United States
Environmental Protection
Agency
Environmental Research
Laboratory
Duluth MN 55804
Research and Development
EPA-600/S3-84-009 Feb. 1984
&EPA Project Summary
Aquatic Toxicity Tests to
Characterize the Hazard of
Volatile Organic Chemicals in
Water: AToxicity Data Summary
N. Ahmad, D. Benoit, L. Brooke, D. Call, A. Carlson, D. DeFoe, J. Huot, A.
Moriarity, J. Richter, P. Shubat, G. Veith, and C. Walbridge
This summary presents acute and
chronic toxicity test data and bioconcen-
tration factors compiled over a two-
year period on fish and invertebrates
exposed to several representative chem-
icals from five chemical classes (chlor-
inated ethanes, chlorinated benzenes,
chlorinated ethylenes, chlorinated di-
enes, and chlorinated propanes).
The fathead minnows and Daphnia
were quite similar in their sensitivities
(acute and chronic) to each chemical
class, while the rainbow trout were
considerably more sensitive to all class-
es during acute tests, except for the
chlorinated diene exposures, where
they were more resistant. The ranking
of acute and chronic sensitivity was
generally the same for each chemical
within each class of chemicals for all
three species tested.
Both the acute and chronic toxicity of
all chemicals within a class increased as
the number of chlorines in the chemical
structure increased.
Bioconcentration factors for fathead
minnows were determined for four of
the chemical classes tested. Hexachlo-
robenzene was bioconcentrated the
most (23,000x), and tetrachloroethane
was bioconcentrated the least (8x).
Again, as with the toxicity experiments,
the greater the number of chlorines on
the molecule the greater the bioconcen-
tration within each class of chemicals.
This Project Summary was developed
by EPA's Environmental Research Labo-
ratory. Duluth, MN. to announce key
findings of the research project that is
fully documented in a separate report of
the same title (see Project Report order-
ing information at back).
Introduction
The objectives of this study were to
evaluate the use of sensitive aquatic
toxicity tests in determining the relative
hazard of volatile organic compounds;
and to compare the sensitivities of three
aquatic species to several homologous
series of organic chemicals. Investiga-
tions were divided into two major areas.
First, preliminary studies were conducted
to determine similarities and differences
in metabolism of selected xenobiotics
between aquatic and mammalian organ-
isms. The second phase, reported here,
represents the largest effort, which was
to develop methods for testing volatile
chemicals and to determine the differ-
ences in toxicity between daphnids (Daph-
nia magna) and fish to selected volatile
chemicals.
Both acute and chronic toxicitv tests
were completed with fathead minnows
(Pimephales promelas) and daphnids.
Only acute tests were performed using
rainbow trout (Salmo gairdneri). Test
chemicals were selected from the follow-
ing classes of compounds: chlorinated
ethanes; chlorinated benzenes; chlori-
nated ethylenes; chlorinated dienes and
chlorinated propanes. Bioconcentration
factors for fathead minnows exposed to
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10 chemicals from the above classes
were also determined.
The full report represents an overview
of the results from these investigations.
Most data have been or are scheduled to
be published in scientific journals.
Methods and Procedures
Bioassay-Chemical
Routine toxicity test met hods and chem-
ical procedures followed closely those
described by Mount and Brungs (1967),
APHA(1975),USEPA(1975), Phippsetal
(1981), and Benoit et al (1982)
All tests, except for the static Daphnia
exposures, were performed using propor-
tional diluters. Lake Superior water was
used for all tests. Nominal water temper-
atures were 12, 20, and 25° C for rainbow
trout, Daphnia, and fathead minnows,
respectively Acute tests were for 96 hrs
forfatheadsand rainbowtroutand48 hrs
for Daphnia. Chronic fish (embryo-larval)
tests were conducted for 32 days, and
chronic Daphnia tests were of 28-day
duration
Chemical characteristics of the control
and experimental waters were as follows:
dissolved oxygen 7.0-9.6 mg/l; pH 6.7-
7.6, total hardness (as CaCO3) 43-57
mg/l, and alkalinity 35-53 mg/l Several
statistical procedures (Spearman-Karber,
ANOV, Probit analyses) were used to test
the significance of the results.
Chemicals selected for testing ranged
in purity from 95-99 percent Solvents
used in the extraction process were of
glass-distilled, gas-chromatography
grade. The effectiveness of the extraction
procedure was examined by determining
the percent recovery of a known amount
of each chemical in the experimental
water. All toxicant concentrations in
water and tissues were analyzed by gas
chromatography Further description of
these analytical procedures, according to
chemical, appear in the full report.
Development of Early Life
Stage, Mini-Diluter Apparatus
In order to successfully and safely test
volatile chemicals, it was necessary to
develop specialized exposure systems
and methods for monitoring air and water
leaving these systems. Interest in develop-
ing fish early life-stage (ELS)toxicity tests
led to the design of a compact continuous
flow mini-diluter exposure system which
accurately delivered as little as 3 liters of
test water per hour to each of five
concentrations plus a control This system
can be used to test the effects of either
single chemicals or treated complex
effluents on young fish in the laboratory
or in the field. The small ELS test appa-
ratus takes less space and requires
smaller volumes of test water, which is a
critical factor when effluents are shipped
to the laboratory or on-site toxicity tests
are conducted. Smaller volumes of test
water also reduce filtration costs for the
removal of hazardous test chemicals
before discharging waste water to the
sewer.
The ELS test system has been tested
and evaluated in the laboratory and on-
site in a mobile trailer (Benoit et al.,
1982). This apparatus has been used to
conduct fathead minnow (Pimephales
promelas) ELS exposures to various toxi-
cants including volatile organic com-
pounds, metals, pesticides, and treated
complex effluents from metal plating, oil
refinery, and sewage treatment plants
The system has also been successfully
used for testing macromvertebrates. All
embryo-larval fish tests described herein
were conducted in this mini-diluter sys-
tem.
Summary and Conclusions
It has long been known that aquatic
animals are extremely sensitive to chemi-
cals at very low /ug/L to mg/L concentra-
tions in the aqueous environment. This
knowledge led us to the position of
evaluating both acute and chronictoxicity
tests with several sensitive aquatic spe-
cies in an effort to deter mi net he range of
sensitivities and the possible application
of the data screening out chemicals,
which after short inexpensive tests with
selected aquatic species are shown to be
extremely toxic, and/or highly bioaccumu-
lable
Phase one of these studies was de-
signedto provide preliminary information
on the metabolic capabilities of rainbow
trout and daphnids These studies, coupled
with earlier studies on mixed function
oxidase activity in mammals and other
animals, indicated the metabolic systems
to be similar qualitatively, therefore, the
mechanisms leading to toxicity and neo-
plasia, for example, are presumed to be
similar in all organisms. Phase two was to
determine the acute and chronic toxicity
of five classes of chlorinated organic
compounds to selected fish and inverte-
brate animals In addition, the bioconcen-
tration potential of these chemicals was
measured to determine possible food-
chain problems involving man.
Daphnia showed generally the same
order of acute and chronic sensitivity as
the fish for all classes of chemicals
tested. A comparison of species acute
sensitivities (Table 1) indicated that Daph-
nia were slightly more resistant in mosl
cases than the fathead minnow, whereas
the rainbow trout was considerably more
sensitive than either the fathead minnow
or Daphnia to all compounds excep!
hexachlorobutadiene
One of the more interesting findings ol
the acute studies was that the toxicity ol
the ethanes, benzenes, and ethylenes
increased as the number of chlorines on
the molecule increased. This was true for
both fathead minnows and Daphnia (Table
1).
These data indicate that either fathead
minnows or Daphnia would provide es-
sentially the same acute values for these
particular chemicals. These chemicals
are not considered to be very toxic tc
aquatic species, since their 96-hr LC50s
are one to two orders of magnitude above
those environmental chemicals consid-
ered as extremely toxic.
Fifteen chronic toxicity tests with fish
were also conducted on chemicals in the
five chemical classes. The order of sensi-
tivity for all chemicals tested chronically
(Table 2) was the same as that established
for acute toxicity (Table 1). Six chronic
values were also determined for Daphnia
and in most cases the sensitivity was
similar except for the ethanes, where
there seemed to be considerable variation
between the fathead and Daphnia results.
Again, chronic toxicity increased consid-
erably for both species as the number of
chlorines on the molecule increased
(Table 2).
The bioconcentration potentials of
these chemicals were determined by
establishing a bioconcentration factor
(BCF) Cp.sh/Cwater) for fathead minnows
exposed for 32 days to each chemical
during an early life-stage toxicity test.
These BCFs were then compared to BCF
values for other species of fish found in
the literature (Table 3). In the two classes
where comparisons were possible (chlori-
nated benzenes and ethanes), bioconcen-
tration as well as toxicity increased as the
number of chlorines on the molecule
increased. The literature values for other
fathead minnow studies as well as studies
with bluegill, sunfish (Lepomis macro-
chirus), and guppies (Poecilia reticulata]
(Table 3) all agree very closely with the
BCFs generated here. This indicates that
age, size, and species of fish have little
effect on the BCF generated over a 32-day
period of water exposures. Based on BCF
values, hexachlorobenzene, hexachloro-
butadiene, and 1,2,3,4-tetrachloroben-
zene are the chemicals from the groups
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Table 1. Summary of Acute Toxicity Data for Fathead Minnows, Rainbow Trout, and Daphnia
Compound
Fathead Minnow
96-hr LCSO
fmg/IJ
Rainbow Trout
96-hr LCSO
(mg/lj
Daphnia
48-hr LCSO
(mg/l)
Chlorinated Ethanes
Hexach/oroethane
Pentachloroethane
1,1,2,2-Tetrachloroethane
1,1,2-Tnchloroethane
1,2-Dichloroethane
Chlorinated Benzenes
Hexachlorobenzene
Pentachlorobenzene
1,2,3,4- Tetrachlorobenzene
1,2,4 • Tnchlorobenzene
1,3-Dichlorobenzene
1,4-Dichlorobenzene
Chlorinated Ethylenes
Tetrachloroethylene
1,1,2-Tnchloroethylene
Chlorinated Propanes
1,2-Dichloropropane
1,3-Dichloropropane
Chlorinated Butadienes
Hexachlorobutadiene
1.53
730
2030
81 70
7 77 SO
1 07
•276
779
4 re
1350
44 10
139 30
131 10
0.10
084
1.52
1.61
1.12
499
290
732
62.10
186.0
268.0
2.09
7.43
18 10
032
"Not tested
"Not toxic at water saturation
Table 2. Summary of Fathead Minnow and Daphnia Chronic Toxicity Data
Compound
Fathead Minnow
32-day (ELS) MA TC
Daphnia
28-day" Chronic
Chlorinated Ethanes
Hexach/oroethane
Pentachloroethane
1,1,2,2-Tetrachloroethane
1,1,2-Trichloroethane
1,2 -Dichloroethane
Chlorinated Benzenes
Hexachlorobenzene
Pentachlorobenzene
1,2,3,4- Tetrachlorobenzene
1,2,4-Tnchlorobenzene
1,3-Dichlorobenzene
1,4-Dichlorobenzene
Chlorinated Ethylenes
Tetrachloroethylene
1,1,2-Tnchloroethylene
Chlorinated Propanes
1,2-Dichloropropane
1,3-Dichloropropane
Chlorinated Butadienes
Hexachlorobutadiene
69-207
900-1,400
1,40O-4,OOO
6,000-14,800
29,000-59,000
4.76"
549"
245-412
499-1,008
1.000-2,267
565-1,040
500-1,400
6,000-11,000
8,000-16.000
65-13.0
6,850-14,4OO
13,200-26,000
10,600-20,700
363-694
689-1,450
505-1.110
"Effect—no effect concentrations
"Water saturation—no effects noted
tested that pose the greatest bioconcen-
tration threat to the environment.
The acute toxicity tests run with both
fish and invertebrates established a rela-
tive order of toxicity for the individual
chemicals that was nearly identical to the
order seen in the more sensitive
chronic exposures. Therefore, the short
96-hr LCSO fish exposures or the 48-hr
Daphnia exposures could be used to
establish a ranking of chemicals found in
water that will permit the researcher to
initially concentrate the expensive chronic
testing on the more toxic materials
The Daphnia acute test may be better
than a fish acute test, since it takes only
48-hrs and does not require the more
difficult flow-thro ugh system required for
a fish 96-hour LC50 estimate, yet it gives
the same relative order of chemical
sensitivity for these classes of chemicals
(Tables 1 and 2).
Early life-stage toxicity tests with fish
(32-days) and/or 28-day Daphnia chron-
ics would provide a more sensitive test
and the fish exposures would also allow
the determination of a BCF, which would
provide further information on the envi-
ronmental impact of a chemical.
The usefulness of aquatic tests as an
early warning system for toxic chemicals
in these classes may be somewhat limit-
ed, because of their low toxicity and low
ambient water concentrations. However,
this approach for more toxic chemicals
has considerable promise as an early
warning system for higher animals includ-
ing man.
References
American Public Health Association, A-
merican Water Works Association, and
Water Pollution Control Federation.
1975. Standard methods for the exami-
nation of water and wastewater 14th
ed. American Public Health Associa-
tion, Washington, DC 20036.
Benoit, D. A., V. R Mattson, and D L.
Olson 1982. A continuous-flow mini-
diluter system for toxicity testing. Water
Res 16-457-464.
Mount, D I. and W. A. Brungs. 1967. A
simplified dosing apparatus for fish
toxicology studies. Water Res., Vol 1,
pp 21-29.
Phipps, G. L., G. W. Holcombe, and J. T.
Fiandt. 1981. Acute toxicity of phenol
and substituted phenols to the fathead
minnow. Bull. Environ. Contam. Toxicol.
26:585-593.
U.S. Environmental Protection Agency,
Committee on Methods for Toxicity
Tests with Aquatic Organisms. 1975.
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Table 3. A Comparison of Bioconcentration Factors for Chemicals Tested in Present Study in
Fathead Minnows vs. Other Species of Fish in Other Studies
Chemicals
Present Study
Fathead Minnows °
BCF Log BCF
Literature Values
Fathead
Minnow* Bluegilf Guppyc
Chlorinated Ethanes
Hexach/oroethane
Pentachloroethane
1 ', 1 ,2,2-Tetrachloroethane
Chlorinated Ethylenes
Tetrachloroethylene
Chlorinated Butadienes
Hexachloro- 1,3-butadiene
Chlorinated Benzenes
Hexachlorobenzene
1 .2,3,4- Tetrachlorobenzene
1 ,2,4- Trichlorobenzene
1 , 3-Dichlorobenzene
1 ,4-Dichlorobenzene
703.4
60.4
8.1
61.5
6,988.6
23,391 5
2,567.3
398.5
97.9
112.4
285
1.78
0.91
1.79
3.84
4.37
3.41
260
1.99
205
138
68
8
49
-
21,878
1,820
1,698
66
60
-
-
-
-
14,454
3.631
646
91
^32-day exposure ELS toxicity test.
^30-day old fish exposed for 30 days.
1Adult fish exposed for 30 days.
Standard practice for conducting acute
toxicity tests with fishes and macro-
invertebrates, and amphibians. EPA-
660/3-75-009. Duluth, MN. 67 p.
N. Ahmad, J. Huot, and A. Moriarity are with the University of Wisconsin,
Superior, Wl 54880; the EPA authors D. Benoit, L. Brooke. D. Call, A.
Carlson, D. Defoe, J. Richter. P. Shubat, G. Ve/th, and C. Walbridge are with
the Environmental Research Laboratory, Duluth, MN 55804.
J. M. McKim and R. A. Drummond are the Co-Project Coordinators (see below).
The complete report, entitled "Aquatic Toxicity Tests to Characterize the Hazard
of Volatile Organic Chemicals in Water: A Toxicity Data Summary," (Order No.
PB 84-141 506; Cost: $13.00, subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The Co-Project Coordinators can be contacted at:
Environmental Research Laboratory
U.S. Environmental Protection Agency
6201 Congdon Blvd.
Duluth, MN 558O4
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