile stages
constitute the critical "sensitive links" in the life cycles of many animal
species (1, 24). It appears plausible that reproductive potential of natural
animal populations may be severely restricted or abolished by trace levels of
toxicants which are harmless or sublethal to most adult organisms. Accordingly,
protective environmental standards and pollution abatement policies, which have
been based to a considerable extent on tolerances of adult animals, may not
provide adequate protection for embryonic development and reproduction of many
species.
Numerous organic toxicants are known to inhibit animal reproduction (1-18).
For example, Bowes et a]_. (16) have found high levels of DDE and PCB in avian
eggs, with PCB reaching egg concentrations of 300 ppm wet weight, and 3700 ppm in
lipid fraction. These occurrences were correlated with embryonic death and reduced
hatchability.
Also, PCB has been shown to adversely affect reproduction of fish and certain
invertebrates. In continuous flow bioassays, exposure to 1.3 ppm Aroclor 1254
for three weeks produced 50% reproductive impairment in cultures of Daphnia magna
(18), and a concentration of 0.32 ppb reduced survival of early fry of the sheeps-
head minnow to 62%, compared with 89% for controls (3). Nebeker et_ al_. (5)
recently investigated the effects of several PCB compounds on survival and
reproduction of the fathead minnow. Aroclor 1254 at 4.6 ppb produced 100%
mortality of eggs, and 0.52 ppb resulted in 59-67% hatchability, compared to
71-76% in control populations. Early fry and embryos were the most sensitive
stages in this chronic life cycle study. Jensen et^ a]_. (6) reported that PCB
residues of 0.4-1.9 ppm in salmon eggs resulted in mortality ranging from 16-100%.
As the accumulation of PCB in fish eggs and tissues may be 75,000-200,000 times
the concentration found in water (1, 19), environmental levels as low as 2-20 parts
per trillion may inhibit egg hatchability in salmonid fishes.
Though generally less toxic than PCB compounds, DDT is known to concentrate
in fish eggs and sperm, affecting hatchability and survival of fry (1, 7-9, 11-13).
Egg concentrations of 0.4 to 4.75 ppm or more produce high levels of embryonic
mortality in trout and salmon (1, 8, 9), and treatment of early salmon embryos
with 50-100 ppb DDT retards behavioral development and impairs balance (1.2).
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Furthermore, the exposure of adult flounder to DDT at a sublethal concentration
of 2 ppb reportedly produces appreciable frequencies of anomalies among resulting
offspring (11).
Various other chlorinated hydrocarbons, including 2,4-D, dieldrin and
endrin are known to produce mortality of fish eggs and fry (10, 11, 20, 21).
In static tests with bluegill, Hiltibran (10) proposed a threshold for develop-
mental stages of approximately 2 ppm 2,4-D, and in chronic bioassays on the
sailfin molly, 0.75 ppb dieldrin was found to reduce growth rate and reproduction
(22). Working with endrin, Johnson (23) reported sublethal exposure to adult
fish which resulted in egg concentrations sufficient to produce mortality of
developing fry. Though many organic toxicants remain to be investigated, it
is evident that animal reproduction is one of the principal target sites of
chlorinated hydrocarbons and numerous other organic pollutants.
Numerous inorganic toxicants also exert adverse effects on animal repro-
duction, particularly aquatic species. Eggs, embryos and/or early juvenile
stages of fish are substantially more susceptible than adults to such metals as
cadmium, copper, mercury and zinc (24-30). For example, continuous flow treat-
ment of rainbow trout eggs with 0.1 ppb inorganic mercury produced 100% embryonic
mortality in 10-14 days (39). In the same investigation, trout and channel cat-
fish embryos treated for 5 days at 0.1-3.0 ppb mercury were found to accumulate
tissue mercury at 500-2000 times the exposure levels. In addition, frog and
chick embryos are up to 1000-10,000 times more sensitive to mercury than are
adult stages (24, 31-34).
Bioassays with boron compounds provide further evidence that developmental
stages are appreciably more sensitive to environmental toxicants than are adult
organisms. Working with the adult mosquito fish (Gambusia affinis). Wall en et al.
(35) reported 96-hr TL values of 3600 and 5600 mg/1 for sodium borate and boric
acid, respectively. Using the figure given for boric acid, this amounts to an
approximate dosage of 980 ppm boron. Turnbull et. al_. (36) determined a 24-hr
TL of 15,000 mg/1 for bluegill treated with boron triflouride administered at
20° C in Philadelphia tap water, and a 48-hr LDgo of 339 ppm boron has been
reported for 15-month old rainbow trout (37, 38). In addition, Sprague (37) has
given safe limits*of 30 ppm and 33 ppm for largemouth bass and bluegill, respectively.
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By comparison, low concentrations of boron produce high frequencies of mortality
and teratogenesis in populations of fish, amphibian and avian embryos (40-45).
Using continuous flow treatment from fertilization through 4 days posthatching,
we obtained boron threshold (LC1) values of 0.001-0.1 ppm, 0.2-1.4 ppm and 0.2-
5.5 ppm for developmental stages of the rainbow trout, goldfish, and channel
catfish treated with boric acid or borax at water hardness levels of 50-200 ppm
CaC03 (45). Depending on test conditions, boron LC5Q values for developmental
stages ranged from 27-100 ppm for rainbow trout, 46-75 ppm for goldfish, and 22-155
ppm for catfish. Concerning avian species, which share a number of developmental
characteristics with piscine forms (.§_• 3.., megalecithal eggs, meroblastic cleavage,
primitive streak gastrulation), embryonic stages also are impaired by low dosages
of boron compounds (43-45). Combining frequencies of embryonic mortality and
teratogenesis in chick embryos treated with boric acid and borax, we obtained
boron threshold and LC5Q values of 0.01 ppm and 0.5-1.0 ppm, respectively.
In contrast to the high boron sensitivity of chick embryos, laying hens have
been found to tolerate a daily ration containing 0.5% boric acid (875 ppm boron).
Though egg laying ceased after 6 days of exposure, normal egg production resumed
within 2 weeks after treatment was discontinued (46).
Operating upon the premise that environmental quality standards for trace
contaminants should be established at levels which are safe for the most suscep-
tible stages in the life cycles of living organisms, consistent with the recom-
mendations of the NTAC, NAS-NAE Committee on Water Quality Criteria, and others
(1, 31, 47-50), we contend that sensitive developmental stages should be included
among the test organisms used for the bioassay analysis of environmental pollutants.
Under support from the Department of Interior (Office of Water Research and
Technology), Environmental Protection Agency (Office of Toxic Substances) and
the National Science Foundation (RANN), we have developed a series of bioassay
and bioindicator procedures for use in evaluating the effects of environmental
toxicants on eggs, embryos and early juvenile stages of fish, amphibian and avian
species. These test systems include a yolk sac injection technique for the chick
embryos (33, 34), as well as static rapid-scan (24, 31, 51), continuous flow,
and full life-cycle bioassays for aquatic vertebrates (39). Attention here will
be restricted primarily to a continuous flow procedure for use with developmental
and early juvenile stages of fish and amphibians, where exposure is maintained
continuously from fertilization through 4-7 days posthatching. Resulting dose/
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response data may be used to define tolerances of sensitive life cycle stages
(£.£., mortality, embryonic anomalies, behavioral impairments) or to screen
compounds for teratogenic properties.
ADVANTAGES OF FISH AND AMPHIBIAN EMBRYOS AS BIOASSAY TEST ORGANISMS
Developmental stages present a broad spectrum of target sites for the toxic
action(s) of trace contaminants, including the biochemical and physiological
mechanisms associated with fertilization, genie action, cell proliferation and
growth, cellular differentiation, basic metabolism and systemic functions, as
well as the hatching process and the initial accommodation to a free-living
existence. As a consequence, bioassay screening with developmental stages is
unlikely to produce "false negative" results, providing exposure to selected
toxicants is maintained continuously from fertilization through 4-7 days
posthatching.
Fish and amphibian developmental stages also display high levels of sensitivity
to environmental contaminants. Concerning more toxic elements or compounds (e..g_.,
Hg, Cd, chlorinated hydrocarbons), developmental stages of trout or other
sensitive species usually exhibit irreversible test responses, including mortality,
teratogenesis or locomotor impairment, at ppb to ppt exposure levels. The
sensitivity of such bioassays often exceeds analytical detection limits. Less
toxic substances (£.£., boron compounds) generally produce test responses at
ppm to ppb concentrations. With respect to metallic toxicants (e_.g_., As, Hg,
Pb, Zn), we usually detect mortality and/or teratogenesis of piscine developmental
stages at exposure levels below those which initiate avoidance or other behavioral
responses in adult fish (39, 45, 52-54). As they exhibit both high sensitivity
and clearly detectable test responses, developmental stages are especially
suitable for short-term bioassay studies. The schematic in Figure 1 indicates a
possible means of using aquatic embryos and larvae for bioassay screening purposes.
Piscine developmental stages also possess a number of other advantages as
test organisms. They are less subject than adult fish to physiological or
behavioral variables induced by crowding or other test conditions. Also, they
require no feeding and excrete minimal waste products, exerting far less burden
on culture water than adult organisms. Test populations of 100-200 fish eggs can
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FIGURE 1
AQUATIC EMBRYOS AND LARVAE AS SENSITIVE, BROAD SPECTRUM TEST ORGANISMS FOR
BIOASSAY SCREENING OF ENVIRONMENTAL POLLUTANTS
TEST MATERIALS
MANUFACTURER
NO GO
Excessive
Risk
FISH & AMPHIBIAN EMBRYOS, JUVENILES
1. Rapid scan static bioassays
2. Continuous flow bioassays
(Toxicity, Teratogenesis)
Minimal
Risk
MANUFACTURER
GO
Probable Risk
Appeal
Excessive Risk
FURTHER TESTING
WITH OTHER SYSTEMS
I
BIOLOGICAL IMPACT
1. Life cycle bioassays
2. Bioaccumulation
3. Biomagnification
4. Biodegradation
5. Environmental half
life, etc.
I
Periodic Review
t
en
Use, Possible Regulations
HEALTH EFFECTS (MAMMALS)
1. Acute toxicity
2. Chronic toxicity
3. Carcinogenesis
4. Teratogenesis
5. Mutagenesis
6. Pharmacodynamics
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be maintained in a compact, closed culture system, providing better control
of exposure level and mixing pattern of the toxicant, and more precise regulation
of other test variables (e_.g_., pH, NH3, 02, water hardness, flow rate). This
is particularly important in testing compounds of low solubility or high volatility,
which are difficult to stabilize in open fish tank cultures. As generous samples
of fish eggs can be maintained under continuous flow conditions in culture
chambers of 0.5-1.0 liter capacity, it becomes feasible to use synthetic or
reconstituted culture water. This eliminates significant problems with back-
ground contamination found in many sources of natural water (e..(j.., heavy metals,
chloramines, phthalates, chlorinated hydrocarbons), and allows formulation
of desired water quality parameters (e..c[., pH, total alkalinity, water hardness).
In egg hatchability tests, the use of synthetic culture water and the omission
of feeding preclude the two major sources of background contamination which plague
bioassays with adult fish. For example, in performing chronic bioassays with
0.1-1.0 ppb cadmium and mercury, one of our principal problems has been to secure
contaminant-free fish food. We have analyzed numerous lots of commercial food
which contained 10,000-20,000 ppb lead, 500-700 ppb cadmium, 20-30 ppb mercury,
and appreciable levels of other metallic and organic toxicants.
In recent studies with mercury and cadmium, we have found threshold values
obtained with trout egg cultures equal or approximate to those determined in
chronic life cycle bioassays (39). Also, in an evaluation of 11 Central Kentucky
streams and rivers, egg hatchability indices for fish and amphibian species
correlated closely with independent ecological indicators of water quality (51; Table 19)
In particular, reductions in egg hatchability closely paralleled decreases in
density and diversity of piscine populations. The above findings further emphasized
the value of the egg culture technique for the initial screening and characteri-
zation of environmental toxicants. Due to their simplicity of design and economy
of maintenance, continuous flow bioassays on egg cultures may be performed in
many laboratories which lack adequate space, facilities or water supply necessary
for maintaining and testing populations of adult fish.
BIOASSAY PROCEDURES
Bioassay system. Our present continuous flow bioassay system is illustrated
in Figures 2 and 3* Fish eggs are cultured through 4-7 days posthatching in pyrex
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FIGURE 2
DIRECTION OF FLOW
CULTURE CHAMBER
FLOW
METER
CULTURE
MEDIUM
PERISTALTIC
PUMP
MIXING CHAMBER
FLOW
METER
CONTINUOUS FLOW BIOASSAY SYSTEM
SYRINGE
PUMP
TOXICANT
Peristaltic pump delivers culture water to mixing chamber at a prescribed flow rate.
Toxicant is administered to the mixing chamber by graduated flow from a syringe pump,
providing toxicant/culture water mixing ratios down to 1:1000 or less. The final
mixture, agitated with a teflon-coated stirring bar, is pumped through the egg culture
chamber under positive pressure. Mixing baffles in the culture chamber facilitate
uniform flow. High resolution liquid flow meters are used to determine flow rates
from syringe and peristaltic pumps.
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chambers through which test water is perfused at a prescribed flow rate. The
toxicant is administered to a mixing chamber situated ahead of each culture
dish, using graduated flow from a syringe pump. Synthetic culture water is
delivered to the mixing chamber by regulated flow from a peristaltic pump.
Flow rates from both syringe and peristaltic pumps are monitored by liquid
flow meters (Figure 3). Flow rates up to 2000 ml/hr or more are obtainable for
300-500 ml culture chambers, and syringe/peristaltic pump mixing ratios down
to 1:1000 or 1:10,000 can be maintained with accuracy. Syringe and peristaltic
pumps are provided with calibrated, continuously variable speed controls. The
concentration of toxicant delivered to the mixing chamber is regulated by
adjusting the mixing ratio from the pumping units and/or by varying the concen-
tration of toxicant delivered from the syringe pump. For example, a toxicant
at 0.1 ppm can be delivered from the syringe pump to a mixing chamber at 0.3
ml/hr, and diluted to a final test concentration of 0.1 ppb by culture water
metered through the peristaltic pump at 300 ml/hr. Solutions from the two
channels are mixed with a teflon-coated magnetic stirring bar, and delivered to
the culture chamber under positive pressure. Culture chambers are provided
with mixing baffles to insure uniform circulation of culture medium.
We normally maintain cultures in banks of 6 to 8, with each bank involving
a single syringe/peristaltic pump combination (Figure 3). Using syringes selected
for equal stroke volume, and fitting the peristaltic pump channels with tubing
of matched diameters, highly consistent performance is obtained. Two control
populations are maintained in each culture bank. Presently, we are using Brinkmann
(model 131900) and Gilson (model HP8) multichannel peristaltic pumps and Sage
syringe pumps (model 355). The latter are fitted with modified syringe holders
(Figure 3.3), and operated using 10 ml double-ground, glass syringes. Tuberculin
1 ml or micro!iter syringes may be used for toxicant delivery when mixing ratios
above 1:1000 prove necessary.
We have used methylene blue dye to check the accuracy of a 1:1000 syringe/
peristaltic pump mixing ratio, and to allow visual inspection of mixing and flow
patterns through the culture system. Results are summarized in Table 1. Also,
the reproducibility of flow rates, dilution ratios and exposure levels for several
boron bioassays is treated in Table 2. Accuracy of flow rates from syringe and
peristaltic pumps*is given in Table 5. We have used the above system extensively
for bioassay measurements on boron compounds and on numerous metallic toxicants, using
treatment levels down to 0.1 ppb. Monitoring of culture water for toxicant levels
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TABLE 1
PERFORMANCE OF THE CONTINUOUS FLOW SYSTEM
Reproducebility of 1:1000 Mixing Ratio for an Aqueous Solution of Methylene
Blue Dye (1000 ug/ml) Delivered at 0.3 ml/hr by a Sage Syringe Pump to Culture ,
Chambers Receiving 300 ml/hr of Culture Water Provided by Gil son Peristaltic Pump
CULTURE ACTUAL DYE CONCENTRATION2'3>4
CHAMBER NO. (ug/ml)
1 1.00 ±0.03
2 0.98 ±0.03
3 0.99 ±0.03
4 1.00 ±0.01
5 0.99 ±0.03
6 0.96 ±0.02
The pumping rates stated above were set to give a final, theoretical dye concentra-
tion of 1 ug/ml culture water. Dye concentrations were determined spectrophoto-
metrically (Gary model 17 recording spectrophotometer).
2
Each concentration given as the mean value ± standard error for 20 measurements
taken at regular intervals over a seven day period of continuous operation.
3
A single Sage syringe pump (model 355) was used to operate all six syringes, and
one multichannel Gil son peristaltic pump (model HP8) provided 6 separate supply
lines for culture water.
4
Sections of tygon tubing used for the different peristaltic pump channels were
selected for matching diameters. Six double ground, 10 cc syringes were mounted
on the syringe pump, using a modified syringe holder.
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TABLE 2
SYRINGE PUMP (BORON) AND PERISTALTIC PUMP (CULTURE WATER) FLOW RATES WITH RESULTING
DILUTION RATIOS OBTAINED IN TREATMENT OF TROUT EMBRYOS AT 1 AND 10 PPB BORONl
TROUT
COMPOUND/HARDNESS
BORIC ACID
50 ppm CaC03
200 ppm CaC03
BORAX
50 ppm CaC03
200 ppm CaCO,
3
BIOASSAYS
NOMINAL BORON
LEVEL (yg/1)
1.0
10.0
1.0
10.0
1.0
10.0
1.0
10.0
FLOW
(pl/hr)
330 ± 3
330 ± 3
318 ± 7
306 ± 1
318 ± 4
325 ± 3
320 ± 1
320 ± 1
SYRINGE PUMP
(BORON)
BORON (uq/1)
THEORETICAL ACTUAL
600 594 ± 4
6000 6050 ± 90
600 614 ± 4
6000 6130 ± 60
600 588 ± 11
6000 5930 ± 80
600 594 ± 4
6000 5870 ± 70
PERISTALTIC PUMP
CCULTURE WATER)
FLOW BORON
(ml/hr) DILUTION RATIO
198 ± 2.4
198 ± 4.8
183 ± 4.0
192 ± 3.7
188 ± 2.9
190 ± 2.8
194 ± 1.9
197 ± 1.3
600 ± 7
600 ± 15
575 ± 13
627 ± 8
591 ± 14
585 ± 11
606 ± 7
616 ± 5
FINAL
BORON
CONCENTRATION^
(yg/D
0.99
10.08
1.07
9.78
0.99
10.14
0.98
9.53
Mean values ± standard errors, where n was 10 for syringe pump boron analyses and 20 for all other determinations.
2
Final boron concentration delivered to the culture chamber was determined as actual syringe pump boron
concentration/boron dilution ratio. The latter was calculated as peristaltic pump flow rate/syringe pump
flow rate.
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has shown good reproducibility (Tables 2, 6-8).
Modifications of present bioassay system. The above system may be modified
in several important respects in order to accommodate testing of organic chemicals
of low solubility or high volatility.
A closed culture chamber may be used to eliminate the air/water interface,
minimizing volatility as a test variable. Such culture chambers may be constructed
from 3" pyrex pipe provided with clamp-locking, teflon 0-ring joints. Using
standard glass blowing techniques, the glass pipe should be cut and sealed to
give a capacity of 0.5 liter, and to position the 0-ring-sealed joint as close
as possible to the "top" of the culture dish. Access to the test organisms
may be obtained by opening the watertight joint and removing the chamber cover.
An outlet port should be annealed to the cover, with an inlet port positioned
near the bottom of the chamber. The culture chamber may be divided by a teflon
screen, positioned horizontally at mid-depth. Test eggs and embryos should be
maintained above the screen, while a glass or teflon-coated, magnetic stirring
bar may be used below the screen to provide moderate agitation of the culture water.
The second principal modification concerns the mixing chamber shown in
Figure 2. This unit should be equipped with a high capacity variable speed
stirring mechanism, suitable to provide a uniform suspension of insoluble toxicant
in culture water, and flow rate through the culture system should be adjusted to
minimize partitioning of the toxicant in the egg chamber. Some experimentation
may be required to develop an adequate mixing technique which will fulfill bioassay
requirements,without excessive foaming or other conditions unsuitable to the
design of the continuous flow procedure. Mechanical mixing, if properly adapted
to the system, may eliminate the need for carrier solvents or emulsifiers which
introduce undesirable test variables. We are now in the process of testing and
perfecting the above culture modifications, performing bioassays with a series of
organic toxicants which vary in solubility and volatility.
Culture water. We have given considerable attention to the development of
a synthetic culture water suitable for bioassays. Synthetic water provides more
stable test conditions than natural water, as the latter is subject to substantial
geographic and seasonal fluctuations in composition (e_.£., total dissolved solids,
hardness, pH). Also, problems with background contaminants are minimized when
prepared water is used for culture purposes. However, it is essential to use a
formulation for synthetic water which gives chemical and physical characteristics
similar to natural water.
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In the event that an ample supply of natural water of high quality is not
available, the synthetic water described below may be used. This formulation has
been used extensively during the past 2 years, and is known to give toxicity
responses with metals (e_.g_., Cd, Cu, Hg, Zn) which are closely comparable to
results obtained using natural water of similar composition. Also, we have
performed hatchability tests with 4 species of fish and 5 species of amphibians,
comparing our synthetic culture water to natural water from Steele's Run, a
local stream (limestone bed) which supports a healthy aquatic ecosystem.
Frequencies of hatchability were indistinguishable for the two water sources,
averaging 92% for fish and 96% for amphibians.
Synthetic culture water should be prepared from distilled, double deionized
water, having a conductivity of 0.25 pmhos or less. Routine monitoring should
be conducted for background contaminants. Calcium, magnesium, sodium and potassium
salts should be added as given in Table 3. This basic stock is prepared to give
a water hardness level of 200 ppm CaCOg and a pH of 7.5-8.0. Different levels
of hardness may be achieved by dilution of the basic stock water, according to
directions given in Appendix II. Should regulation of pH prove necessary, adjust-
ments may be made by altering the concentration of sodium bicarbonate and/or by
addition of dilute sulfuric acid or sodium hydroxide (55, 63).
This basic stock solution (Table 3) has essentially the same pH, conductivity
and water hardness as given for Mount's test water, which consisted of a mixture
of natural spring water (emerging from limestone strata) and carbon-filtered,
deionized tap water (55). Also, the content of calcium, magnesium and potassium
is in close agreement. The concentrations of cations and anions listed in Table
3 fall within ranges published for freshwater resources in Arizona (56), Kentucky
(57), and other areas of the U.S. (58). Total chloride content, total dissolved
solids, and the concentration of sodium plus potassium are well under maximum
levels of 170 mg/1, 400 mg/1 and 85 mg/1, observed in 95% of U.S. waters found to
support good, mixed fish fauna (59). Also, specific conductivity of 300 pmhos
falls within the range of 150-500 recommended for fish propagation (58), and is
typical of values recorded for freshwater resources in Kentucky (57). In addition,
osmolarity is well within the maximum limit of 50 mOsm/Kg water suggested for
freshwaters in the U.S. (47). Total alkalinity exceeds the minimum recommended
level of 20 mg/1 CaC03, and closely approaches the range cited as optimum to
support a diversified aquatic life (47, 58). As maintained in the culture system
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TABLE 3
SYNTHETIC CULTURE WATER
DISSOLVED SALTSl (mg/1 )
CaCl2
MgS04'7H20
NaHC03
KC1
150
150
100
5
CHEMICAL COMPOSITION (mg/1)
Ca
Mg
Na
K
Cl
HC03
so,
Total dissolved solids
54.2
14.8
27.4
2.6
98.2
72.6
58.5
328.3
PHYSICOCHEMICAL CHARACTERISTICS2
Hardness (as mg/1 CaC03)
Total alkalinity (as mg/1 CaC03)
Conductivity (vmhos/cm)
Osmolarity (mOsm/Kg H20)
PH
200.0
82.0
300.0
12.0
7.9
'Prepared in distilled, deionized water (specific conductivity
of 0.2 ymhos).
Measurements made at 22° C. Above figures represent mean
values for six measurements.
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described herein (continuous aeration), the dissolved oxygen level is approxi-
mately 10 mg/1 at the culture temperature used for trout embryos (13° C). A
minimum of 7 mg/1 has been recommended for trout and salmon spawning waters
(47). The pH of our culture medium also is well within the range found in
natural waters. Bass Becking et_ al_. (60) observed a pH range of 6-8 for 85% of
347 freshwater rivers and lakes surveyed, and pH values of 6.5-8.5 generally
are found in the more productive freshwater resources in the U.S. (47).
Test conditions and monitoring procedures. In the initial characterization
of an environmental toxicant, water quality parameters discussed above (Table
3) should be held constant, while the concentration of the toxicant is varied
to determine dose/response data. Using the bioassay system as recommended, the
commonphysicochemical parameters of the test water should fall within ranges
prevailing for most U.S. freshwaters, facilitating extrapolations from test
data. After initial dose/response studies are completed for a selected toxicant,
certain water quality variables (e_-g_., pH, hardness, temperature) may be manipulated
independently to determine whether they affect test responses exerted by the
toxicant.
In initial investigations, if only one water hardness level is to be selected,
we recommend using 100 or 200 ppm CaCOo, with a pH of 7.5-8.0 (Table 3; Appendix II).
Culture water should be given continuous aeration (filtered air) supplied either
to the mixing chamber or to the reservoir serving the peristaltic pump (Figure 2).
The latter is advised when more volatile toxicants are used. Temperature should
be maintained within the range considered optimum for development of the selected
animal test species, preferably holding overall fluctuations to 1° C. It should be
noted that the rate of embryonic development for fish and amphibians will vary
with temperature. Significant increases in temperature will shorten hatching
time, providing the tolerance limit of the species is not exceeded. Concerning
test species which we have used most frequently in the bioassay system described
above, average hatching times with preferred temperatures are 24 days for rainbow
trout (13° C), 6 days for channel catfish (22° C), 5 days for goldfish (19° C),
4 days for black bass (18° C) and 6-3.5 days for the leopard frog (18-25° C).
Data for additional species are given in Table 10.
Bioassay cultures preferably should be maintained in walk-in, temperature
regulated environmental rooms. All cultures must be monitored once or twice
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- 15 -
daily for temperature, dissolved oxygen, ammonia, water hardness and pH. For
these measurements we use a YSI tele-thermometer with thermocouple, YSI oxygen
meter (model 51 A), Orion ammonia and water hardness electrodes, and a Corning
digital pH meter (model 110, with expanded millivolt scale). The accuracy with
which general water parameters may be regulated within this test system is
illustrated in Table 4, which summarizes results for a series of 18 bioassays
conducted with boron compounds at water hardness levels of 50 and 200 ppm CaCO^
and pH levels varying from 7.5-8.5.
Particular attention must be given to trout embryos, especially during the
"green stage." Harmful exposure to artificial light should be precluded (61,
62) and it is advisable to maintain cultures under semi-sterile conditions to
minimize occurrences of soft egg disease or fungus. Prior to each use, culture
rooms are disinfected and irradiated with UV for 12 hours, and culture dishes and
other glassware are chemically cleansed and autoclaved. Culture rooms may be
maintained under positive pressure to minimize contamination. In the event of
fungus or soft egg disease, periodic treatment may be given with formaldehyde
(1/1000 for 10 minutes), acriflavine (1/2000 for 20 minutes), or other procedures
given in Appendix II.
Flow rates from peristaltic and syringe pumps must be monitored at least
twice daily, and the toxicant exposure level should be analyzed at regular,
12-24-hr intervals, as discussed below (Monitoring of Toxicant). Test organisms
must be examined daily to gauge extent and frequency of development, and to remove
dead specimens. Control eggs should be cultured simultaneously with experimentals
and under identical conditions, except for the omission of the selected toxicant.
In bioassays with metallic toxicants, we generally have maintained flow rates
at 200, 300 and 600 ml/hr, giving turnover times for 300 ml cultures of 1.5, 1.0 and
0.5 hours, respectively. As seen in Table 11, we have not found flow rate to
constitute a substantial test variable, providing it is sufficiently high to prevent
deterioration of culture water. For water soluble toxicants of low volatility,
we recommend a flow rate which will provide a culture turnover time of 0.5-1.0 hour.
However, flow rate requirements may differ significantly for certain organic
toxicants. Accordingly, some experimentation may be required to determine effects
of flow rate on selected organics. In particular, higher flow rates may be
required to prevent hydrophobic organics from partitioning in the culture chambers.
Monitoring of toxicant. It is essential that actual exposure levels be
-------
- 16 -
determined by direct chemical analysis of culture water. Each culture,
including controls, should be analyzed daily, using duplicate or triplicate
samples, and mean exposure ± standard error should be determined for the
bioassay treatment period. The continuous flow test system should be operated
24-72 hours prior to the introduction of test organisms. During this period,
toxicant exposure levels and other test parameters should be stabilized.
Particular care should be given to toxicant levels during the first 24
hours following initiation of the egg cultures. When toxicants of high
bioaccumulation potential are administered at ppb levels, the exposure concen-
tration may drop sharply when eggs are first added to the test system, and
adjustments in the toxicant injection rate may be required.
The method of chemical analysis to be used for culture monitoring should be
chosen carefully, considering detection limits, reproducibility, sample volume
requirements, and operational efficiency. Simultaneous operation of 20-30
bioassay units will impose a considerable burden on analytical facilities.
However, culture assays are essential on a daily basis, to confirm exposure
levels and to provide necessary data for adjustments in the test systems.
Generally, exposure levels can be held to an overall variation of 5-10%.
Proficiency in analytical techniques should be achieved before bioassay tests
are undertaken, and alternative procedures should be used to confirm the reliability
of the chosen monitoring method.
Considering the vast array of environmental toxicants which may require
screening, the bioassay technician may have recourse to a wide variety of
analytical procedures. Consultation with analytical chemists may help insure
proper choice and execution of detection methods. Two forms of instrumentation
which we have used extensively in bioassay studies include atomic absorption
spectrophotometry and gas chromatography. The former provides reliable
detection for a wide range of elements which may appear as trace contaminants
in water used for toxicological studies. In a present investigation on coal
toxicology, a Perkin-Elmer model 503 AAS unit, equipped for digital readout
and data recording, is being used to monitor inorganic trace elements. When
operated with a cold-vapor kit or graphite furnace, low detection limits are
achievable for mercury, cadmium and many other elements (Table 12).
In the bioassay evaluation of a series of organic toxicants, we are
-------
- 17 -
using gas chromatography (Packard 7400 with data integrator) as the principal
monitoring technique. Table 13 gives detection limits and recommended GC
procedures for several classes of organics.
Backup analyses for organic compounds are performed by GC-mass spectroscopy
(Finnigan model 3300 F). The latter is particularly useful for monitoring
control and experimental test water for background contaminants.
Test concentrations and exposure time. Depending upon the degree of
anticipated toxicity, solubility and analytical detection limits, bioassay
tests generally are initiated at 10-100 parts per million (10-100 mg/1)
toxicant and continued at 10-fold dilutions down to 0.1-1.0 part per billion
(0.1-1.0 yg/1). If necessary, this range of test concentrations may be extended
to provide adequate delineation of threshold and LC50 values, or to reflect
levels of toxicants reported for natural water resources. Usually, 5 to 6
test concentrations are sufficient for the initial characterization of a selected
toxicant. It is often desirable to repeat the exposure series for two or more
water hardness levels (.§_.£., 50 and 200 ppm CaCOo).
Generally, toxicants should be administered continuously from fertilization
through 4-7 days posthatching. At 4 days posthatching, using recommended temper-
atures, exposure periods will approximate 28, 10, 9 and 8 days for trout, catfish,
goldfish and bass, respectively. In bioassays with fish eggs, it appears that
certain environmental contaminants (e..£., Hg, B) are more toxic to early embryos,
while other pollutants (e..c|_., Cu, Zn, PCB) are more lethal to late embryonic stages
and/or early fry (3, 5, 24-30, 45). Exposure times noted above cover the full
range of embryonic events, as well as hatching and the sensitive early juvenile
period, and are advisable to adequately evaluate the effects of environmental
toxicants on the development and growth of immature piscine stages. However,
shorter exposure periods may be used for initial rapid-screening procedures.
Sample size and test responses. Control eggs should be cultured simul-
taneously with experimental and under identical conditions, except for the
omission of the selected toxicant. With proper daily maintenance, viable,
fertilized eggs should give hatching frequencies in control populations of 85%
or more (e..£., Table 14). In the event control survival drops below 75%,
attention should be given to the source of eggs and sperm, as well as to culture
conditions (Appendix II).
A sample size of 100-150 eggs (2-3 replicates) is recommended for both
-------
- 18 -
experimental and control cultures. Quantification of test responses generally
should be limited primarily to lethality, teratogenesis and locomotor impairment,
expressed in each case as frequency among experimental populations/frequency in
controls. Dose/response data should be plotted for continuous exposure from
fertilization through hatching and 4 days posthatching (e..£., Figures 4, 5;
Table 8). Threshold and LC5Q values should be expressed for individual and
combined test responses for embryonic and postembryonic stages, as well as for
the entire exposure period from fertilization to 4-7 days posthatching (£.5..,
Tables 8, 9). Log-probit analysis or other statistical procedures should be
used to determine significance of test responses (.e.cj.., Table 9).
Observations on teratogenesis may be restricted to debilitating anomalies
affecting those animals which survive the hatching process. Examples of normal
and teratogenic trout alevins are given in Plates I-VI of Appendix III. In the
analysis of teratogenic defects, particular attention should be given to the
skeletal system, especially the vertebral column. The latter appears to be the
single most reliable indicator of teratogenic action in fish. This is apparent
in Table 15, where we have summarized frequencies of boron-induced anomalies
for several aquatic species. As seen in Plates 11-IV, development of the vertebral
column, probably sclerotomial differentiation, also is affected by a number of
metallic toxicants (e..5_., Cd, Hg). Rainbow trout and the channel catfish are
particularly suitable test species for screening teratogenic compounds. As seen
in Table 16, trout embryos exhibit especially high frequencies of teratogenesis
for a wide range of metallic toxicants.
Selection of animal test species. Over the past 4 years we have worked with
approximately 20 fish and amphibian species. Most are adaptable for use with
the bioassay system described above. The fish which we have used most extensively
include the black bass (Micropterus salmoides), goldfish (Carassius auratus).
channel catfish (Ictalurus punctatus). and the rainbow trout (Salmo gairdneri).
Amphibian species, all of which are Anurans, include the red-spotted toad
(Bufo punctatus), southern gray treefrog (Hyla chrvsocephala), narrow-mouthed
toad (Gastrophryne carolinensis). squirrel treefrog (Hyla squirella). pig frog
(Rana grylio) and the leopard frog (Rana pipi ens). Hatching times, egg production,
seasonal availability and possible sources of supply are summarized in Table 10.
Numerous other Species also are suitable for test purposes, including the fat-
head minnow (Pimephales promelas), bluntnose minnow (Pimephales notatus). bluegill
-------
- 19 -
(Lepomis macrochirus) and the zebra fish (Brachydanio rerio).
In selecting test species, consideration should be given to availability,
ease of handling in the laboratory, geographical and ecological distribution,
pattern of reproduction and development, and sensitivity to toxicants. Sensitivity
of eggs and embryos may vary with hatching time, egg yolk volume, and certain
other developmental features. For example, in rapid-scan bioassays in which
culture water and toxicant were renewed at 12-hr intervals, we observed up
to 100-fold variations in mercury tolerances of various fish and amphibian eggs
(Figure 6; Tables 17, 18). Generally, mercury sensitivity of fish stages increased
with hatching time and/or egg yolk volume. However, species sensitivity does not
remain constant for all classes of toxicants. Thus, for in-depth bioassay
investigations, we recommend selecting 2 to 3 test species which differ in important
developmental characteristics. Such tests will provide a more reliable and
representative index to the embryopathic effects produced by environmental trace
contaminants.
In comparing the sensitivity of developmental stages of different fish and
amphibian species to particular toxicants, it is important to regulate exposure
period to cover essentially the same embryonic and post-embryonic stages. This
is especially important as both the order and degree of sensitivity of different
developmental stages may vary substantially for different toxicants. The
recommended exposure period largely encompasses all developmental processes from
fertilization through yolk sac resorption, providing a basis for comparative
toxicological studies. As developmental (exposure) time may vary for different
test species, it should be treated appropriately as a significant test variable.
-------
- 20 -
APPENDIX I
SUPPORTING DATA
FIGURES 3-6
TABLES 4-19
-------
- 21 -
FIGURE 3
CONTINUOUS FLOW BIOASSAY SYSTEM
3.1 Series of egg culture dishes (A) which receive toxicant
and culture water from mixing chamber (B). Culture water
is supplied to mixing chambers by peristaltic pump (C).
Liquid flow meters (D) are used to measure flow rate.
3.2 Enlarged view of culture room showing culture dishes (A),
mixing chambers (B), peristaltic pumps (C), and flow
meters (D). Culture dishes exhaust water into plexiglass
drain troughs which are connected to waste reservoirs.
3.3 Syringe pump used to supply toxicant to mixing chamber.
Syringe pumps are mounted on outside culture room wall
to avoid mechanical problems resulting from culture room
temperature and humidity. Note the modified holder which
supports six 10 ml syringes. A modified syringe mounting
bracket is shown inverted on the syringe drive carriage
(arrow).
-------
-------
FIGURE 4
100 •' -
80
60
§
40
20
0-01
N
ro
ro
0-1
I
10
100
1000
BORON CONCENTRATION (ppm)
TOXICITY OF BORIC ACID TO GOLDFISH EMBRYOS
-------
FIGURE 5
100
80
60
40
20
o-
Zn*+
Cd**
0-001
0-01
0-1
I
10
METAL CONCENTRATION (ppm)
ro
CO
100
TOXICITY OF METALS TO GOLDFISH EMBRYOS
-------
FIGURE 6
100
80
60
40
20
A-
o
I
•A—2
\
^^
\
A-GOLDFISH
o-CATFISH
•-TROUT
A-ALL SPECIES
ro
i
•A—A
A-
0-001 0-01
0-1
I
10
100
Hg++ CONCENTRATION (ppm)
TOXICITY OF INORGANIC MERCURY TO FISH EMBRYOS
-------
TABLE 4
TEST PARAMETERS OBSERVED DURING BIOASSAYS WITH BORON COMPOUNDS
(EMBRYONIC &
TEST
SPECIES
CATFISH
BIOASSAYS
POSTEMBRYONIC STAGES)
COMPOUND/WATER HARDNESS
BORIC ACID @
50 ppm CaCOo
200 ppm CaCOo
BORAX @
50 ppm CaCOo
200 ppm CaC03
FOWLER'S TOAD BORIC ACID @
GOLDFISH
LEOPARD FROG
TROUT
50 ppm CaCOo,
200 ppm CaC03
BORIC ACID @
50 ppm CaCOo
200 ppm CaCOa
BORAX @
50 ppm CaC03
200 ppm CaCOa
BORIC ACID @
50 ppm CaCOo
200 ppm CaCOa
BORAX @
50 ppm CaCOa
200 ppm CaCOa
BORIC ACID 0
50 ppm CaCOa
200 ppm CaCOa
BORAX 0
50 ppm CaCOo
200 ppm CaCOa
OBSERVED TEST PARAMETERS (MEAN ± STANDARD ERROR)
TEMPERATURE
(°C)
25.0 ± 0.4
24.7 ± 0.6
29.4 ± 0.3
29.4 ± 0.3
23.7 ± 0.6
23.7 ± 0.6
24.8 ± 0.3
24.8 ± 0.3
27.0 ± 0.4
27.0 ± 0.4
25.0 ± 0.3
25.0 ± 0.3
25.3 ± 0.2
25.3 ± 0.2
13.7 ± 0.1
13.3 ± 0.1
14.0 ± 0.1
13.0 ± 0.1
DISSOLVED
OXYGEN (ppm)
7.3 ± 0.1
7.6 ± 0.1
6.4 ± 0.1
6.5 ± 0.1
6.8 ± 0.1
6.8 ± 0.1
7.4 ± 0.0
7.5 ± 0.0
7.5 ± 0.2
7.5 ± 0.2
7.7 ± 0.2
7.8 ± 0.2
7.7 ± 0.2
7.8 ± 0.2
9.2 ± 0.1
9.6 ± 0.1
10.1 ± 0.2
10.3 ± 0.2
WATER HARDNESS
(ppm CaC03)
51. 8 ± 2.0
212.0 ± 11.0
46.7 ± 2.3
194.7 ± 3.2
57.4 ± 4.2
207.5 ± 10.0
54.4 ± 4.2
207.5 ± 10.0
46.2 ± 1.8
194.8 ± 16.0
52.5 ± 1.4
212.3 ± 5.4
46.2 ± 4.9
203.0 ± 2.3
54.1 ± 3.5
204.0 ± 4.0
49.0 ± 1.4
191.0 ± 4.0
PH
7.5 ± 0.1
7.6 ± 0.0
8.5 ± 0.0
8.2 ± 0.1
7.6 ± 0.1
7.6 ± 0.1
7.9 ± 0.1
7.6 ± 0.1
8.3 ± 0.1
8.1 ± 0.1
7.7 ± 0.0
7.7 ± 0.0
8.3 ± 0.1
8.4 ± 0.1
7.7 ± 0.1
7.9 ± 0.1
7.9 ± 0.1
7.8 ± 0.1
FLOW RATE
(ml/hr)
194.2 ± 4.3
199.7 ± 2.7
188.8 ± 4.1
191.0 ± 1.8
185.3 ± 2.8
189.8 ± 4.6
184.6 ± 5.7
180.4 ± 5.4
208.0 ± 4.5
224.2 ± 3.0
193.4 ± 2.3
189.2 ± 4.4
180.0 ± 4.1
195.6 ± 4.1
195.0 ± 1.2
189.4 ± 2.1
190.4 ± 2.9
189.4 ± 2.1
ro
01
-------
- 26 -
TABLE 5
TABLE 5A. ANALYSIS OF FLOW RATES FOR ,
BRINKMAN PERISTALTIC PUMP (MODEL 131900)1
Flow Rate
(ml/hr)
50
100
150
300
1
49.8*0.3
99.4*0.4
150.8*2.4
299.9*2.0
2
49.5*0.2
97.4*0.5
146.6*1.0
294.6*2.7
Channel
3
49.2*0.1
98.6to.3
150.2*0.8
296.1*2.0
Number^
4
50.0*0.6
101.5*0.4
150.9*0.9
304.6*2.0
5
50.8*0.2
101.6*0.4
149.1*1.2
302.2*2.1
6
50.1*0.2
99.7±0.3
151.4*1.2
297.7*2.3
1 Tygon tubing - 1/16" ID, 1/8" OD.
2 Mean * standard error; each value based on 20 reolicatesover a 7 day
period.
TABLE 5B. ANALYSIS OF FLOW RATES FOR
GILSON PERISTALTIC PUMP (MODEL HP8)1
Flow Rate
(ml/hr)
50
100
175
275
1
48.7*0.4
96.7*0.4
170.4*0.6
267.3*0.8
2
50.4*0.7
100.5*0.5
173.6*0.6
273.9*2.0
Channel
3
50.8*0.6
99.9*0.5
177.2*0.5
278.5*0.9
Number^
4
48.7*0.4
100.1*0.5
173.8*0.6
274.1*0.6
5
48.3*0.0
96.5*0.4
172.4*0.4
268.5*0.7
6
48.3*0.0
96.5*0.4
169.4*0.5
265.5*9.7
1 Tygon tubing - 1/16" ID, 1/8" OD.
2 Mean * standard error; each value based on 20 replicates over a 7 day
period.
TABLE 5C. ANALYSIS OF DELIVERY RATES FOR
SARE SYRINGE PUMP (MODEL 355)
Flow Rate
(ml/hr X 10(
Syrinoe Number^
1
10.002 10.07*0.24 9.80*0.17 10.00*0.16 10.21*0.21 9.53*0.13 10.42*0.19
30.002 31.40*0.46 31.80*0.48 32.00*0.37 31.50*0.48 31.50*0.50 32.60*0.36
30.OO3 30.14*0.57 30.09*0.67 32.05*0.76 31.13*0.82 30.50*0.58 30.14*0.61
60.OO3 64.00*1.24 57.11*2.20 60.38*1.31 63.79*1.24 61.67*2.16 64.00*1.15
1 Mean * standard error; each value based on 20 reolicates over a 7 day neriod.
2 10 ml glass syringe (sinale ground).
3 10 ml p!astickdisposable syrinoe.
-------
TABLE 6
CORRELATION OF PERCENT SURVIVAL WITH EXPOSURE TIME
FOR TROUT EMBRYOS TREATED WITH INORGANIC MERCURY IN A CONTINUOUS FLOW SYSTEM
CULTURE
NUMBER
i
2
•j
% SURVIVAL
ppb Hg*2
% SURVIVAL
ppb Hg
515 SURVIVAL
ppb Hg
1
100
0.1
100
0.1
100
0.1
2
99
0.1
97
0.1
100
0.1
EXPOSURE TIME IN DAYS
3456
95
0.1
94
0.2
97
0.1
82 59 41
0.1 0.1 0.1
79 55 39
<0.1 0.1 0.1
03 57 49
<0.1 <0.1 0.1
MEAN EXPOSURE1
7 8
23 0
0.1 0.2 0.11 ± 0.01
26 0
i
"" " ro
0.2 0.3 0.14 ± 0.03 ,
26 0
0.2 0.2 0.10 ± 0.03
1
Mean exposure ± standard error for the 8 day period.
-------
TABLE 7
EXPOSURE OF RAINBOW TROUT EMBRYOS AND ALEVINS (SALMO GAIRDNERI)
CONCENTRATION1
CADMIUM (PPB)
5.16 ± 0.44
11.52 ± 0.82
27.95 ± 1.70
50.95 ± 2.51
79.52 ± 3.97
103.20 ± 4.09
254.00 ± 7.00
TO CADMIUM IN WATER OF 100
TEST RESPONSES AT
% HATCHABILITY2
94(2)
93(3)
86(8)
76(9)
72(10)
78(23)
55(100)
PPM CaC03
HATCHING
% NORMAL
H
92
90
79
69
65
60
0
HARDNESS
(H) AND 4 DAYS
SURVIVAL3
PH
92
89
67
63
62
55
0
POSTHATCHING
(PH)
% DEAD OR TERATOGENIC
H
8
10
21
31
35
40
100
PH
8
11
33
37
38
45
100
Concentration given as mean ± standard error as determined by atomic absorption spectrophotometry.
2
Percent of surviving population bearing gross teratogenic defects is given parenthetically.
3Percent of initial population surviving with normal morphology.
ro
oo
-------
TABLE 8
EXPOSURE OF CATFISH EMBRYOS AND FRY (ICTALURUS PUNCTATUS)
TO BORON (BORIC ACID) IN WATER OF 200 PPM CaC03 HARDNESS
TEST RESPONSES AT HATCHING (H) AND 4 DAYS POSTHATCHIN6 (PH)
CONCENTRATION
BORON (PPM) i c, uATpHflR
NOMINAL ACTUAL1 * HATCHAB
0.01
0.05
0.50
0.75
1.00
2.50
5.00
7.50
10.00
25.00
50.00
75.00
150.00
300.00
[LITY2 % NORMAL
H
100(2) 98
100(1) 99
0.53 ± 0.02 100(2) 98
0.77 ± 0.01 100(2) 98
0.96 ± 0.01 96(3) 93
2.33 ± 0.12 99(2) 97
4.90 ± 0.07 95 t
7.40 ± 0.10 95 ^
9.43 ± 0.23 94 I
25.10 ± 0.27 90 2
48.30 ± 0.84 83 2
77.70 ± 0.54 71 4
140.00 ± 5.00 57 2
302.00 ±29.00 0
I 91
L 91
J 87
!8) 65
)0 58
\3 40
!2 44
0
SURVIVAL3
PH
98
98
94
98
86
87
82
75
65
40
28
9
2
0
% DEAD OR
H
2
1
2
2
7
3
9
9
13
35
42
60
56
100
TERATOGENIC
PH
2
2
6
2
14
13 ^
18
25
35
60
72
91
98
100
TConcentration given as mean ± standard error as determined by curcumln method.
2Percent of surviving population bearing gross teratogenlc defects is given parenthetically.
3Percent of Initial population surviving with normal morphology.
-------
TABLE 9
LOG PROBIT ANALYSIS OF THRESHOLD (LC^ VALUES IN PPM
FOR AQUATIC EMBRYOS EXPOSED TO BORON COMPOUNDS
1
BORIC ACID
TEST
SPECIES
CATFISH
FOWLER'S TOAD
GOLDFISH
LEOPARD FROG
TROUT
50 ppm
H
1.0
25.0
0.6
26.0
0.1
CaC03
PH
0.5
25.0
0.6
13.0
0.1
200 ppm
H
0.3
5.0
0,2
23.0
0.001
CaC03
PH
0.2
5.0
0.2
22.0
0.001
50 ppm
H
5.5
-
1.4
6.0
0.07
BORAX
CaC03
PH
5.5
-
1.4
5.0
0.07
200 ppm
H
1.7
-
0.9
3.0
0.07
CaC03
PH
1.7
-
0.9
3.0
0.07
threshold values were calculated at hatching (H) and 4 days posthatching (PH) for two
water hardness levels (50 ppm CaC03, 200 ppm CaC03). Grossly anomalous survivors were
counted as lethals.
to
o
-------
TABLE 10
SPECIES SELECTED FOR USE AS EMBRYONIC BIOINDICATORS
SPECIES
Largemouth Bass
(Micropterus salmoides)
Goldfish
(Carassius auratus)
Channel Catfish
(Ictalurus punctatus)
Rainbow Trout
(Sal mo gairdneri)
Pig Frog
(Rana grylio)
Leopard Frog
(Rana pi pi ens)
Red-spotted Toad
(Bufo punctatus)
Narrow-mouthed Toad
(Gastrophryne carol inensis)
Southern Gray Treefrog
(Hyla chrysocephala)
Squirrel Treefrog
(Hyla squirella)
Developmental
Time in Days
4 (Hft)
5 (19°C)
6 (22°C)
24 (13°C)
6 (22°C)
6 (19°C)
3 (22°C)
3 (22°C)
3 (22°C)
3 (22°C)
Number of Eggs*
Per Female
10,000-15,000
15,000-25,000
10,000-15,000
6,000-8,000
6,000-10,000
2,000-4,000
2,000-4,000
1,000-3,000
100-500
80-150
Breeder's
Availability
April -June
March-July
May-August
Sept. -April
March-June
Dec. -May
April -August
May-August
May-June
May-August
Source
National Fish Hatchery
Frankfort, Kentucky
National Fish Hatchery
Frankfort, Kentucky
National Fish Hatcheries
Frankfort, Ky. and
Senacaville, Ohio
National Fish Hatchery
Wytheville, Virginia
Charles D. Sullivan
Nashville, Tennessee
Amphibian Facility,
Univ. Michigan, Ann Arbor
Charles D. Sullivan
Nashville, Tennessee
Charles D. Sullivan
Nashville, Tennessee
Charles D. Sullivan
Nashville, Tennessee
Charles D. Sullivan
Nashville, Tennessee
CO
__1
I
''Egg production dependent upon size and maturity.
-------
TABLE 11
EFFECT OF FLOW RATE ON
METAL TOXICITY TO CATFISH EMBRYOS AND ALEVINS
FLOW RATE
(ml/hr)
50
125
250
400
TURNOVER RATE PERCENT SURVIVAL AT HATCHING (H)
(per hour)1 AND 4 DAYS POSTHATCHING (PH) FOR METAL CONCENTRATIONS IN PPM
Hg+2 H PH Cd*2 H PH Zn+2 H PH
0.2 0.0021 36 17 0.23 40 13 0.42 57 40
0.5 0.0027 45 23 0.25 48 22 0.50 59 38
1.0 0.0025 46 25 0.29 54 32 0.50 60 34
1.6 0.0024 43 25 0.30 50 27 0.59 61 35
Values represent fraction of a 250 ml culture chamber renewed per hour.
CO
ro
-------
- 33 -
TABLE 12
COAL TRACE ELEMENTS SELECTED FOR BIOASSAY EVALUATIONS
Trace
El ement
Al umi num
Antimony
Arsenic
Cadmium
Cobalt
Chromium
Copper
Germanium
Lanthanum
Lead
Manganese
Mercury
(Al)
(Sb)
(As)
(Cd)
(Co)
(Cr)
(Cu)
(Ge)
(La)
(Pb)
(Mn)
(Hg)
Molybdenum (Mo)
Nickel
Silver
Selenium
Strontium
Thallium
Tin
Tungsten
Vanadium
Zinc
(HI)
(Ag)
(Se)
(Sr)
(Tl)
(Sn)
(W)
(V)
(Zn)
Concentration
in Coal (ppm)
10,440-12,900
0.50-1.26
4.45-14.02
0.47-2.52
2.90-9.57
13.75-18.00
8.30-15.16
1.00-6.59
3.80-10.00
4.90-34.78
33.80-49.40
0.12-0.20
5.00-7.54
16.00-21.07
0.03-0.12
2.08-2.20
10.00-23.00
0.29-2.00
0.03-4.79
0.10-3.00
28.50-32.71
46-272
Bioassay
Test Compound
AlClg
SbCl3
NaAs02
CdCl2
CoCl3
CrCl2
CuCl2
Ge02
LaCl3
PbCl2
MnCl2
HgCl2
Mo02Cl2
NiCl2
AgN03
Na2Se04
SrCl2
T1C13
SnCl2
wo2ci2
V2°5
ZnCl2
Detection
Limit (AAS)
0.20 ppb
2.00 ppb
0.80 ppb
0. 01 ppb
1 . 60 ppb
0.40 ppb
0.20 ppb
0.20 ppm
2.00 ppm
0.20 ppb
0.04 ppb
0.10 ppb
2.00 ppb
2.00 ppb
0.02 ppb
2.00 ppb
1 . 00 ppb
0.40 ppb
4.00 ppb
1 . 00 ppm
0.02 ppm
0.004 ppb
1
The majority of values are from Ruch et. a]_. (9), Fulkerson et. al_. (10), and
Carter (11), and represent means for multiple coal samples taken largely
from Western Kentucky and Southern Illinois. Lower means for Ag, Tl, Sn
and W are from Lloyd (39), and the upper mean for Ag is from Vaughan et al_.
(4).
-------
TABLE 13. PHYSICAL-CHEMICAL CHARACTERISTICS AND ANALYTICAL PROCEDURES FOR SELECTED ORGANIC CHEMICALS
ORGANIC COMPOUND
Aniline *
Atrazine
Benzidine
Chlorobenzene
Chloroform
2,4-D
Dieldrin
D1-2-ethylhexyl-
phthalate
Endrin
Hexachloro-
benzene
Malathion
Phenol
Toxaphene
CLASS OF SOLUBILITY
COMPOUND mg/1
aromatic amine
chlorinated
hydrocarbon
aromatic amine
chlorinated
hydrocarbon
chlorinated
hydrocarbon
chlorinated
hydrocarbon
chlorinated
hydrocarbon
carboxylic acid
chlorinated
hydrocarbon
chlorinated
hydrocarbon
organic phosphate
aromatic
hydrocarbon
chlorinated
hydrocarbon
35000
33
400
Insol.
7.2
620
pract.
Insol .
Insol.
0.23
pract.
Insol.
144
67000
pract.
insol.
VAPOR PRESSURE
mm Hg @ (°C)
1.0 (34)
3xlO-7 (20)
1.0 (126)
10.0 (22)
100.0 (10.4)
0.4 (160)
3x1 O"6 (20)
1.3 (200)
2x10-7 (25)
IxlO"5 (20)
4x1 O"5 (30)
1.0 (40)
0.2-0.4 (25)
ANALYTICAL PROCEDURES
EXTRACTION DETECTION1
SOLVENT METHOD
acetone, benzene
methyl ene chloride,
hexane
chloroform, hexane,
acetone
methyl ene chloride,
hexane
cyclohexane, ether,
hexane
methyl ene chloride,
hexane
chloroform, hexane,
acetone
methyl ene chloride,
hexane
methyl ene chloride,
hexane, acetone
methyl ene chloride,
hexane
ethyl acetate, hexane
chloroform
methyl ene chloride,
hexane, acetone
A
B,C
D
B
E,F
B
B
B
B
B
G
H
B
DETECTION
LIMIT ug/1
0.01-.1
0.01-.1
0.01-.1
0.01-.1
0.01-.1
0.01-.1
0.01-.1
0.01-.1
0.01-.1
0.01-.1
0.01-.1
2
0.01-.1
REFERENCES2
1,2,3,5
4,6,9,12,14,15
2,7,8
1,6,7,9,14,15
2,7,10
CO
4,6,9,14,15 '
3,4,6,9,12,14,15
6,7,13
3,4,6,9,11,12,14
4,6,9,12,14,15
4,9,12,14,15
1,9
4,6,9,12,14,15
following page.
-------
- 35 -
TABLE 13 CONTINUED
Key to Organic Detection Methods
A. GC Flame ionization detector E. GC Electron capture detector
Column - 1% SE-30 Column - 20% Carbowax, 20M with
B. GC Electron capture detector microcoulometric detector
Column - 10% DC-200 Anakrom ABS F- GC Electron capture detector
C. GC Flame ionization detector Column - 3% OV-17
Carbowax 20M on Gas-chromosorb Q G. GC Electron capture detector
D. GC Alkali flame ionization detector Column - 4% SE-30 and 6% QF-1
Column - 3% OV-17 H. Colorimetric-spectrophotometric
technique- 4-aminoantipyrine reaction
^References to Methods of Organic Analysis
1. The Merck Index, 8th Edition. 1968. (P.G. Stecher, ed., M. Windholz, D.S. Leahy,
assoc. eds.), Merck and Company, Inc., Rahway, N.J. p. 84.
2. Handbook of Chemistry and Physics, 55th Edition. 1974-1975. (R.C. Weast, ed.),
Chemical Rubber Company, Cleveland, Ohio.
3. Chesters, G., H.B. Pionke, and T.C. Daniel. 1974. Ir[ Pesticides in Soil and
Water. University of Wisconsin, Madison, WisconsTff. pp. 451-550.
4. Martin, Hubert and Charles R. Worthing. 1974. Pesticide Manual, 4th Edition.
British Crop Protection Council, Boots Company Ltd., Nottingham, Eng. 565pp.
5. Fales, Henry M. and John J. Pisano. 1964. In Biomedical Applications of Gas
Chromatography. (H.A. Szymanski, ed.), Plenum Press, N.Y. pp.31-87.
6. Hurley, John T. 1974. American Water Works Association Journal, 66: 27-31
7. Condensed Chemical Dictionary, 8th Edition. 1971. (G.C. Hawley, ed.),
Van Nostrand Reinhold Company, N.Y. 971 pp.
8. Bruser, D. 1976. Department of Chemistry, University of Kentucky, Personal
Communication.
9. Standard Methods for the Examination of Water and Wastewater, 13th Edition.
1971. APHA, AWWA, WPCF, Washington, D.C. pp. 100-107.
10. Cuopra, N.M. and L.R. Sherman. 1972. Anal. Chem. 44(6): 1036-1038.
11. Veith, G.D. and V.M. Comstock. 1975. J. Fish. Res. Bd. Canada 32(10): 1849-1851.
12. Official Methods of Analysis of the Association of Official Analytical Chemists,
12th Edition. 1975. (W. Horwitz, ed.), Association of Official Analytical
Chemists, Washington, D.C. 1094 pp.
13. Bryant, H. 1976. Department of Entomology, University of Kentucky, Personal
Communication.
14. Pesticide Analytical Manual, Vol. 1. 1975. (B.M. McMahon and L.D. Sawyer, eds.),
U.S. Department of Health, Education, and Welfare. Food and Drug Administration,
Rockville, Md.
15. Pesticide Analytical Manual, Vol. 2. 1974. (J.R. Markus and B. Pumz, eds.),
U.S. Department of Health, Education, and Welfare. Food and Drug Administration,
Rockville, Md.
-------
TABLE 14
SURVIVAL FREQUENCIES FOR CONTROL POPULATIONS OF AQUATIC EMBRYOS
EMBRYONIC BIOASSAYS
TEST SPECIES
CATFISH
#
FOWLER'S TOAD
GOLDFISH
LEOPARD FROG
TROUT
COMPOUND/WATER HARDNESS
BORIC ACID @
50 ppm CaC03
200 ppm CaCCh
BORAX @
50 ppm CaCO*
200 ppm CaC03
BORIC ACID 0
50 ppm CaCO?
200 ppm CaC03
BORIC ACID 0 ^~
50 ppm CaC(h
200 ppm CaCOs
BORAX @
50 ppm CaCOo
200 ppm CaCOs
BORIC ACID @
50 ppm CaCOo
200 ppm CaCOa
BORAX @
50 ppm CaCOs
200 ppm CaC03
BORIC ACID @
50 ppm CaCOo
200 ppm CaCOs
BORAX @
50 ppm CaCOo
200 ppm CaC03
COMBINED
POPULATION SIZE
304
258
371
368
443
410
294
294
249
249
268
233
268
233
242
242
252
252
%
AT HATCHING
93
94
97
98
92
94
94
93
93
93
90
89
90
89
91
91
92
92
CONTROL SURVIVAL1
AT 4 DAYS POSTHATCHING
91
93
97
98
92
94
94
93
92
92
89
87
89
87
91
91
87
87
CO
0>
«,,fc.^ tests at the two water hardness levels (50 and 200 ppm CaCOa) were conducted simultaneously,
experimental results were gauged against the same populations of control animals.
-------
TABLE 15
TERATOGENESIS IN AQUATIC EMBRYOS SURVIVING TREATMENT WITH BORIC ACID AND BORAX
BIOASSAYS
SPECIES
CATFISH
GOLDFISH
TROUT
FOWLER'S
TOAD
LEOPARD
FROG
TOXICANT &
HARDNESS
BORIC ACID @
50 ppm CaCOo
200 ppm CaC03
BORAX @
50 ppm CaC0.3
200 ppm CaCOs
BORIC ACID @
50 ppm CaCOo,
200 ppm CaC03
BORAX @
50 ppm CaOh
200 ppm CaC03
BORIC ACID (<>
50 ppm CaCO?
200 ppm CaC03
BORAX @
50 ppm CaCOg
200 ppm CaC03
BORIC ACID @
50 ppm CaC03
200 ppm CaC03
BORIC ACID @
50 ppm CaCOg
200 ppm CaC03
BORAX G>
50 ppm CaCOo
200 ppm CaCO 3
NO. ANIMALS
ANOMALOUS1
77
125
84
107
65
26
44
68
58
278
40
33
810
544
209
190
155
151
PERCENT DISTRIBUTION OF
DWARFED
BODY
6
2
38
21
-
-
2
-
2
3
10
3
-
-
-
-
CRANIUM
3
5
2
2
2
-
-
-
10
8
25
13
1
1
-
1
1
VERTEBRAL
COLUMN
53
93
55
75
93
100
93
91
84
80
40
63
16
12
6
33
49
92
ANOMALIES
FINS
38
-
-
-
5
-
-
2
-
2
3
6
-
-
-
-
1
BY AREAS AFFECTED
NERVOUS YOLK SAC &
SYSTEM ABDOMEN
-
_
5
2
-
-
5 ^
7 ^
i
4
2 5
5 17
15
83
87
94
67
50
6
"'includes anomalous survivors for all exposure levels
-------
TABLE 16
TERATOGENIC EFFECTS OF METALS ON TROUT EMBRYOS
PERCENT SURVIVAL AT HATCHING1
:ORCEN
in
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
1.
2.
5.
7.
10.
TRATION*
ppm CHoHg Hg ^
001 90 3) 90 2
002 75 9) 72 8
005 57 10) 59 11
_LO -4-O .LO
Cd ^ Cu As ^
) 98(2) 97(4)
1 94 2)
?) 97 7 90(10)
Zn+2*
99
-
100(1)
007 46(17) 46 19) 97(4)
010 33(20) 34(22) 97(6) 92(7) 100
025 29(35) 24(4.
050 0 0
075
100 0 0
250
500 0 0
750
000
500
000
500
000
3) 96 7) 82(8) 100(2)
91 9) 65(11) 99(1)
82 6) 65(5) 98
58(11) 63(4) 96 5)
38(32) 45(9) 83 11)
32(28) 24(31) 67 8)
28(26) 0 47(18)
5 0 30(27)
002
0 - 1
0
0 - 0
TL50 0.005 0.005 0.10 0.10-0.25 0.50-0.75
98(1)
-
98(3)
98(3)
99(7)
-
91(2)
-
56(4)
25(8)
1(100)
0
—
1.00
Se+6
99(2)*
-
99(1)*
-
90(2)*
-
91(1)*
-
90(2)*
-
85(4)
-
82(7)
75(6)
52(11)
50(10)
41(15)
5.00
GJ
00
Percent survival includes all embryos which completed hatching. Frequencies of survivors
exhibiting gross anomalies are given parenthetically.
^Concentrations based on content of metal added to Holtfreter's culture water.
Asterisks designate cultures where NaCl content was reduced to 0.1 g/1.
-------
TABLE 17
TOXICITY OF INORGANIC MERCURY (HgCl2) TO FISH EMBRYOS
PERCENT SURVIVAL AT HATCHING1
CONCENTRATION
(ppm)
0.001
0.002
0.005
0.007
0.010
0.025
0.050
0.075
0.100
0.250
0.500
1.000
5.000
10.000
Ti-50
TROUT (24)
89
66
51
37
26
13
0
-
0
-
0
0
0
0
0.005
CATFISH (6)
100
97
92
91
84
60
30
26
20
14
1
0
0
0
0.025
GOLDFISH(3)
100
100
99
99
98
96
94
66
50
25
13
0
0
0
0.10
BASS (4)
99
96
95
92
86
83
81
70
68
39
14
0
0
0
0.10-0.25
I
co
Hatching time in days is given parenthetically.
-------
TABLE 18
TOXICITY OF INORGANIC MERCURY TO AMPHIBIAN EMBRYOS
PERCENT SURVIVAL AT HATCHING
CONCLNIRAI10N
(ppm) Gastrophryne Hyla
carol inensis[3] squirel!
0.001
0.002
0.005
0.007
0.010
0.025
0.050
0.075
0.100
0.250
0.500
0.750
1.000
51
35
22
0
0
0
0
0
-
-
-
_
—
80
-
44
-
0
_
0
-
0
_
_
-
—
Hyla chr
a|_3] cephala[
82
64
44
13
0
0
0
0
_
_
_
_
-
yso- Rana
3] pipiens
96
87
72
62
47
34
9
1
0
_
0
_
0
Bufo
;[6] fowl eri [
99
100
100
100
98
79
18
8
0
0
0
-
0
Bufo
.3] punctatu;
100
100
100
99
92
83
35
9
0
0
0
_
0
Rana Rana
;[3] gryTTo[6] heckscheri[6]
100
_
99
96
94
89
75
56
17
0
0
0
0
100
-
100
100
94
90
64
48
23
0
0
0
0
1
.£»
O
1
TL
50
0.001
0.005
0.005
0.010 0.025-0.05 0.025-0.05
0.075
0.075
-------
TABLE 19
CORRELATION OF PERCENT EGG HATCHABILITY WITH
POLLUTION-INDUCED REDUCTION IN DIVERSITY OF PISCINE FAUNA
MONITORING
SITE1
Cane Run
Town Branch
Wolf Run
East Hickman Creek
West Hickman Creek
Gainesway Branch
Hickman Creek
Shelby Branch
Kentucky River
Elkhorn Creek
Steel e's Run
FISH
0
0
40
56
63
70
74
81
83
86
91
PERCENT EGG
AMPHIBIANS
0
0
18
45
58
74
82
90
89
92
97
HATCHABILITY2
FISH & AMPHIBIANS
0
0
24
48
60
73
80
87
87
89
94
DIVERSITY OF
I SPECIES/EXPECTED^
0/15
0/15
2/15
8/17
11/16
7/10
15/22
14/17
21/25
43/48
15/15
FISH FAUNA3
% SPECIES REMAINING
0
0
13
47
69
70
68
82
84
90
100
''streams and rivers selected to represent various levels of water pollution resulting from recent increases
in urbanization and industrialization in the Kentucky Bluegrass region.
2Egg hatchability data combined and averaged for four species of fish (rainbow trout, largemouth bass, goldfish,
channel catfish) and five species of amphibians (squirrel treefrog, gray treefrog, narrow-mouthed toad,
.pig frog, red-spotted toad).
3Data on fish fauna for past 22 years taken from Carter (70), Jones (71), Kuehne (72), Laflin (73), MacGregor
.and Andre (74), Westerman and Westerman (75), and Small (76).
^Number species persisting at monitoring sites/number of species reported in earlier censuses.
-------
- 42 -
APPENDIX II
SYNOPSIS OF BIOASSAY PROCEDURES
-------
- 43 -
I. Preparation of Culture Water.
If an adequate supply of natural water free of background contamination is
not available, reconstituted water may be used. A synthetic culture water which
has been found satisfactory for bioassay procedures is given in Table 3, and
possesses ionic composition and physicochemical characteristics similar to Mount's
spring water (55). Synthetic culture water should be prepared from charcoal
filtered, distilled-deionized or double deionized water having a conductivity of
0.25 ymhos or less. Routine monitoring should be conducted for possible background
contaminants (.6.3.., heavy metals, chlorinated hydrocarbons).
Culture water may be prepared from the following three stock solutions.
Reagent grade chemicals should be used.
1. NaHC03 100 g/1
KC1 5 g/1
2. CaCl2 60 g/1
3. MgS04-7H20 60 g/1
To prepare culture water with hardness levels of 50-200 ppm CaCOs, stock
solutions should be blended in the following ratios and diluted to a final volume
of 20 liters using distilled-deionized water.
Stock Reagent Water Hardness Level (ppm CaC03)
50 100 200
NaHC03 + KC1 20.0 ml 20 ml 20 ml
CaCl2 12.5 ml 25 ml 50 ml
MgS04-7H20 12.5 ml 25 ml 50 ml
II. Setup of Bioassay System.
Continuous flow bioassays should be set up and operating at least 24-72 hours
prior to introducing test organisms, allowing sufficient time for adjustments and
stabilization of toxicant level and other parameters (.§_.£., temperature, dissolved
oxygen, pH). During this period all test parameters should be monitored at regular
8-12 hour intervals.
-------
- 44 -
A. Preparation for testing.
Bioassays preferably should be conducted in walk-in, temperature-controlled
environmental rooms, disinfected before use with a microbicide and 12-24 hours of
ultra-violet irradiation. Bioassay glassware should be chemically cleaned using
the procedure given below or some suitable alternative.
1. Rinse toxicant solutions from all glassware.
2. Scrub with Alconox detergent and rinse with tap water.
3. Rinse thoroughly in tap distilled water and allow to dry.
4. Soak in acetone for 15-20 minutes and allow to dry.
5. Rinse and soak in tap distilled water.
6. Soak in concentrated nitric acid for 15-30 minutes.
7. Rinse in glass-distilled water.
8. Soak for several hours in glass-distilled or deionized water.
9. Dry at 300° F or autoclave.
10. Allow cooling to room temperature and cover with parafilm.
11. Store in closed dust-free cabinet.
B. Installation of peristaltic and syringe pumps.
Prior to performing bioassays, tests should be conducted on all pumping units
to calibrate and evaluate reproducibility of flow rates. The variable speed
Brinkmann (model 131900) and Gilson (models HP 8 and HP 16) peristaltic pumps.fitted
with tygon tubing, generally give satisfactory performance. Periodic adjustments
should be made in pumping rate to offset effects of wear on tubing. Tygon tubing
should be replaced after 2-3 weeks of operation.
Syringe pumps should be equipped with up to six double-ground glass syringes.
For this purpose we have used a model 355 Sage syringe pump equipped with a modified
holder (Fig. 3). Generally, more satisfactory performance is obtained when syringe
pumps are mounted outside the environmental room, obviating effects of reduced
temperature and/or high humidity. Each set of syringes should be matched for equal
diameter and stroke volume.
C. Setup of mixing and culture chambers.
Each cultuVe chamber (e..£., 300 ml modified deep etri dish) is connected to
a mixing chamber (e.&., 250 ml sidearm Erlenmeyer flask) using 1/4" i.d. polyethylene
or teflon tubing. A separate peristaltic pump channel should be connected to each
-------
- 45 -
mixing flask, using tygon tubing. A liquid flowmeter (size no. 12, Roger Gilmont
Instruments, Great Neck, N.Y.) should be installed in each line to calibrate and
monitor flow. Each mixing flask should receive its own syringe pump line. Teflon
tubing (e_.£., McMaster-Carr Co., Chicago, 111.) should be used for this purpose, and
a flowmeter (Gilmont Microflowmeter) should be inserted in each syringe pump line.
Teflon rapid-disconnects may be used in connecting pumping lines to flow meters.
Culture water should receive continuous aeration supplied either to the
mixing chamber or the culture water reservoir which feeds the peristaltic pump.
The latter is advised when testing more volatile toxicants. The air line to the
culture room should be equipped with a filter and moisture trap (e_.£., McMaster-
Carr Co., 40 y or less).
D. Maintenance of syringe pumps.
While syringes may be removed individually from the pump and replaced with
a fresh set, it is generally more expeditious to leave them mounted, retract the
drive carriage and refill them in place. In either case, the syringe lines must
be clamped securely to prevent toxicant discharge into the mixing chambers. Solutions
used to fill syringes should be analyzed to confirm concentration of the toxicant
to be delivered to the mixing flask. The rate of toxicant injection should be
calculated to dilute culture water to desired exposure levels.
III. Acquisition and Care of Test Animals.
The investigator should be familiar with the basic biology of his selected
test species, including some knowledge of (1) taxonomy, (2) geographical distribution,
(3) ecology and natural history, (4) life cycle and reproductive behavior (e_.c[.,
optimum spawning temperature, hatching time), and (5) embryonic development
(e_.c[., cleavage, gastrulation, organogenesis). Such background information is
essential to the selection of test species and the use of developmental stages for
bioassay purposes.
A. Selection of animal test species.
As noted above, we have found sensitivity to toxicants to vary substantially
for eggs of different fish and amphibian species. Therefore, where possible, we
recommend the use of 2-3 test species, selected for different developmental character-
istics (e_.£., egg type, yolk volume, hatching time, cleavage pattern). The fish
and amphibian species we have used most extensively are listed in Table 10, together
-------
- 46 -
with data on hatching time, egg production, seasonal availability and source of
supply.
B. Obtaining gravid animals.
Gravid animals should be collected from sources reasonably free of environmental
contaminants. Particular consideration should be given to chlorinated hydrocarbons
and other toxicants with high bioaccumulation potential and which may concentrate
to high levels in eggs and gonadal tissues, affecting hatchability. Federally
supported investigators may coordinate through the Director of Sport Fisheries and
Wildlife to obtain gravid animals from a number of Federal Hatcheries.
C. Maintenance of adult animals.
Once obtained, gravid animals may be maintained for short periods of time in
large capacity non-flow, temperature-regulated fish tanks (e_.£., 100 gal Living
Stream fiberglass tank, Frigid Units, Toledo, Ohio). However, for longer storage,
150-300 gal flow-through, temperature-regulated, fiberglass tanks should be used.
If an adequate supply of well or spring water is not available, charcoal filtered
dechlorinated tap water generally proves satisfactory. The source of water should
be monitored for possible background contaminants. If available water supply will
not support an adequate flow-through culture, fish tanks may be equipped with
high capacity recirculating filters (e_.£., activated charcoal, filter floss). Such
filters are particularly useful for removing food debris and other particulates
from the culture water. Adequate aeration should be supplied to all fish tanks.
A high quality fish food should be used to support fish populations (e_.£.,
Purina; Silver Cup). However, as certain lots of food may contain high levels of
trace contaminants, it is advisable to analyze for suspected toxicants (£.£., heavy
metals, chlorinated hydrocarbons).
D. Egg spawning techniques.
In the event freshly spawned eggs cannot be obtained in the laboratory or from
local hatcheries, artificial spawning may be used. Animals should be selected in
optimum spawning condition, as underripe or overripe eggs may exhibit substantially
reduced hatchability (64, 65). The general appearance of the eggs and external
features of the adult animal (.§.£., body size and shape, breeding color, extension
of the urogenital papilla) may be used as indicators of optimum spawning conditions.
For best results,*eggs maintained in control cultures should give at least 75%
hatchability. Lower hatching frequencies generally indicate an inadequate source
of eggs and sperm or faulty culture technique.
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- 47 -
Egg and sperm collecting techniques vary with different test species. Using
the method of Leitritz (64), the artificial stripping technique works well for the
rainbow trout. Gravid females should be artificially spawned as soon as eggs can
be obtained with a gentle to moderate stripping action. Males, which are usually
ripe concurrent with the females, may be spawned several times during the breeding
season. Eggs from the goldfish and numerous other species may be collected by
similar procedures. Temperature change and/or hormonal injection (300 IU HCG/454 g
body weight, administered I.P.) may be used to initiate spawning in such species as
the goldfish. When artificial stripping is not feasible, eggs sometimes can be
collected by the incision method (64). We have used the latter satisfactorily for
both channel catfish and largemouth bass, despite a modest reduction in egg hatchability.
Once eggs and sperm have been collected, they should be mixed in spawning
trays or large glass finger bowls for 10-15 minutes, avoiding exposure to fluorescent
light. Preferably, eggs from several females should be mixed prior to setting up
test sub-samples, to minimize individual variations in hatchability or sensitivity to
toxicants.
It should be noted that eyed trout eggs (i_.£., 12-15 developmental days) can
be obtained by air express from a number of hatcheries and are particularly useful
for initial bioassay screening studies. In addition, fresh catfish spawn can be
successfully shipped by air express. It should be noted, however, that sensitivity
of the bioassay may be somewhat diminished if early developmental stages are omitted
from the test.
IV. Performance of Bioassay Tests.
A. Introduction of eggs to chambers.
Egg samples (100-150) should be introduced into each culture chamber, using a
graduated beaker or measuring scoop. Final egg tallies may be delayed until all
bioassay cultures have been established. Care should be taken not to overcrowd
or unnecessarily contaminate cultures.
B. Monitoring of bioassay cultures.
Toxicant exposure levels should be confirmed by direct chemical analyses
performed on all bioassay cultures at regular daily intervals. It is particularly
important to stabilize toxicant concentration before introduction of eggs into the
bioassay system, and to monitor exposure levels carefully during the first 24-48 hours
after egg treatment is initiated.
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- 48 -
Cultures also should be monitored daily for the following test parameters:
temperature, pH, alkalinity, conductivity, dissolved oxygen, water hardness, and
ammonia. Also, flow rates from both the syringe and peristaltic pumps should be
measured at least twice daily.
C. Observation of test animals.
Test animals and culture conditions should be observed daily to gauge extent
of development and remove dead specimens. Lack of embryonic development, discoloration
of eggs and vascular degeneration are useful criteria for early recognition of
embryonic mortality. Where necessary, reference may be made to standard embryological
staging guides (64, 66-69).
Contamination of the cultures should be minimized by maintaining semi-sterile
test conditions. In the event of soft egg disease or fungus, cultures may be treated
according to the procedures recommended below:
1. Malachite green procedure.
a. Prepare stock solution of malachite green by dissolving 100 g of
dye in 100 ml distilled water.
b. Dilute stock solution 1:15,000 (v/v) with distilled water.
c. Decant the culture water from the exposure chamber.
d. Add 100 ml of malachite green solution to the dish and let stand
for 30 seconds.
e. Decant malachite green solution.
f. Rinse eggs carefully with fresh culture water to remove excess dye.
2. Betadine procedure.
A 1:5 solution of Betadine (povidone-iodine, Purdue Frederick Co., Norwalk,
Conn.) may be substituted for malachite green and used as given above.
3. Formaldehyde (1/1000 for 10 minutes) or acriflavine (1/2000 for 20 minutes)
may be used as alternative methods of treatment.
As such treatment may introduce undesirable test variables, the most practical course
may be to discard contaminated cultures and repeat the bioassay experiment.
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- 49 -
APPENDIX III
NORMAL AND TERATOGENIC STAGES OF RAINBOW TROUT
ALEVINS AND FRY
PLATES I-VI
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- 50 -
PLATE I
NORMAL STAGES OF TROUT ALEVINS AND FRY
Trout alevin at 1 week posthatching, showing heart
(A), lateral line (B), dorsal soft-ray fin (C),
caudal fin (D), mandible (E), iris of eye (F), yolk
sac with vitelline vessels (6), and urogenital
papilla (H). 7.5X.
2. Normal trout fry at 21 days posthatching, showing
external nares (A), operculum (B), dorsal soft-
ray fin (C), adipose fin (D), caudal fin (E),
mandible (F), pectoral fin (G), parr marks (H),
pelvic fin (I), urogenital papilla (J), anal fin
(K), and caudal peduncle (L). Parr marks represent
a characteristic pattern of skin pigmentation
appearing in juvenile stages at about 2 weeks.
10X.
-------
E F
H
J K L
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- 51 -
PLATE II
CADMIUM-INDUCED TERATOGENESIS IN TROUT
Figures 1-6 show congenital deformities induced in trout embryos by
cadmium treatment. Photographs were taken 1-2 weeks subsequent to hatching
at a magnification of 7.5-10X. The anomalies shown illustrate various
degrees of Siamese twinning and other defects (e_.£., immovable lower jaw,
anomalous or absent eyes, truncated upper jaw).
1. Twinning of head and trunk extends to posterior border
of dorsal fin (arrow). Left eye of lower twin is reduced.
2. Dorsal view of dicephalous alevin. Partial twinning
extends to dorsal fin (arrow).
3. Twinning with partial duplication of yolk sac (arrow).
The upper jaws are truncated, with immobile lower jaws.
4. Complete Siamese twins conjoined only by the yolk sac
(right arrow). The upper twin displays relatively
normal morphology, while the lower twin possesses a
truncated upper jaw and immobile lower jaw (left arrow).
5. Conjoined assymetrical twins, with the upper arrow
Indicating the host. The parasite head (lower arrow)
is defective, lacking normal eyes, upper jaw, and
operculum. Both partial twins possess dorsal fins.
6. A further example of partial twinning. The less
developed head has defective eyes (arrow).
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- 52 -
PLATE III
TERATOGENIC DEVELOPMENT IN TROUT ALEVINS
Figures 1-6 show teratogenic development induced by boron compounds
and heavy metals. Figures 1-4 show various degrees of acute, inflexible
curvatures of the spinal column. Photographs were taken 1-2 weeks sub-
sequent to hatching at a magnification of 7.5-1 OX.
1. Acute curvature of the spine (arrow) and reduced upper
and lower jaws resulting from boron treatment.
2. Defective curvature of the spine produced by a 1:1
mercury-copper mixture.
3. Anomalous cephalic and spinal structure induced by
exposure to a mercury-selenium mixture.
4. Cadmium-induced anomalies including a truncate upper
jaw (arrow), curvature of the spine and defective iris
of the eye.
5. Treatment with a 1:1 mercury-cadmium mixture resulting in
a grossly reduced caudal peduncle (upper arrow),
truncated upper jaw, immobile lower jaw (lower arrow)
and a dwarfed trunk.
6. Boron-induced congenital defects including a reduced
scoliotic caudal peduncle (upper arrow), defective
eyes (lower arrow), absent upper jaw and immobile
lower jaw.
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0
•'. '•$!
©
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- 53 -
PLATE IV
TERATOGENIC DEVELOPMENT IN TROUT ALEVINS
Figures 1-6 show teratogenic defects produced by boron and heavy
metals. Photographs were taken 1-2 weeks posthatching at a magnification
of 7.5-10X.
1. Acute ventral flexion of spinal column
induced by treatment with boron.
2. Acute lateral flexion of spinal column (scoliosis)
produced by a mercury-copper mixture.
3. Cadmium-induced flexion of the spinal column.
4. Acute, inflexible curvature and irregular structure
of spinal column resulting from treatment with
cadmium.
5. Kyphosis of the spine (arrow) and dwarfed trunk
resulting from an exposure to a mercury-selenium
mixture.
6. Anomalous spinal column and dwarfed trunk (arrow)
produced by treatment with a mercury-copper mixture.
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- 54 -
PLATE V
MERCURY-INDUCED TERATOGENESIS IN TROUT
Photographs 1-7 show congenital deformities frequently observed in
trout alevins. Photographs were taken 1-2 weeks subsequent to hatching
at a magnification of 7.5-10X. The anomalies most frequently encountered
included defective vertebral columns, immovable lower jaw, absent or
reduced fins, absent eyes, hydrocephalous brain development, Siamese
twinning, and retarded yolk sac reabsorption.
1. Twinning of head and anterior trunk, ventral view.
Single yolk sac (arrow) possesses paired yolk stalks,
one serving each partial twin.
2. Dorsal view of dicephalous alevin. Twinning extends
to anterior border of dorsal fin (arrow).
3. Inflexible C-shaped curvature of tail, accompanied by
hydrocephaly (arrow).
4. Skeletal anomalies including an acute lordotic spine
and a defective, immovable lower jaw (arrow). Yolk
sac reabsorption also is retarded (YS).
5. Acute flexion of spinal column, resulting from defective
vertebral development. Retarded yolk sac reabsorption
also is evident (YS).
6. Anomalous, immobile curvature of spinal column (arrows).
7. Defective dorsal fin and sharp angular flexion of spinal
column.
-------
YS
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- 55 -
PLATE VI
BORON-INDUCED TERATOGENESIS IN TROUT
Photographs 1-6 show congenital deformities frequently observed
in trout alevins which survived treatment with boron compounds. Photo-
graphs were taken 1-2 weeks subsequent to hatching at a magnification of
7.5-10X. The anomalies most frequently encountered included various acute
defects of the vertebral column, amphiarthrotic (immobile) jaw articulation,
absent or reduced fins, anomalous or absent eyes, dwarfed or irregular trunk
morphology, Siamese twinning, and retarded yolk sac resorption.
1. Skeletal anomalies including an acute, lordotic
spine, attenuated tail fin, and a defective,
immobile lower jaw (arrow).
2. Twinning of head and trunk, with a single tail. A
single yolk sac possesses paired yolk stalks, one
serving each partial twin.
3. Acute scoliosis of the vertebral column and cephalic
twinning. Secondary head (arrow) is only partially
formed.
4. Kyphosis of the vertebral column with irregular,
dwarfed trunk structure.
5. Anomalous, inflexible curvature of spinal column,
irregular, truncate upper jaw, immobile lower jaw,
and retarded yolk sac resorption. Also, as seen
in Figures 1 and 6, incomplete closure of the choroid
fissure has resulted in coloboma of the retina. This
condition is discernible as a vertical cleft at the
mid-inferior border of the eye.
6. Irregular, fixed curvature of the vertebral column
similar to that shown in Figure 5.
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©
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- 56 -
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%
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-------
1. REPORT NO. "" |:
EPA- 5 6 O/S^TTjJK) 2 [
fECHNICAL REPORT DATA
,/ ,'fjsi nvJ Imuritciiwis on the revets l>t lor-1 i'i
J. RECIPIENT'S ACCf SSION-NO.
4. TITLE AND SUBTITLE
"A Continuous Flow System Using F1sh and Amphibian
Eggs for Bioassay Determinations on Embryonic Mortal
and Teratogenesis."
5 REPORT DATE
APRIL 1977
7. AUTHOR(S)
Wesley J. B1rge and Jeffrey A. Black
OTPERFORMING ORGANIZATION NAML AND ADDRESS
University of Kentucky
Lexington, Kentucky
8 I tHFOHMING ORGANIZATION REPORT NO.
10. PROGRAM ELEMENT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
Office of Toxic Substances
U.S. Environmental Protection Agency
Washington, D. C. 20460
ORGANISATION CODE
11. CONTRACT/GRANT NO.
EPA 68-01-4321
13, TYPt OF REPORT AND PERIOD COVERED
_.£!MALJECUIiICAL.REEflRI I
14. SPONSORING AGENCY CODE
16. SUPPLEMENTARY NOTES
16. ABSTRACT
A procedure is described for continuous flow bioassays on sensitive develop-
mental stages of fish and other aquatic species. Environmental toxicants may be
screened for teratogenic and other embryopathic effects (e_.g_., mortality, locomotor
impairment) on eggs, embryos, and early juvenile stages. Toxicant concentration
may be regulated down to 1.0-0.1 ppb for an exposure period extending from fertili-
zation through 4-7 days posthatchlng. Bioassays may be performed with either natural
or synthetic water, and standard physlcochemical parameters (e.g., temperature,
water hardness, pH) can be manipulated independently to determine effects on toxicity
The procedure 1s suitable for use with a wide range of fish and amphibian species,
including rainbow trout, channel catfish, largemouth bass, leopard frog and others.
Concerning the toxic effects of many environmental trace contaminants, embryonic
and early juvenile stages constitute the critical "sensitive links" in the life
cycles of many aquatic animal species. Reproductive potential of acquatic animals
may be severly restricted or abolished by trace levels of toxicants which are harm-
less or sublethal to most adult organisms.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
Bioassay
Aquatic Pollutants
F1sh Eggs
Embryonic Mortality
Teratogenesis
b.lDENTIFIERS/OPCN ENDED TERM:,
C. COSATI I irlil/Ciroup
19. DISTRIBUTION STATEMENT
UNLIMITED
Hi. SECURITY CLASS (Ihi* Hepiirt)
_ UNCLASSIFIED
20. SECURITY CLAGC jrV/ii'i page/
UNCLASSIFIED
21. NO. OF PAGES
22TPR7CE
EPA Form 2220-1 (9-73)
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