United States
Environmental Protection
Agency
Environmental Research
Laboratory
Duluth MN 55804
Research and Development
EPA-600/S3-83-097 Jan. 1984
SERA Project Summary
Toxicological Studies with
Herbicides, Selected EPA
Priority Pollutants and Related
Chemicals in Aquatic
Organisms
Daniel J. Call, Larry T. Brooke, Raymond J. Kent, Nasim Ahmad, and Joseph E.
Richter
Toxicological studies were conducted
in two areas: (1) the toxicity, biocon-
centration potential and metabolism of
five herbicides in fish; and (2) the
toxicity and/or metabolism of priority
pollutants and related chemicals in
various aquatic organisms.
The test herbicides included alachlor
[2-chloro-2',6'-diethyl-N- (methoxy-
methyl) acetanilide], bromacil (5-
bromo-3-sec-butyl-6-methyluracil), din-
oseb [2-(5ec-butyl)-4,6-dinitrophenol],
diuron [3-(3,4-dichlorophenyl)-1,1 -
dimethylurea], and propanil (3,4-di-
chloropropionanilide). Acute toxicity
(through 192 hr), early life-stage
toxicity (58-64 day), and bioconcentra-
tion studies were conducted with
fathead minnows (Pimephales promelas)
in Lake Superior water. Herbicide
metabolism was investigated in rainbow
trout (Salmo gairdneri] both in vivo and
in vitro.
Twenty-two chemicals from the EPA
priority pollutant list were studied for
their acute and/or chronic toxicity to
selected freshwater organisms. These
included 1,2-dichloroethane, 1,1,2-
trichloroethane, 1,1,2,2,-tetrachloro-
ethane, tetrachloroethylene, 1,2-
dichlorobenzene, 1,3-dichlorobenzene,
1,4-dichlorobenzene, hexachloroben-
zene, hexachlorobutadiene, di-n-butyl-
phthalate, pentachlorophenol, hep-
tachlor, chlordane, toxaphene, arsenic*3,
chromium*6, lead+2, mercury+2, nicker2.
silver*', selenium*4, and cyanide. Fresh-
water species tested included the
fathead minnow, rainbow trout, bluegill
sunfish (Lepomis macrochirus). flagfish
(Jordanella floridae). Daphnia magna.
scud (Gammarus pseudolimnaeus),
midge (Tanytarsus dissimilis) and green
alga (Selenastrum capricornutum).
Toxicity tests were also conducted with
pentachloroethane, hexachloroethane,
1,2,4-trichlorobenzene, pentachloro-
benzene, methanol and dimethylforma-
mide. The uptake by fish of di-n-
butylphthalate from water, its metabo-
lism and elimination were investigated.
Comparative metabolism of 1,1,2-
trichloroethane, chlorobenzene, 1,1,2-
trichloroethylene, chloroform, and
carbon tetrachloride was studied in rain-
bow trout and Daphnia.
This Project Summary was developed
by EPA's Environmental Research
Laboratory, Duluth. MN. to announce
key findings of the research projects that
are fully documented in separate reports
of the same title (see Project Report
ordering information at back).
Introduction
Many different agricultural and indus-
trial chemicals enter aquatic ecosystems
each year. For the protection of aquatic
biota, it is important to evaluate the
hazard potential of these chemicals.
Toxicological studfes collectively repre-
-------
sent one component of a hazard evalua-
tion scheme.
Extensive toxicological investigations
have been carried out for only a small
fraction of the thousands of chemicals in
production. Generally, the most extensive
aquatic toxicological data bases have
been generated on a select few chemicals,
where a combination of use, production
volume, frequency of occurrence in water
and/or biota, environmental persistence,
and known adverse effects have dictated
a need for toxicological data.
It is important to develop data bases on
such chemicals to the extent that
judicious environmental risk documents
can be prepared. The major focus of this
research project consisted of toxicological
studies on individual chemicals where
data bases were considered insufficient
for hazard evaluations.
Methodology
Flow-through toxicity tests with fish
and scuds were conducted in proportional
diluters using either Lake Superior water
or dechlormated city water. Toxicity tests
with Daphnia magna were static tests
using Lake Superior water in which the
toxicant solutions were renewed at 24 hr
intervals. Midge acute toxicity tests were
conducted in chambers in which toxicant
solutions in Lake Superior water were
continuously replaced from reservoirs
above the test chambers at a slow drip
rate.
In the acute tests, observations were
made at selected time intervals for
mortalities and gross behavioral effects.
LCso and ECso values were calculated by
several methods (trimmed Spearman-
Karber, probit, moving average, or
binomial) depending upon the character-
istics of the data. In the fish early life-
stage toxicity tests, observations were
made for mortalities, abnormal develop-
ment, and growth. Data were analyzed by
one-way analysis of variance in conjunc-
tion with Dunnett's procedure to deter-
mine "no-effect" concentration ranges
In Daphnia chronic life-cycle studies,
observations were made of mortalities,
number of offspring produced, and
growth. Data were analyzed to determine
"no-effect" concentration ranges.
Algal toxicity tests were run in flasks
under uniform conditions of light, tem-
perature, and aeration. Dry weights were
measured after 96 hr of exposure, and the
concentration that inhibited growth by 50
percent was determined by the trimmed
Spearman-Karber method.
Studies on uptake, bioconcentration,
elimination, and metabolism of selected
organics in fish and Daphnia were
conducted using 14C radiolabeled com-
pounds. Five herbicides (alachlor, brom-
acil, dinoseb, diuron, and propanil) and
the plasticizer, di-n-butylphthalate, were
studied in fish. The compounds 1,1,2-
trichloroethane, chlorobenzene, 1,1,2-
trichloroethylene, chloroform, and carbon
tetrachloride were studied for their
relative tendencies to be metabolized by
fish and Daphnia.
Results and Conclusions
Results of the acute toxicity, early life-
stage toxicity, bioconcentration studies
for the five herbicides are summarized in
Table 1 Dinoseb was the most toxic
herbicide on an acute basis, while
propanil was most toxic on the basis of a
longer exposure encompassing the early
lifestages. Parent herbicides did not
appreciably accumulate in fish tissue.
Metabolism studies indicated that fish
quite readily metabolized the herbicides.
Mass spectral analysis of extracts from
the bile of fish exposed to diuron and
propanil aided in the characterization of
metabolites
Toxicity data for EPA priority pollutants
are presented in Table 2. LC5o values
were not determined for hexachloroben-
zene due to insufficient mortalities at
concentrations in water approaching
solubility.
Radiolabeled di-/?-butylphthalate was
rapidly accumulated and metabolized by
fathead minnows. It was concentrated in
fish tissue approximately 600 times its
concentration in the water. The radiolabel,
which consisted largely of metabolites,
was slowly eliminated from the fish upon
transfer to clean water.
Chloroform was more readily metabol-
ized in vitro by rainbow trout and Daphnia
than was carbon tetrachloride, chloro-
benzene, 1,1,2-trichloroethylene or
1,1,2-trichloroethane. Metabolism by
enzyme systems from both species
resulted in similar percentages of total
14C that were hexane-unextractable for
carbon tetrachloride, chlorobenzene, and
1,1,2-trichloroethylene. A greater per-
centage of total 14C-chloroform was
hexane-unextractable with the Daphnia
enzyme system than with the trout
system; while the converse applied for
1,1,2-trichloroethane. Higher percentages
of total 14C were protein bound with trout
systems than with Daphnia systems for
all compounds. Assays indicated that
both species possess active mixed
function oxidase systems.
Table 1. Toxicity and Bioconcentration Potential of Five Herbicides With the Fathead Minnow
Herbicide
Acute Toxicity
(96 Hr. LCso. and
95% C.I.. ma • L~'l
Early Life-Stage Toxicity
(58-64 Day "No Effect-
Concentration Range.
ua -Z.'V
Bioconcentration
Factor
Alachlor
Bromacil
Dinoseb
Diuron
Propanil
5.0(4.5-5.6}
182(177-188)
0.7 (0.6-0.7)
14.2(13.4-15.0)
8.6(7.7-9.5)
520-1.000
1.000
14.5-48.5
33.4-78.0
0.4-0.6
6X
<3X
1X
2X
2X
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Table 2. Toxicities of Selected EPA Priority Pollutants to Freshwater Organisms
Chemical
1,2-Dichloroethane
1, 1 ,2-Trichloroethane
1. 1 ,2,2,-Tetrachloroethane
Tetrachloroethylene
"
Tetrachloroethylene/ DMF
1,2 -Dichlorobenzene
1 , 3 -Dichlorobenzene
1,4-Dichlorobenzene
Hexachlorobenzene/DMF
"
"
Hexachlorobutadiene
Di- n -butylphthalate
Pentachlorophenol
"
Heptachlor
Chlordane
Toxaphene
Arsenic*3
Arsenic *3
Selenium**
Chromium*6
"
Lead*2
"
Mercury*2
Nicker
Si/ver^
"
"
"
CAT
Organism
Daphnia magna
Daphnia magna
Daphnia magna
Daphnia magna
Tanytarsus dissimilis
Salmo gairdneri
Salmo gairdneri
Tanytarsus dissimilis
Salmo gairdneri
Daphnia magna
Tanytarsus dissimilis
Salmo gairdneri
Tanytarsus dissimilis
Salmo gairdneri
Lepomis macrochirus
Salmo gairdneri
Lepomis macrochirus
Daphnia magna
Tanytarsus dissimilis
Gammarus pseudolimnaeus
Selenastrum capricornutum
Daphnia magna
Selenastrum capricornutum
Gammarus pseudolimnaeus
Daphnia magna
Pimephales promelas
Jordanella floridae
Tanytarsus dissimilis
Tanytarsus dissimilis
Gammarus pseudolimnaeus
Tanytarsus dissimilis
Gammarus pseudolimnaeus
Pimephales promelas
Daphnia magna
Tanytarsus d'ssimilis
Gammarus pseudolimnaeus
Pimephales promelas
Jordanella floridae
Tanytarsus dissimilis
/-Cso
Concentration
mg • L"1
(Test Duration)
268,315*
186,174*
62.1,56.9*
18.1,9.09*
308
4.99
5.84
12.0
1.58
7.43,7.23*
13.0
1.12
>H2O Sol.
>H2O Sol.
>H2O Sol.
0.320
0.324
3.7
46.0
0.280
0.0381,
0.0282t>-c
0.035
0.38"
0.875
1.54. 4.83"
14.2
14.4
42.5
57.3
0.0671
224
0.140
0.150
0.915
3.17
0.0045
0.0107
0.0092
2.36. 2.49"
(48 hr)
(48 hr)
(48 hr)
(48 hr)
(48 hr)
(96 hr)
(96 hr)
(48 hr)
(96 hr)
(48 hr)
(48 hr)
(96 hr)
(48 hr)
(96 hr)
(96 hr)
(96 hr)
(96 hr)
(48 hr)
(48 hr)
(96 hr)
(96 hr)
(48 hr)
(96 hr)
(96 hr)
(48 hr)
(96 hr)
(96 hr)
(48 hr)
(48 hr)
(96 hr)
(48 hr)
(96 hr)
(96 hr)
(48 hr)
(48 hr)
(96 hr)
(96 hr)
(96 hr)
(48 hr)
"No Effect-
Concentration
Range
mg • L'1
(Test Duration)
10.6-20.7
13.2-26.0
6.85-14.4
0.505-1.11
0.689-1.45
0.63-1.32
2.1-4.3
2.1-4.1
<0.00021
(28 days)
(28 days)
(28 days)
(28 days)
(28 days)
(28 days)
(30 days)
(30 days)
(35 days)
"Results from assays in which organisms were unfed and fed, respectively.
''Calculated concentration that inhibited algal growth by 50 percent.
cECso values for two separate tests.
"ECso values from assays in which organisms were unfed and fed, respectively.
"Expressed as HCN and free CN~, respectively.
-------
D, J. Call, L T Brooke, P. J. Kent, N. Ahmad, and J. E. Richter are with the
University of Wisconsin, Superior, Wl 54880.
John Teasley is the EPA Project Officer (see below).
This Project Summary covers two reports, entitled:
"Toxicity and Metabolism Studies with EPA Priority Pollutants and Related
Chemicals in Freshwater Organisms," (Order No. PB 83-263 665; Cost:
$14.50, subject to change)
"Toxicity, Bioconcentration, and Metabolism of Five Herbicides in
Fresh water Fish,'' (Order No. PB 83-263681; Cost: $ 13.00, subject to change
The above reports are available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Environmental Research Laboratory
U S Environmental Protection Agency
6201 Congdon Blvd.
Duluth, MN 55804
*US. GOVERNMENT PRINTING OFFICE' 1984-759-015/7278
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
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