TECHNICAL REPORT DATA
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1 REPORT NO 2
EPA-uOO/J-80-194 Journal Article
a. rec"
4, Tt rt_E ANOSUBT1TLE
Chronic Toxicity of Hexavaient Chromium to the
Fathead Minnow (Pimephales promelas;
5. REPORT DATE
e. PERFORMING ORG ANIZATION COOE
7 AUTHOR(S)
Q. H. Pickering
8. PERFORMING ORGANISATION RLPORT NO
9 PERFORMING ORGANIZATION NAME AND ADDRESS
U.S. Environmental Protection Agency
Newtown Fish Toxicology Station
341 . Church Street
Cincinnati, OH 45244
10. PROGRAM ELEMENT NO.
1BA820
tt CONTRACT/GRANT NO
12 SPONSORING AGENCY NAME AND AODRESS
U.S. Environmental Protection Agency
Environmental Research Laboratory-Duluth
6201 Congdon Boulevard
Du]uth, MN 55804
13, TYPE OF REPORT AND PERIOD COVERED
Journal article
14. SPONSORING AGENCY COHE
EPA-600/03
15 SUPPLEMENTARY NOTES
REFERENCE: Arch. Environ. Contam. Toxicol. 9, 405-413 (1980).
16 ABSTRACT - "
The chronic effects of hexavaient chromium on the fathead minnow (Pimephales
promelas) were investigated. Survival was affected only at the high test concen-
tration of 3.95 mg Cr/L. All chromium concentrations, including 0.018 mg/L, the
lowest tested, retarded the early growth of first-generation fish, but this effect
was only temporary. Growth of second-generation fish was not affected at concen-
trations of 1.0 mg/L or lower. Reproduction and hatchability of eggs were not
affected a1" any chromium concentration tested.
The maximum acceptable toxicant concentration (MATC) for fathead minnowa in hard
water (209 mg/L as CaCOj at pH 7.7) was based on survival and lies between 1.0 and
3.95 mg Cr/L, respectively. The application factor (MATC/96-hr LC50) is between
0.03 and 0.11.
>7 KEY WORDS AND DOCUMENT ANALYSIS
a DESCRIPTORS
b. IDENTIFIERS/OPEN ENDED TERMS
c COSATl J icId/Croup
Blo&ssay Fish
Acute tonicity
Fathead minnow Aquatic life
Chronic tcxicity
Survival
Hexavaient chromium
Growth
Reproduction
Metala
Freshwater
18 DISTRIBUTION STATEMENT
19. SECURITY CLASS (ThU Report)
21. NO. OF PAGES
UNCLASSIFIED
9
70 SECURITY CLASS fThltpagrl
72. PRICE
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EPA-600/J-80-194
Journal Article
Arch. Fnvircnm. Contain Toxicol 9.405-413 (1980) Archive* oi EnvirOnmSfltSl
Contamination
and Toxicology
Chronic_Toxicity_of-Hexa.valentjChromiuni_to_the_Fathead
Minnow (Pimephales promelas)
Q. H. Pickering
U.S. Environmental Protection Agency, Environmental Research Laboratory-Duluth,
Newtown Fish Toxicology Station, Cincinnati, Ohio 45244
Abstract. The chronic effects of hexavalent chromium on the fathead min-
now (Pimephales promelas) were investigated. Survival was affected only
at the high test concentration of 3.95 mg Cr/L. All chromium concentra-
tions, including 0.018 mg/L, the lowest tested, retarded the early growth of
first-generation fish, but this effect was only temporary. Growth of second-
generation fish was not affected at concentrations of 1.0 mg/L or lower.
Reproduction and hatchability of eggs were not affected at any chromium
concentrator, tested.
The maximum acceptable toxicant concentration (MATC) for fathead
minnows in hard water {209 mg/L as CaCOn at pH 7.7) was based on sur-
vival and iies between 1.0 and 3.95 mg Cr/L, respectively. The application
factor (MATC/96-hr LC50) is between 0.03 and 0.11.
Hexavaient chromium is a trace metal in natural water, and its presence at high
concentrations usually indicates discharge from industrial and municipal
effluents. In a 5-year study of 1,577 samples from rivers and lakes of the United
Slates, Kopp and Kroner (1969) found dissolved chromium in 24.5% of the
samples; the mean concentration was 9.7 jig/L, and the range was 1 to 121 ptg/L
based on total chromium. Most measurements of environmental samples of
chromium are on the basis of total chromium. Criteria developed for chromium
(U.S. Environmental Protection Agency 1976) for both domestic water suppK
and for freshwater aquatic life were developed on the basis of total chromium.
Because or its widespread use and toxicity, chromium can be a hazard to
aquatic life and is one of the 65 toxic pollutants listed in a consent decree court,
order (National Resources Defense Council et al. vs Train 1976). A review of
the environmental effects of chromium (Towill et al. 1978) indicates that the
environmentally important forms are trivalent and hexavalent chromium. Hexa-
valent chromium is the more toxic form to mammals and fish.
Most 96-hr LC50 values for hexavalent chromium for fish range from 10 to
130 mg/L. Adverse effects are produced, however, at much lower concentra-
tions with long-term exposure. Olson and Foster (1956) studied long-term ef-
fects of hexavalent chromium on Chinook salmon (Oncorhynchus tshawytscha)
0090- 4341/80/0009 - 0405 $01.80
1980 Springer-Verlag New York Inc.
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406
Q, H. Pickering
?nd rainbow trout (Sohno f>airdneri). They reported that survival was affected
at V jxg/L, and growth was reduced at 13 /xg/L. Benoit (1976) examined the
chronic effects of hexavalent chromium on brook trout [Sohelinus fontituilis)
and rainbow trout and found that brook trout alcvin survival was decreased at
350 ^ig/L, and growth in weighFwas retarded at lO pt^ITcJurirTg the 8-month
O^posure. Sauter et al. (1976) studied the effects of hexavalent chromium on
the eggs and fry of seven fish species. These embryo-larval studies gave esti-
mated maximum acceptable toxicant concentration (MATC) values that varied
from 51 to 105 jig/L for rainbow trout to more than 2,167 fig/L for walleye
iStizostedion vitreum vitreum).
The present chronic toxicity study is one of a series of studies designed to
evaluate the direct effects of metals on fish. The objective of this study was to
determine the MATC (Mount and Stephan 1967) of hexavalent chromium in
hard water for the fathead minnow (Pimephales protnelas). The MATC is es-
tablished on the basis of a chronic exposure; survival, growth, and reproduc-
tion are used as measures of effect. In addition, acute toxicity studies were
made to estimate an application factor (Mount and Stephan 1967) for chromium
toxicity.
Methods and Experimental Conditions
Exposure $\ stem
The design of th'irstudy wns~simil^(o thTirdescn&ecn}y~Mounraruf Sfeph3rrn967). A serial diluler
wuh a dilution factor of 0 25 was used to provide the control and five chromium concentrations
This dilution factor was used to obtain a wide range of concentrations, because long-term effects on
survival were anticipated (Olson and Foster 1956). The randomly arranged all-glass exposure
chambers (60 x 30 x .10 cm) contained a 15- x 15-cm compartment in the outlet corner for
second-generation 30-day survival and growth studies. In addition, stainless steel chambers (60 x
15 x 20 cm) were used for the 30- to 60-day second-generation studies. The water volume of each
adult spawning chamb-r was maintained ai 31 L, and the flow ra'.e averaged 200 L per day The
dilution water was a nnxlure of pond water originating from a spring and carbon-filtered, de-
mineralized Cincinnati tap water. Six spawning substrates, consisting of half-tiles, were placed in
each chamber, and eggs from these tiles wete incubated in nylon-screen-bottom cups suspended
from a rocker-arm that oscillated two times per minute. Light and photoperiod were provided by
cool-white fluorescent ceiling lights and t>0-W incandescent light bulbs suspended above each
chamber
BhiIohkuI
The chronic exposure was started ir> November when thirty-five 4-week-old juveniles reared from
four groups of eggs spawned in the laboratory were randomly assigned to each exposure concen-
tration After nine weeks of exposure the number of fish was reduced :o 20, except for the high
concentration in which only I3 fish survived. Excess males were removed during the spawning
season, which began in June, to reduce territorial conflict. The fish were fed a dry trout food daily
and cladocerans-weekly—Live-organisms-in-the-watef-supply-supplemented-this diet,
During the spawning season all spawning substrates were examined for eggs in ihe early
afternoon. These eggs were removed and counted, and some were placed in egg cups for hatchabil-
ity deteiminations. Usually, 100 eggs from each spawning, 25 eggs per cup, were exposed. Hatch-
ability was calculated as the percentage of larvae hatched seven days after (he spawning L
F.-'ty of these second-generation larvae from each concentration were placed tn the larval
growth chambers for 30 days from time ol spawning. The fish were then measured and transferred
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Toxicity of Chromium (o Fathead Minnows
407
lo the stainless sieel chambers for an additionaL30_days,_AHJarval studies were started from eggs
that were spawned within three days of each other
f'livuial ami Chtmuol Conditions
Routine water analyses were made weekly with procedures described by the American Public
Health Association et al (1965) Oxygen was measured in nil chambers, and hardness, pH, alkalin-
ity, and acidity in two chambers The mean and Standard deviation for hardness, alkalinity, and
acidity were 209 * 5 mg/L, 159 * 20 mg/L, and 9.5 ± 3.5 mg/L as CaCOj, respectively. The pH
varied from 7 5 to 8 2 The pH measurements were converted to ion concentration, and the mean
ion concentration v.as calculated. Encoding this to pH, the mean pH was 7.73. The mean of the pH
measurements, as sitch, was 7 75 Dissolved oxyp.cn had a mean concentration and standard
deviation of 7 5 ± I 5 mg/L Dissolved oxygen concentiations in (he two high chromium concen-
iranons wt,e corrected for (he positive influence of dichromate in the Winkler titration
Reagent-grade potassium dichromate was* introduced from a constant-level funnel maintained
by a float valve via a toxicant-metering system as described by Mount and Warner (1965) Each
weekday, water samples were measured for hcxavalent chromium with the diphenylcarbazide
method (American Public Health Association et al. 1965) A calibration curve was prepared at
periodic intervals, and the reproducibility of the curve was excellent The curve was linear, com-
plying with Beer's law, with the mean absorbaiice readings ofO 782. 0.548,0.394, 0.156, and 0 078
for 200, 140. 100, 40, and 20 CrJL, respectively. Higher test concentrations were quantitatively
diluted to maintain a small range of concentrations Control blanks showed no positive interfer-
ences Mean chromium lest concentrations ranged from 3.95 to 0.018 mg/L (Table I)
No attempt was made to conduct the ch7onic~study~ara~con.stanttempcrature. Mean weekly -
temperature was I6'C during the first fall and winter months and then slowly increased to 24°C in
June, where it remained until the end of the test. The usual weekly fluctuation was about 3°C. and
the minimum and maximum temperatures were and 27®C, respectively. Temperature was
recorded continuously with a 7-day indicating and recording thermograph. A natural photopenod
(Evansville. Ind ) was maintained, and adjustments were made bimonthly. For spawning purposes,
a 16-hr light cycle was maintained from late June to (he end of the test.
At uir tini< ilv
Sidf by-si
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Table 1. Survival and growth of fathead minnows exposed to hexavdent chromium
First generation
Measured
concentration
mg/L"
9 weeks
Survival
We
81
Second generation
ght
Final (412 days)
Ma'es Females
Length Weight Length Weight
30 days
mm'
E'
mm"
gc
Survival
%
60 days
Length
mm"
Survived
%
Length
mm"
3.95' ± 0.20
1.00 ± 0.07
0.26 ± 0.016
0.066 ± 0.008
0.018 ± 0.002
Control
37
94
86
97
97
100
0.11
0.15
0.14
0.15
0.19
9.7 ± 0.8
15.4 ± 1.8
15.1 ± 1.3
13.7 - 1.2
11.9 ± 1-2
15.1 ± 2.4
12
98
94
82
80
72
11.5 - t.6
24.7 ± 3.5
26.5 ± 3.2
23.9 ± 3.6
24.6 * 3.7
24.7 ± 3.6
a Mean and standard deviation
" Each concentration value is the mean of 217 measurements
r Mean I
d Significantly different (P = 0.05) from control
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Tonicity of Chromium to Fathcnd Minnows
409
1), and only 13% survived to the .termination of the exposure. Survival of
second-geneiaMon fish also was greatly reduced at 3.95 mg Cr/L (Table 1).
After 30 days, survival was 38%, and aftei 60 days only 12% survived. Survival
of both first- and second-generation fish at concentrations of 1.0 mg/L and
lower was similar to that of the control fish.
Survival of the eggs that were spawned and incubated at all concentrations
was not adversely affected by chromium (Table 2). Hatchability varied from 86
to 96%.
The 96-hr LC50 values of hexavalent chromium for the two static bioas-
says were 39.7 and 32.7 mg/L. These two values were not significantly different
(P = 0 05), sc the data were combined giving a 96-hr LC50 value of 36.2 mg/L
(27.2 - 45.4 mg/L) The 96-hr LC50 values of the two consecutive flow-through
bioassays were 37.7 mg/L (29.5 - 57.5 mg/L) and 37.0 mg/L (27.4 - 52.6 mg/L).
The 96-hr L7C50 valueoftheth"iFd~fIow-through"bioassay was 3579mg/L (29.1
45.9 mg/L). The mean of the three flow-through tests was 36.9 mg/L.
Growth
All concentrations of chromium reduced growth of first-generation fish after
nine weeks of exposure (Table 1). Analysis of variance indicated that chromium
had a significant effect (P =0.01) on the weight of fish. Duncan's multiple range
test indicated that the weight of fish exposed to 1.0 mg/L was significantly
different (P = 0.05) frotn the three low concentrations and the control. The
three low-test concentrations were homogeneous and significantly different
from the control. At the end of the chronic exposure, 412 days, the length and
weight of the females exposed to chromium were similar to control fish (Table
1). At the sublethal concentrations of chromium, the length and weight of the
males were similar to that in the control (Table 1). The length and weight of the
males in the lethal concentration of 3.95 mg/L was less than that in the control.
However, small excess males were removed from the sublethal concentrations
and control to reduce teiritorial conflict. So it was not possible to statistically
analyze these growth data. The mean length of the second-generation fish
exposed to 3.95 mg/L was reduced more than 50% bflov the control after 60
days' exposure. However, the final lengths of the fish at 1.0 mg/L and lower
were similar to that of the control fish.
Reproduction
Sublethal concentrations of chromium did not adversely affect egg production,
and even at the highest concentration, which caused some deaths, the surviving
fish spawned (Table 2).
Discussion
The results of this study clearly show that the high concentration of 3.95 mg
Cr/L was lethal to the fathead minnow This concentration had an adverse
effect on survival of both first- and second-generation fish. The first-generation
fish started dying after three weeks of exposure, and 63% were killed after nine
-------
410
Q. II Pickering
Tabic 2. Spawning results and hatchiibility of fiilhend minnow eggs in the chronic exposure
lo hcxuvalent chromium
Mean measured
Mean
Number of
eunc«nir?tion
Number of Number of
Tout number
Total number
eggs per
Percentage
eggs
mg/L
males
fi:male^
of ejgs
of spawning*
female
halch
inrubuted
3 95
IS
2
711
3
356
86
135
1 00
8
9
10.) f9
43
1.132
95
600
0 26
7
8
7.285
36
910
9^
625
0 066
7
8
4,889
21
611
93
364
0 018
9
11
2.559
15
269
96
547
Control
4
7
2.632
17
376
95
525
weekv The fish in this concentration were visually smaller than fish in other
concentrations, and the smallerones-were-thcfirsHodie—Fish continued to die
for about f»'o more months, but no adult fish died during the spawning season.
Second generation and first-generation fish were very similar in their sensitiv-
ity to the lethal effects of chromium.
Fathead minnows tested in hard water were more resistant to long-teim
lethal effects of chromium tha.i salmonoids exposed in softer water. Olson and
Foster (1956) studied the effects of long-term exposure to chromium on the
various developmental stages of rainbow trout and chinook salmon. They
found that exposure to 0.17 mg/L caused the death of rainbow trout fry within a
few days, and fish continued to die over a 2-month period to a maximum
mortality or 94% . For chinook salmon, they found that after the end of the fry
stage significantly fewer fish survived at 0.18 mg/L, and mortality increased
during the fingerling stage. Benoit (1976) reported that mortality of alevin rain-
bow trout exposed to 0 34 mg/L for 12 weeks was 100%, and mortality ot
young brook trout was 72% under similar conditions. Sauter et cil (1976) re-
ported that exposure to 0.82 mg/L for 60 days significantly reduced survival of
rair.bow trout fry when compared controls. They also found that concentra-
tions between 1.4 and 11.6 mg/L appeared to reduce survival of lake trout fry,
but because of variability among replicates only concentrations of 6.0 and 11.6
mg/L significantly reduced survival (P = 0.05).
— Fathead minnow einbryos-werenot-sensitivetochromiunrexposu-e; Sur-
vival of eggs that were spawned and incubated in all concent:ations of
chromium was similar to survival of control eggs. Hatchability varied from 86
to 96%, all within the normal variability found at our laboratory. Bencit (1976)
found that hatching success of brook trout eggs spawned >ind incubated in 0.35
mg Cr/L and control embryos transferred to 0.76 and 1.56 mg Cr/L was as good
as control eggs. Sauter et al, (1976) reported on the toxicity of chromium to the
eggs and fry of seven fish species. In all tests, survival and/or growth of fry
were affected at concentrations lower than those that affected hatchability.
The 96-hr LC50 values calculated during the present study were similar to
96-hr LC50 values reported by Adelnan and Smith (1976). Tliey found a mean
96-hr LC50 value of chromium for ihe fathead minnow of 26 mg/L, when the
desired concentrations were immediately obtained, and a mean 96-hr LC50
value of 48 mg/L when the desired concentrations were not obtained for 3 to 4
hr. Ruesink and Smith (1975) reported a 96-."ir LC50 value of 37 mg/L for the
fathead minnow tested at 25°C. However, the fathead minnow tested in haid
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Toxicity of Chromium lo Fathend Minnows
411
water had a lower 96-hr LC50 value than that reported by Benoit (1976) for the
brook trout exposed in soft water. Pickerhg and Henderson (1966) found that
hexavalent chromium was more toxic to the fathead minnow and bluegill
(Lepomis macrochirus) in soft water than in nard water of high alkalinity and
pH. Trama and Benoit (i960) reported that hexavalent chromium was less toxic
when potassium chromate was used than when potassium dichromate was
used. They attributed this difference in toxicity to the greater concentration of
hydrochromate ions in the more acidic dichromate salt solution.
Decreased growth is considered an indication of sublethal toxicity. Growth
of first-generation fish was significantly (P = 0.05) affected at all chromium
concentrations after nine weeks of exposure. The mean weight of fish exposed
to 1.0 mg/L was about 60% that of control fish, and the mean weight of fish in
the three low concentrations was about 80% of the weight of controls. This
adverse effect on growth was only tempoury, however, as the final weight of
these fish was similar to the we'ght of the controls, and the return to normal
weight suggests recovery from the toxic effects of chromium. This effect of
chromium on growth parallels t h a tfou nd~by~B enoi t(l 976)rH
-------
Q. H Pickering
based on a MATC of-b0-^-3795-mg-er/L-and-a-96=hrLC50 value of 36.9 mg
Cr/L. Benoit (1976) reported an application factor of 0.003 - 0.006 derived
from a MATC of 0.20 - 0.35 mg Cr/L. He used survival as the endpoint and a
96-hr LC50 value of 59 mg Cr/L. Temporary adverse effects of chromium on
growth were not used to derive the MATC in either of these studies. If these
temporary effects on growth were used to derive the MATC, the application
factor for chromium would be more similar, but much smallei. Macek and
Sleight (1977) reported an application factor for hexavalent chromium of 0,02 -
0.05 based on an embryo-larval study. This application factor is similar to that
found in the present study. The MATC for their study was based on reduced
weight uf rainbow trout after 60 days' exposure of the larval stage.
At present, it does not seem possible to recommend an application factor
for chromium based on chronic studies reported here and by Benoit. The
MATC for chromium, when the fathead minnow and brook trout are used as
test animals, is based on lethal concentrations. In the acute tests, chromium in
hard water killed fathead minnows at lower concentrations than chromium in
soft water killed brook trout. In the chronic tests, chromium killed brook trout
at lower concentrations. Death of the test animal is the most significant end-
point of toxicitv, and as a criterion of toxicity is unambiguous and final. Re-
duced growth is also an endpoint. However, the ecological significance of a
temporary effect on"growth~ofTirst-gen"eration nslris~uncertain.
References
Adelmnn. I R . anil L L Smith, Jr.- Standard lest fish development. Part 1 rathead minnows
iPinicphtilcs i>ruinclnirtliieri) Water Res 4, 4?? (1976).
Dixon, W J Biomedical computer programs Univ California Publications in Automatic Com-
putation, Umv California Press, Berkeley. CA <1974).
Harris, E K Confidence limits for the LD50 using the moving average angle method Biometri
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Hermanutz, R O Endnn and malathion toxicity lo flagfish (Jordanellti llnru/ae) Arch Environ
Contam Toxicol 7,159(1978)
Kopp, J. F , and R C Kroner. Trace metals in water of the United States A five year summary of
(race metals in rivers and lakes of the United States (Oclober 1, 1962-September 30, 1967).
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evaluaiing hazards associated with the chronic toxicity of chemicals to fishes In F. L Mayer
and J L Hamelink (eds )- Aquatic toxicology and hazard evaluation, p. 137 American Soci-
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the butoxyethanol ester of 2,4-D. Trans. Am. Fish. Soc. 96, 185 (1967)
Mount, D. I , and R. E. Warner. A serial dilution apparatus for continuous delivery of various
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(1965)
National Rewuna Dejense Council el a! r Tram 8 ERC 2120, Settlement Agreement, D D C
(1976)
-------
Toxicity of Chromium 10 Fathead Minnows
41?
Olson, P. A., nnd R F Foster. Effect of chronic exposure 10 sodium dichromate on young chinook
snlmon and rainbow iroul. In Hanford biological research annual report for 1955. HW-4I500,
pp. 35-47. Richland, WA (1956).
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warmwater fishes Air Water Pollut. Int. J. 10, 453 (1966)
Ruesink, P G , and L. L Smith, Jr.: The relationship of the 96-hour LC50 to the lethal threshold
concentration of hexavalent chromium, phenol, and sodium pentachlorophenate for fathead
minnows U'n>ici>lwlci i>r
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