December 1976
Ecological Research Series
EFFECTS OF AROCLOR
1254 ON BROOK TROUT,
Salvelinus fontinalis
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Duiuth, Minnesota 55804
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into five series. These five broad
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environmental technology. Elimination of traditional grouping was consciously
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The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ECOLOGICAL RESEARCH series. This series
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influences. Investigations include formation, transport, and pathway studies to
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This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/3-76-112
December 1976
i
EFFECTS OF AROCLOITV 1254 ON BROOK TROUT, SALVELINUS FONTINALIS
by
Virginia M. Snarski
Frank A. Puglisi
Environmental Research Laboratory-Duluth
Duluth, Minnesota 55804
ENVIRONMENTAL RESEARCH LABORATORY-DULUTH
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
DULUTH, MINNESOTA 55804
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DISCLAIMER
This report has been reviewed by the Environmental Research Laboratory-
Duluth, U.S. Environmental Protection Agency, and approved for publication.
Mention of trade names or commercial products does not constitute endorsement
or recommendation for use.
XI
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FOREWORD
Our nation's freshwaters are vital for all animals and plants, yet our
diverse uses of water for recreation, food, energy, transportation, and
industry physically and chemically alter lakes, rivers, and streams. Such
alterations threaten terrestrial organisms, as well as those living in water.
The Environmental Research Laboratory in Duluth, Minnesota develops methods,
conducts laboratory and field studies, and extrapolates research findings
—to determine how physical and chemical pollution affects aquatic life
—to assess the effects of ecosystems on pollutants
—to predict effects of pollutants on large lakes through use of models
—to measure bioaccumulation of pollutants in aquatic organisms that
are consumed by other animals, including man
This report describes the effects of a long-term exposure of brook trout
to the polychlorinated biphenyl, Aroclor® 1254, at extremely low concentra-
tions in the water (0.01 - 0.94 pg/1). Because of the persistence of these
chlorinated hydrocarbons and their tendency to bioaccumulate, the measurement
of tissue concentrations was an important part of the study.
Donald I. Mount, Ph.D.
Director
Environmental Research Laboratory
Duluth, Minnesota
111
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ABSTRACT
No adverse effects were observed on survival, growth, and reproduction
of brook trout exposed for 71 weeks to 0.94 yg/1. and lower concentrations of
the polychlorinated biphenyl Aroclof® 1254 (P = 0.05). Survival and growth
to 90 days of alevin-juveniles from exposed parents were also unaffected
(P = 0.05). Polychlorinated biphenyl concentrations in the brook trout were
directly proportional to the water exposure concentration (P = 0.05). The
PCB tissue concentrations appeared to have reached a steady state by the first
sampling after 14 weeks of exposure. The PCB residues (wet-tissue basis) in
chronically exposed fish were approximately 2 yg/g in the fillet and 9 yg/g
in the "whole body" (entire fish minus one fillet and the gonads) at the
highest water concentration, 0.94 yg/1. The higher residue in the whole body
compared to the corresponding fillet was due to the higher fat content of the
former.
iv
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CONTENTS
Foreword iii
Abstract iv
Tables vi
Acknowledgment vii
1. Introduction . 1
2. Conclusions 2
3. Recommendations 3
4. Materials and Methods , ,4
Bioassay , 4
Residue sampling and analysis 5
5. Results 8
Bioassay , ,....,. 8
PCB tissue residues , 8
6. Discussion 14
References 17
Appendix
A. Recommended bioassay procedure for brook trout Salvelinus
fontinalis (Mitchill) partial chronic tests ........ .20
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TABLES
Number Page
Arocloi® 1254 Concentrations (yg/1.) in Duplicate Brook Trout
Adult and Alevin-Juvenile Tanks Measured by Gas Chromatography. 6
Survival, Growth, and Reproduction of Brook Trout Exposed to
Aroclor®1254 for 16 Months 9
Hatchability, Survival, and Growth of Brook Trout Alevin-
Juveniles from Parents Exposed to Aroclo^S* 1254 for 14 Months
Before Spawning 10
Concentration of PCB G-ig/g wet weight) and Fat Content (%) of
Fillets from Brook Trout Exposed to Aroclor^ 1254 for Various
Time Periods 12
Concentration of PCB (yg/g wet weight) and Fat Content (%) of
Whole Body (Entire Fish Minus One Fillet and Gonads) of
Brook Trout Exposed to Aroclor^ 1254 for Various Time Periods . 13
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ACKNOWLEDGMENTS
The authors thank Mr. C. Walbridge for collecting PCB water samples, for
making routine water analyses, and for daily assistance; Mr. L.F. Mueller for
assisting in PCB water analysis and constructing test equipment; and the
Environmental Research Laboratory-Duluth's staff for technical assistance,
advice, and manuscript review. We would also like to thank the Monsanto Com-
pany for kindly supplying the Aroclor® 1254 used in this experiment.
VII
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SECTION 1
INTRODUCTION
The objective of this study was to determine the effects of 1 yg/1. ..and
lower water concentrations of the polychlorinated biphenyl (PCB) Aroclor®
1254* on the life cycle of the brook trout, Salvelinus fontinaj-is (Mitchill).
Widespread PCB contamination of the environment has occurred (Jensen, 1966;
Koeman _et_ al_., 1969; Duke e^ al_., 1970, Veith, 1972; Giam _et, al_., 1973; and
others). Although laboratory studies have shown adverse effects on survival
and reproduction and biological accumulation from low (yg/1.) PCB concentra-
tions on other fish species (Hansen et_ jil_., 1971, 1973; Stalling and Mayer,
1972; Nebeker et_ al_., 1974; Schimmel &t_ ai_., 1974), no studies on the effects
of long-term exposure to known concentrations of PCB's on salmonids could be
found in the literature. Because of similarities in chemical properties and
biological activity, demonstrated particularly in birds, between PCB's and
DDT (Risebrough and Brodine, 1970), this experiment was designed to determine
if sublethal PCB concentrations might have effects on salmonids similar to
those observed with DDT (Burdick e^ al_., 1964, 1972; Allison et_ _al., 1964;
Macek, 1968) . The uptake of PCB residues from chronically exposed females
and transfer to their ova as well as the survival of embryos and alevins at
the yolk-sac absorption stage were of particular interest.
^Registered trademark of Monsanto Co.. St. Louis, MO.
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SECTION 2
CONCLUSIONS
No adverse effects were observed on survival and growth of first-genera-
tion brook trout during 71 weeks of exposure or on their progeny exposed for
90 days to Aroclor® 1254 concentrations of 0.01-0.94 yg/1. (P = 0.05).
Polychlorinated biphenyl concentrations in fillets and whole bodies
(entire fish minus one fillet above the lateral line and gonads) of the brook
trout reached an apparent steady state by the first sampling period after 14
weeks of exposure.
Exposure of brook trout to 0.01-0.94 yg/1. AroclorR 1254 resulted in mean
PCB residues from less than detectable (<0.2) to 2 yg/g in fillets and from
0.5 to 9 yg/g in whole bodies. The differences in PCB concentration between
fillet and whole body samples at a given water concentration were directly
related to their fat content.
Linear regression analyses showed the PCB concentration in the tissue to
be directly proportional to the concentration in the water. Concentration
factors in whole bodies of brook trout of 10,000-42,000 times the water con-
centration agree with the concentration factors of 20,000-70,000 for other
species of fish observed by several other investigators.
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SECTION 3
RECOMMENDATIONS
It is recommended that further research be conducted at higher PCS
concentrations to determine if increased residues in parental fish cause
effects on their offspring.
It is recommended that additional studies be conducted to determine
factors that might affect PCB residue levels in different species of fish.
Concentration factors observed in this study with brook trout ranged from
10,000 to 42,000, whereas those in fathead minnows were around 200,000
(Nebeker et_ _al. , 1974). The roles of such factors as feeding habits, water
temperatures, and water chemistry and possibly other behavioral differences
deserve attention.
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SECTION 4
MATERIALS AND METHODS
BIOASSAY
The long-term exposure to assess the effect of Aroclorw 1254 on survival,
growth, and reproduction of brook trout was conducted according to the recom-
mended procedures of the Environmental Research Laboratory-Duluth [APPENDIX],
except as noted below.
Lake Superior water passed through an ultraviolet light sterilizer was
used throughout the study. Mean water quality characteristics (± standard
deviation) in the exposure tanks were: acidity, 6.0±2.0 mg/1.; alkalinity,
43.1±1.4 mg/1.; total hardness, 45.8±1.8 mg/1. (as CaCC>3); dissolved oxygen,
78±14 percent saturation; pH, 7.2 (mode). Aroclor© 1254 had no measurable
effect on any of these parameters.
A proportional diluter (Mount and Brungs, 1967) delivered 4 1./cycle of
each of five Aroclor® 1254 concentrations and a lake-water control. The flow
rate was maintained at approximately 100 1. of each concentration every hour.
A flow splitter (Benoit and Puglisi, 1973) was used to divide the flow be-
tween duplicate spawning tanks, A and B, and later among duplicate spawning
tanks and duplicate incubation-growth tanks. Because the solubility of
Aroclor® 1254 in water is low, acetone was used as a carrier in the stock
solution. Acetone was also added to the control water so that it received
the same concentration (0.004 ml/1.) as the high PCB concentration. An
injector, designed to hold two 50-ml. glass syringes, delivered the PCB stock
solution and acetone simultaneously to appropriate diluter chambers at each
cycle.
Young-of-the-year brook trout obtained from Cedar Bend Hatchery in
Scandia, Minnesota, were acclimated to the water supply and beginning test
temperature for 2 months. Forty 10-month-old fish were then distributed by
stratified random assignment to each duplicate spawning tank. In addition,
a random sample of 40 fish was taken, and the fish were weighed, measured,
and stored at -20° C for later determination of background PCB tissue
residues. Mean weight and total length of these trout were 3.93 g and 7.3 cm,
respectively.
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The fish were fed PR-6 trout food for 11 months until a formulation
change necessitated changing to a gelatin-based purified diet (Castell at al.,
1972) to minimize pesticide residues that might complicate interpretation of
the PCB experiment. The progeny were fed EWOS Salmon Starter$.
Total lengths and weights were recorded as the fish were sampled (see
below). Analysis of variance of the logarithmic transformation of body weight
was used to detect treatment difference. Survival data for each sampling
period were subjected to analysis of variance and Dunnett's test when required.
During spawning two substrates (Benoit, 1974) were provided in each
tank. Because viability was low in many spawnings at all exposure levels,
eggs in addition to those recommended in the bioassay procedure [APPENDIX]
were incubated in hatchability cups to provide sufficient larvae for obser-
vation of possible toxicant effects. Analyses of variance of eggs per female,
percent viability, and percent hatchability (arc sin ^""percentage transforma-
tion used for viability and hatchability data) were made.
Ninety-day survival and growth studies of the offspring were also
conducted [APPENDIX]. Data were subjected to analysis of variance and Stu-
dent's t-test (Steel and Torrie, 1960).
Water concentrations of PCB in the test tanks were measured on 5-day
composite water samples by gas chromatography (Table 1). Each day the PCB's
were extracted from 1-1. water samples onto polyurethane foam plugs (Gesser
_e_t a.l_., 1971). The PCB residues were composited for 1 week and were then
extracted from the foam plugs with aliquots of redistilled acetone and
hexane. Before gas chromatographic measurement, a Florisil column clean-up
was used to reduce background contaminants that co-extracted with the PCB's.
Quantitation was based upon the height of the major AroclorE'1254 peak com-
pared to the same peak of the standard.
RESIDUE SAMPLING AND ANALYSIS
To determine the uptake of PCB's from the water, fish were sampled after
14, 27, 36, 41.5, 48, 55.5, 60, and 71 weeks of exposure. Five fish were
randomly selected from each tank during the first six samplings. The
seventh sampling, at 60 weeks, consisted of the fish discarded after thinning
each tank to two males and four females in preparation for spawning [APPENDIX],
After 71 weeks of exposure, 2 weeks after the last spawning, the remaining
brook trout were sampled. A fillet from the left side of the body above the
"'"Manufactured by Glencoe Mills, Glencoe, Minn. 55336.
A product of EWOS of Sweden, sold in the United States by Astra Pharmaceuti-
cal Co., Worcester, Mass.
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TABLE 1. AROCLOIT I2$k CONCENTRATIONS (pg/1.) IN DUPLICATE BROOK
TROUT ADULT AND ALEVIN-JUVENILE TANKS MEASURED BY GAS
CHROMATOGRAPHY (CORRECTED FOR RECOVERY)
Nominal
concentration
A
Control
B
A
0.012
B
A
0.036
B
A
0.11
B
A
0.33
B
A
1.0
B
Adult tanks
N
35
35
37
37
39
37
36
36
39
32
39
33
Mean
0.00 :
0.00
0.01
0.01
0.03
0.03
0.08
0.08
0.2k
0.25
1.01
0.86
Standard
deviation
1 0.01
0.01
0.01
0.02
0.01
0.02
0.03
0.03
0.0k
0.05
0.23
0.29
Alevin- juvenile tanks
N
6
6
7
8
8
8
9
8
7
8
9
9
Mean
0.00 H
0.00
0.01
0.01
0.03
0.02
0.07
0.08
0.28
0.23
1.23
1.07
Standard
deviation
H 0.00
0.00
0.00
0.01
0.02
0.01
0.03
0.03
0.11
0.06
0.36
0.27
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lateral line, the gonads, and the remainder of the body were frozen for
residue analysis. Samples from one duplicate at the three highest PCB con-
centrations (0.08, 0.24, and 0.94 yg/1.) at 48 weeks and all samples at 55.5
weeks were individually wrapped in acetone-rinsed aluminum foil for separate
residue analysis to obtain information on biological variability in PCB
accumulation. For each of the other samplings, fish were pooled to make one
composite of each sample type from each tank. Composite samples of newly
spawned eggs from each tank were also frozen for PCB analysis. A sufficient
number of eggs (75-125) were composited to obtain a sample of approximately
5 g.
The frozen fish tissues and eggs were packed in dry ice and shipped to
the Analytical-Biochemical Laboratories in Columbia, Missouri, for analysis.
There the samples were homogenized while still frozen, and approximately 10-g
aliquots were mixed with 30 g of anhydrous sodium sulfate. The mixture was
placed in a chromatographic column and washed with approximately 250 ml of
15% ether and hexane. The fat content was determined gravimetrically on an
aliquot of the extract. The remaining extract was cleaned-up by placing it
on a 20-g Florisil column and diluting it with 150 ml of 15% ethyl ether in
hexane. The eluant was concentrated and was then injected into a gas
chromatograph.
The PCB's were quantitated by summing the peak heights of peak numbers
70, 84, 125, 145, and 174, relative to DDE, and comparing the sum to the
Arocloi® 1254 standard. Concentrations were expressed in micrograms of PCB
per gram of tissue, wet weight.
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SECTION 5
RESULTS
BIOASSAY
No significant difference (P = 0.05) in survival was noted at any
Aroclor® 1254 concentration compared to the control during the first 60
weeks of exposure (Table 2). The increased mortality during the remaining
11 weeks of exposure was not believed to be due to the PCB. During this
latter period a disease of undetermined etiology occurred producing lesions
and other signs suggestive of furunculosis (Bullock et al., 1971). All but
one of the fish that died within this period displayed these pathological
signs. The fish in every tank were treated by incorporation of tetracycline
hydrochloride (at approximately 7.5 g/100 kg body weight per day) into the
food for 14 consecutive days.
No statistically significant difference in growth between any PCB
concentration and the control was observed during any period of the exposure
(P = 0.05). Growth data from the final sampling period only (after 71 weeks)
are presented in Table 2.
Spawning occurred at all PCB concentrations and in the controls after
approximately 14 months of exposure. No significant difference in total
spawning or eggs per female could be detected between the PCB treatments
and the controls (P = 0.05) (Table 2). Viability of eggs was highly variable
at all PCB concentrations and in the controls. Eggs from many spawnings were
entirely nonviable. The reason for this poor viability is not known; how-
ever, since it occurred in controls as well as in the toxicant-containing
tanks, it could not be attributed to the Aroclor^1254. Hatchability of eggs
incubated reflected the erratic viability (Table 3). However, mean hatcha-
bilities of viable eggs were 93-100% for the control and all ArocloiW 1254
concentrations tested. [Viability is defined as the formation of a neural
keel after 11-12 days at 9° C (APPENDIX).]
No significant differences (P = 0.05) in survival and growth between
controls and any PCB concentration tested were noted during any part of the
90-day alevin-juvenile exposure (Table 3).
PCB TISSUE RESIDUES
Concentrations of PCB's in the unexposed brook trout sampled at the
beginning of the experiment were less than detectable (<0.2 yg/g wet weight).
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TABLE 2. SURVIVAL, GROWTH, AND REPRODUCTION OF BROOK TROUT EXPOSED
TO AROCLOR^ 1254 FOR 16 MONTHS
Mean
measured PCB
concentration
(us/1.)
AC
Control
0 . 00 Bc
A
0.01
B
A
0.03
B
A
0.08
B
A
0.21*
B
A
0.9lt
B
Cumulative
mortality
Weeks
60 71
1 1
0 0
1 5
0 1
0 2
0 0
1 2
1 2
1 2
1 It
0 1
0 1
Length (cm) and weight (g)
at termination (71 weeks)
Males
cm g
29.9dp 308.1*
(2.2)6 f(60.l)
ltf
30.1 29!*. 7
(3.6) (92.1)
2
27.1 211.lt
(3.1) (6k. 2)
It
30.3 29>t.O
(0.1) (9.2)
2
27.3 2lt9.0
(2. it) (>*9.5)
3
27.lt 231.3
(2.0) (36.6)
1*
Females
cm g
26. 5d 175.7
(1.8) (21.9)
8
27.9 ao.2
(2.2) (It3. 7)
7
28.lt 237.8
(1.0) (32.0)
6
26.8 185.1
(2.0) (51*. 9)
8
26.5 187.3
(1.7) (37.5)
5
26.3 179.1
(2.3) (35.1+)
6
Sex ratio
in each tank
male/female
2/lt
2/lt
1/2
1/3
1/3
3/2
lA
1A
2/lt
1/3
2/lt
2/2
Total
spawnings
10
15
7
15
lit
17
9
12
13
6
9
5
Mean eggs/
female
6»tl
720
7lt9
9U.
870
1,554
255
579
630
615
1*89
852
Mean viable
eggs/female
1*1*9
160
137
62
32l*
363
10
121*
126
500
231
1*76
Viability
(%)
70
22
18
7
37
23
1*
21
20
81
1*7
56
Represents fish present during spawning period that contributed to spawning data.
A spawning is defined as any egg deposition of 50 or more eggs.
CA and B are duplicate spawning tanks.
Data from duplicate tanks combined.
eStandard deviation in parentheses.
Number of fish.
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TABLE 3. HATCHABILITY, SURVIVAL, AND GROWTH OF BROOK TROUT ALEVIN-JUVENILES
FROM PARENTS EXPOSED TO AROCLORR' 125^ FOR lU MONTHS BEFORE SPAWNING
Mean
measured PCB
concentration
(uS/1.)
Aa
Control
0.00 Ba
A
0.01
B
A
0.03
B
A
0.08
B
A
0.2k
B
A
0.91*
B
Mean
percent
hatch
57 [9]b
5 [15]
1U [7]
5 [10]
19 [12]
13 [15]
8 [9]
8 [11]
15 [10]
66 [7]
>*5 [8]
75 [5]
Ale vin- juvenile
survival (%)
Hatch-
30 days
100 (175 )C
d
100 (1*9)
100 (50)
96 (71)
99 (126)
100 (29)
100 (50)
99 (71*)
100 (100)
100 (156)
98 (ll*8)
30-90 days
91* (50)C
68e(25)
100 (1*6)
88 (50)
81* (1*9)
81* (50)
88 (25)
78 (50)
65 (U6)
60 (50)
81* (50)
91* (1*7)
Ale vin- juvenile
90-day growth
Mean
length (mm)
hi
3h
h3
U2
39
3U
38
1*U
3U
35
33
33
Mean
veight (g)
0.69
0.1*0
0.87
0.72
0.68
0.1*1*
0.57
0.99
0.1*7
0.51*
0.1*1
0.1*2
^Duplicate chambers.
[ ] Number of hatch cups incubated.
( ) Initial number of alevins.
All fish killed before 30 days by temperature increase in this tank.
eOne group of 25 fry transferred from duplicate after the resident fish died.
10
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The results of the PCB residue analyses of fillet and "whole body" (entire
fish roiRus one fillet and the gonads) after various lengths of exposure to
Aroclor® 1254 are presented in Tables 4 and 5, respectively. Because no
biological effects were observed on embryos and alevins, no gonads were
analyzed. Both tables suggest that the brook trout had reached an apparent
steady state by the first sampling after 14 weeks of exposure. Linear
regression analysis for each sampling period showed the PCB concentration
in the tissue to be directly proportional to the PCB concentration in the
water. (Coefficients of correlation ranged from 0.970 to 0.999, all
statistically significant at P = 0.05 with only 60- and 71-week R-values not
significant also at P = 0.01.) The PCB concentrations in the fillet were
below detectability (<0.2 yg/g) at water concentrations of 0.03 yg/1. and
less. Exposure to 0.94 yg/1., the highest water concentrations tested,
resulted in PCB residues of approximately 2 yg/g in the fillet and 9 yg/g in
the whole body.
Limited information on PCB residues in newly spawned eggs was obtained
because of the availability of only a small number of samples and variability
within these samples. Control eggs and those from 0.01 yg/1. had less than
detectable amounts (<0.1 ug/g, N = 2 and 1, respectively). Eggs from brook
trout exposed to 0.03 and 0.08 yg/1. contained PCB's at the lower detection
limit (0.1 yg/g, N = 3 and 2, respectively). Mean residues of 1.8 and 1.7
yg/g were detected in eggs from the 0.24 and 0.94 yg/1. concentrations
(standard deviations = 1.3 and 0.1, N = 3 and 2, respectively). Percentages
of fat ranged from 0.4 to 7.3 with a mean of 1.8% (N = 13). No relationship
was evident between fat content and magnitude of PCB residue in the eggs.
11
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TABLE 4. CONCENTRATION OF PCB (ug/g wet weight) AND FAT CONTENT (%) OF FILLETS FROM
BROOK TROUT EXPOSED TO AROCLOR^ 1254 FOR VARIOUS TIME PERIODS. VALUES REPRESENT
AN ANALYSIS ON A COMPOSITE SAMPLE OF FIVE FISH UNLESS OTHERWISE NOTED.
PCB
concentration
A
Central
0 . OC B
A
C.C1
B
A
O.C3
B
A
O.C8
B
A
0.2lt
B
A
B
Length of exposure
lit weeks 27 weeks 36 weeks ltl.5 weeks I|8 weeks 55.5 weeks 60 weeks 71 weeks
PCB Percent
Pg/g fat
0.2 0.9
0.2 L.k
<0.2 1.5
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TABLE 5. CONCENTRATION OF PCB (ug/g wet weight) AND FAT CONTENT (%) OF WHOLE BODY
(ENTIRE FISH MINUS ONE FILLET AND GONADS) OF BROOK TROUT EXPOSED TO AROCLOK^
1254 FOR VARIOUS TIME PERIODS. VALUES REPRESENT AN .ANALYSIS OF A
COMPOSITE SAMPLE OF FIVE FISH UNLESS OTHERWISE NOTED.
PCS
concentration
(pg/i.)
A
Control
0.00 B
A
0.01
B
A
0.03
B
A
0.08
B
A
0.2k
B
A
0.91*
B
Length of exposure
lit veeks
PCS Percent
Mg/g fat
<0.2 5.7
O.lt 6.8
0.6 6.9
1.3 7.5
2.9 7.7
6.2 5.3
27 weeks
PCB Percent
pg/K fat
O.li 5."l
0.5 6.8
0.6 5.1*
1.3 7.9
2.0 It.l
8.9 7."t
36 weeks
r PCB Percent
UR/g fat
O.I. 8.6
0.6 14.1
0.6 9.3
0.8 5.1
2.8 8.U
8.0 8.2
1.1.5 weeks
PCB Percent
Ug/R fat
0.3 6.9
O.I. 7.0
O.I. 6.3
0.9 6.1
3.3 10.1.
7-9 7.8
1.8 weeks
PCB Percent
VK/K fat
0.5 P.O
0.6 6.1.
0.8 8.0
l.la 6.0
(10)c (1.2)
1.3 7.9
2.5 5.6
3.5a 7.2
(20) (20)
10.7 8.1.
10. 8a 6.0
(21) (11)
55.5 weeks
PCB Percent
pg/g fat
<0.2a 7.0
0.5a 5.2
("40) (1.1)
0.8b 6.0
(16) (1.8)
1.7a 7.2
(30) (15)
It. 9s* 6.3
(20) (35)
3.7 6.5
(28) (30)
10. 9a 6.6
(26) (18)
12. 3a 6.3
(1*2) (31)
60 weeks
PCB Percent
Pg/g fat
-
-
0.8d It. 6
n=l.
2.1 3.6
SV 5.6
n=3
7.2d 3.2
,n=lt
6.8d 6.1.
n=lt
71 weeks
PCB Percent
pg/g fat
-
-
-
0.9 2.3
lf.lt 5.1.
3.5d 2.1
n=3
9.1. 2. It
7.2 2.1.
aMean of individual analyses of five fish.
bMean of individual analyses of four fish; one omitted because of suspected mix-up in chromatograms.
C( ) = relative standard deviation. The RSD is the standard deviation expressed as a percentage of the mean.
Analysis of composite sample of N fish.
-------
SECTION 6
DISCUSSION
In this experiment no adverse effects were noted on survival and growth
of brook trout or their progeny chronically exposed to 0.01-0.94 yg/1.
Aroclor© 1254. Jensen j^t al. (1970) observed mortalities of 16-100% in
field-collected Atlantic salmon (Salmo salar) embryos containing PCB residues
of 0.4-1.9 yg/g wet weight (7.7-34 yg/g of fat). Stalling and Mayer (1972)
subjected the data of Jensen et al. (1970) to a regression analysis and
demonstrated a statistically significant (P = 0.01) direct correlation between
PCB residue and mortality of the embryos. Though some PCB egg residues in
this study (1.8 and 1.7 yg/g from 0.24 and 0.94 yg/1. water concentrations,
respectively) were comparable to those reported by Jensen _et_ aJU (1970), this
experiment provided no evidence that PCB's transferred from parents cause any
adverse effects on brook trout embryos. Also, in this experiment no unusual
mortality occurred at yolk-sac absorption, which took place about 1 month
post-hatch, as was observed in other salmonid alevins from DDT-fed parents
(Burdick _et al., 1964, 1972).
Concentration factors of 10,000-42,000 times the water exposure in whole
bodies of brook trout were comparable to values of 20,000-71,000 for Aroclor?)
1254 reported for bluegills (Lepomis macrochirus) and three species of
estuarine fish by Stalling and Huckins (unpublished data) and Hansen et al.
(1971, 1973). These residue concentration factors are considerably lower
than values of around 200,000 reported by Nebeker et^ al_. (1974) for fathead
minnows (Pimephales promelas)- The reasons for this discrepancy are not
known; however, factors such as water temperatures and differences in fat
content or feeding habits of the species may be partly responsible.
This study has shown, as others have (Reinert, 1970; Hamelink et al.,
1971; Reinert and Bergman, 1974), that the fat content in the tissue plays
an important role in determining the concentration of an organochlorine
compound in various tissues from the same water concentration. The PCB
concentrations in the brook trout fillets (Table 4) and whole bodies (Table 5)
ranged from 2,000 to 3,000 and from 10,000 to 42,000, respectively, times
higher than those in the water. Plotting the residue data as micrograms
of PCB per gram of fat (Figure 1) reduces the differences in concentrations
of PCB's between whole body and the corresponding fillet shown in Tables 4
and 5. As with the wet weight PCB concentrations, concentrations of PCB in
fat were directly proportional to the water concentration (P = 0.01).
The apparent increase in the PCB content of the brook trout between 60
and 71 weeks (Figure 1) can be explained by a decrease in the fat content and
14
-------
400 -r
350 4-
©
(D
©
©
©
oo
CO
300 t
250 4-
200 J.
150 +
100 4-
50
Fillet, 0.94 pg/l.t
Whole body*, 0.94 pg/1.
Whole body, 0.24 ug/1.
Fillet, 0.24 ug/1-
Whole body, 0.08 Mg/1.
Fillet, 0.08 pg/1.
tWater exposure concentration.
*Entire fish minus fillet above lateral
line on left side and gorge's.
10
20
30
60
70
80
40 50
Weeks of Exposure
Figure 1. Concentration of PCB in fat brook trout tissues
after various periods of exposure.
90
100
-------
a further concentration of the PCB residues in the remaining fat. The per-
centage fat values at 71 weeks of both fillets and whole bodies are
approximately one-half to one-third their previous values (Tables 4 and 5).
During this period of exposure when the fish were spawning, increased
activity, physiological stress, and a decreased feeding rate resulted in
utilization of body-fat reserves. The hazards of otherwise sublethal PCB
residues that might result from periods of prolonged mobilization to the
point of complete depletion of fat reserves are not known. With another
organochlorine compound, DDT, Grant and Schoettger (1972) reported high
mortality of DDT-exposed rainbow trout (Salmo gairdneri) during prolonged
exercise and fasting.
Bioconcentration of PCB's is important, not only because of the potential
hazard of the residue to the fish, but also because of its economic and human-
health implications. The U.S. Food and Drug Administration has set an interim
tolerance level for PCB's in food of 5 yg/g, above which food is banned from
the interstate market (U.S. Food and Drug Administration, 1974). In this
experiment, PCB water concentrations of 0.24 and 0.94 yg/1. resulted in
residues in whole bodies of brook trout approaching or exceeding the 5 yg/g
action level. Because of the approximately five-fold lower fat content,
fillets at none of the water concentrations tested exceeded the U.S. Food
and Drug Administration's interim tolerance level. Since the U.S. Food and
Drug Administration defines edible portion as an eviscerated, beheaded fish
(U.S. Food and Drug Administration, 1969), our values for neither whole body
nor fillet exactly represent their defined edible portion PCB concentrations.
16
-------
REFERENCES
Allison, D. T., B. J. Kallman, 0. B. Cope, and C. Van Valin. 1964. Some
chronic effects of DDT on cutthroat trout. U.S. Dept. Int., Bureau of Sport
Fisheries and Wildlife Research Rept. 64. 30 p.
Benoit, D. A. 1974. Artificial laboratory spawning substrate for brook
trout (Salvelinus fontinalis Mitchill). Trans. Am. Fish. Soc. 103:144-145.
Benoit, D. A., and F. A. Puglisi. 1973. A simplified flow-splitting chamber
and siphon for proportional diluters. Water Res. 7:1915-1916.
Bullock, G. L., D. A. Conroy, and S. F. Snieszko. 1971. Diseases of fishes:
Book 2A. Bacterial diseases of fish. T. F. H. Publications, Inc., N.J.
151 p.
Burdick, G. E., H. J. Dean, E. J. Harris, J. Skea, R. Karcher, and C. Frisa.
1972. Effect of rate and duration of feeding DDT on the reproduction of
salmonid fishes reared and held under controlled conditions. N.Y. Fish &
Game J. 19:97-115.
Burdick, G. E., E. J. Harris, H. J. Dean, T. M. Walker, J. Skea, and D. Colby.
1964. The accumulation of DDT in lake trout and the effect on reproduction.
Trans. Am. Fish. Soc. 93:127-136.
Castell, J. D., R. 0. Sinnhuber, J. H. Wales, and D. J. Lee. 1972. Essential
fatty acids in the diet of rainbow trout (Salmo gairdneri): Growth, feed
conversion, and some gross deficiency symptoms. J. Nutr. 102:77-86.
Duke, T. W., J. I. Lowe, and A. J. Wilson, Jr. 1970. A polj^chlorinated
biphenyl (Aroclor 1254) in the water, sediment, and biota of Escambia Bay,
Florida. Bull. Environ. Contain. Toxicol. 5:171-180.
Gesser, H. D., A. Chow, F. C. Davis, J. F. Uthe, and J. Reinke. 1971. The
extraction and recovery of polychlorinated biphenyls (PCB) using porous
polyurethane foam. Anal. Lett. 4:883-886.
Giam, C. S., M. K. Wong, A. R. Hanks, W. M. Sackett, and R. L. Richardson.
1973. Chlorinated hydrocarbons in plankton from the Gulf of Mexico and
Northern Caribbean. Bull. Environ. Contam. Toxicol. 9:376-382.
17
-------
Grant, B. F., and R. A. Schoettger. 1972. The impact of organochlorine
contaminants on physiologic functions in fish. Proc. Tech. Sess. 18th Ann.
Meeting, Inst. Environ. Sci. pp. 245-250.
Hamelink, J. L., R. C. Waybrant, and R. C. Ball. 1971. A proposal: Exchange
equilibria control the degree chlorinated hydrocarbons are biologically
magnified in lentic environments. Trans. Am. Fish. Soc. 100:207-214.
Hansen, D. J., P- R. Parrish, J. I. Lowe, A. J. Wilson, Jr., and P. D. Wilson.
1971. Chronic toxicity, uptake, and retention of AroclorR 1254 in two
estuarine fishes. Bull. Environ. Contam. Toxicol. 6:113-119.
Hansen, D. J., S. C. Schimmel, and J. Forester. 1973. Aroclor® 1254 in eggs
of sheepshead minnows: Effect on fertilization success and survival of
embryos and fry. Proc. 27th. Ann. Conf. Southeastern Assoc. Game Fish Comm.,
420-423.
Jensen, S. 1966. Report on a new chemical hazard. New Scientist and Sci.
J. (Great Britain) 32:612.
Jensen, S., N. Johansson, and M. Olsson. 1970. PCB-indications of effects
on salmon. PCB Conference, Stockholm, September 29, 1970. Swedish Salmon
Research Institute Report LF1 MEDD 7/1970.
Koeman, J. H., M. C. Ten Noever De Braw, and R. H. DeVos. 1969. Chlorinated
biphenyls in fish, mussels, and birds from the River Rhine and the Netherlands
coastal area. Nature (London) 221:1126-1128.
Macek, K. J. 1968. Reproduction in brook trout (Salvelinus fontinalis) fed
sublethal concentrations of DDT. J. Fish. Res. Board Can. 25:1787-1796.
Mount, D. I., and W. A. Brungs. 1967. A simplified dosing apparatus for
fish toxicology studies. Water Res. 1:21-29.
Nebeker, A. V., F. A. Puglisi, and D. L. DeFoe. 1974. Effect of polychlori-
nated biphenyl compounds on survival and reproduction of the fathead minnow
and flagfish. Trans. Am. Fish. Soc. 103:562-568.
Reinert, R. E. 1970. Pesticide concentrations in Great Lakes fishes.
Pesticides Monitoring J. 3:233-240.
Reinert, R. E., and H. L. Bergman. 1974. Residues of DDT in lake trout
(Salvelinus namacush) and coho salmon (Oncorhynchus kisutch) from the Great
Lakes. J. Fish. Res. Board Can. 31:191-199.
Risebrough, R. W., and V. Brodine. 1970. More letters in the wind.
Environment 12:16-27.
Schimmel, S. C., D. J. Hansen, and J. Forester. 1974. Effects of Aroclor®
1254 on laboratory-reared embryos and fry of sheepshead minnows (Cyrinodon
variegatus). Trans. Am. Fish. Soc. 103:582-586.
18
-------
Stalling, D. L., and J. N. Huckins. Unpublished from the U.S. Bureau of Sport
Fisheries and Wildlife, Fish Pesticide Laboratory, Columbia, MO. 65201.
Stalling, D. L., and F. L. Mayer, Jr. 1972. Toxicities of PCB's to fish
and environmental residues. Environ. Health Persp. April: 159-164.
Steel, R. G. D., and J. H. Torrie. 1960. Principles and procedures of
statistics with special reference to biological sciences. McGraw-Hill, New
York. 481 p.
U.S. Food and Drug Administration. 1969. Pesticide analytical manual, Vol.
1, Section 141.12c, July 1, 1969.
U.S. Food and Drug Administration. 1974. Interim tolerance for PCB in fish.
Federal Register 39(6): part 2.
Veith, G. D. 1972. Recent fluctuations of chlorobiphenyls (PCB's) in the
Green Bay, Wisconsin, region. Environ. Health Persp. April: 51-54.
19
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APPENDIX
RECOMMENDED BIOASSAY PROCEDURE FOR
BROOK TROUT SALVELINUS FONTINALES (MITCHILL) PARTIAL CHRONIC TESTS
20
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RECOMMENDED BIOASSAY PROCEDURES
Preface
Recommended Bioassay Procedures are established by Che approval
of both the Committee on Aquatic Bioassays and the Director of the
National Water Quality Laboratory. The main reasons for establishing
them are: (1) to permit direct comparison of test results,
(2) to encourage the use of the best procedures available, and
(3) to encourage uniformity. These procedures should be used by
National Water Quality Laboratory personnel whenever possible,
unless there is a good reason for using some other procedure.
Recommended Bioassay Procedures consider the basic elements that
are believed to be important in obtaining reliable and reproducible
results in laboratory bloassays. An attempt has been made to adopt
the best acceptable procedures based on current evidence and opinion,
although it is recognized that alternative procedures may be adequate.
Improvements in the procedures are being considered and tested, and
revisions will be made when necessary. Comments and suggestions are
encouraged.
Director, National Water Quality Lab (NWQL)
Committee on Aquatic Bioassays, NWQL
21
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Recommended Bioassay Procedure for
Brook Trout Salvelinus fontinales (Mitchill) Partial Chronic Tests
April, 1971
(Revised January, 1972)
A. Physical system
1.. Diluter: Proportional diluters (Mount and Brungs, 19&7) should
be employed for all long-term exposures. Check the operation
of the diluter daily, either directly or through the measure-
ment of toxicant concentrations. A minimum of five toxicant concen-
trations and one control should be used for each test with a dilution
factor of not less than 0.30. An automatically triggered emergency
aeration and alarm system must be installed to alert staff in case of
diluter, temperature control or water supply failure.
2. Toxicant mixing: A container to promote mixing of toxicant
bearing and w-cell water should be used between diluter and
tanks for each concentration. Separate delivery tubes should
run from this container to each duplicate tank. Check to see
that the same amount of water goes to duplicate tanks and
that the toxicant concentration is the same in both.
3. Tank: Each duplicate spawning tank (preferably stainless steel)
should measure 1.3 X 3 X 1 ft. wide with a water depth of 1 foot
and alevin-juvenile growth chambers (glass or stainless steel with
glass bottom) 7 X 15 X 5 in. wide with a water depth of 5 inches.
Growth chambers can be supplied test water by either separate
delivery tubes from the mixing cells described in Step 2 above
or from test water delivered from the mixing cell to each
duplicate spawning tank. In the second choice, test water must
always flow through growth chambers before entering the spawning
tank. Each growth chamber should be designed so that the test
water can be drained down to 1 inch and the chamber transferred
over a fluorescent light box for photographing the fish (see
B.10).
U. Flow rate: Flow rates for each duplicate spawning tank and growth
chamber should be 6-10 tank volumes/2k hr.
5. Aeration: Brook trout tanks and growth chambers must be aerated
with oil free air unless there are no flow limitations and.60%
of saturation can be maintained. Total dissolved oxygen levels
should never be allowed to drop below 60% of saturation.
6. Cleaning: All tanks and chambers must be siphoned daily and
brushed at least once per week. When spawning commences,
gravel baskets must be removed and cleaned daily.
22
-------
7- Spawning substrates: Use two spawning substrates per duplicate
made of plastic or stainless steel which measure at least
6 X 10 X 12 in. with 2 inches of .25 to .50 inch stream gravel
covering the bottom and 20 mesh stainless steel or nylon screen
attached to the ends for circulation of water.
8. Egg cup: Egg incubation cups are made from k-oz. 2-inch OD
round glass jars with the bottoms cut off and replaced with
stainless steel or nylon screen (Uo meshes per inch). Cups
are oscillated in the test water by means of a rocker arm
apparatus driven by a 2 r.p.m. electric motor (Mount, 1968).
9- Light: The lights used should simulate sunlight as nearly
as possible. A combination of Duro-Test (Optima FS)1'2 and wide
spectrum Gro-lux-^ fluorescent tubes has proved satisfactory at the
NVQL.
10. Photoperiod: The photoperiods to be used (Appendix A) simulate
the dawn to dusk times of Evansville, Indiana. Evansville dates
must correspond to actual dates in order to avoid putting natural
reproductive cycles out of phase. Adjustments in photoperiod
are to be made on the first and fifteenth of every Evansville
test month. The table is arranged so that adjustments need be
made only in the dusk times. The dawn and dusk times listed in
the table (Evansville test time) need not correspond to the
actual test times where the test is being conducted. To illustrate
this point, a test started on March first would require the use
of the photoperiod for Evansville test date March first, and the
lights could go on any time on that day just so long as they
remained on for twelve hours and fifteen minutes. Fifteen days
later "bhe photoperiod would be changed to thirteen hours.
Gradual changes in light intensity at dawn and dusk (Drummond
and Dawson, 1970), may be included within the photoperiods shown,
and should not last for more than 1/2 hour from full on to full
off and vice versa.
11. Temperature: Utilize the attached temperature regime (see Appendix
B). Temperatures should not deviate instantaneously from the
specified test temperature by more than 2s C and should not remain
outside the specified temperature ±1° C for more than U8 hours at
a time.
12. Disturbance: Spawning tanks and growth chambers must be covered
with a screen to confine the fish and concealed in such a way
that the fish will not be disturbed by persons continually walking
Mention of trade names does not constitute endorsement.
2 Duro-Test, Inc., Hammond, Ind.
3 Sylvania, Inc., New York, N. Y.
23
-------
past the system. Tanks and chambers must also be shielded
from extraneous light which can affect the intended photoperiod
or damage light sensitive eggs and alevins.
13. Construction materials: Construction materials which contact the
diluent water should not contain leachable substances and
should not sorb significant amounts cf substances from the
water- Stainless steel is probably the preferred construction material.
Glass absorbs some trace organics significantly. Rubber should not
be used. Plastic containing fillers, additives, stabilizers,
plasticizers, etc., should not be used. Teflon, nylon, and
their equivalents should not contain leachable materials and
should not sorb significant amounts of most substances. Un-
plasticized polyethylene and polypropylene should not contain
leachable substances, but may sorb very significant amounts of
trace organic compounds.
lU. Water: The water used should be from a well or spring if at all
possible, or alternatively from a surface water source. Only
as a last resort should water fron a chlorinated municipal water
supply be used. If it is thought that the water supply could be
conceivably contaminated with fish pathogens, the water should be
passed through an ultraviolet or similar sterilizer immediately before
it enters the test system.
B. Biological system
1. Test animals: Yearling fish should be collected no later than March 1
and acclimated in the laboratory to test temperature and water quality
for at least one month before the test is initiated. Suitability of
fish for testing should be Judged on the basis of acceptance of food,
apparent lack of diseases, and 2% or less mortality during acclimation
with no mortality two weeks prior to test. Set aside enough fish to
supply an adequate number for short-term bioassay exposures used in
determining application factors.
2. Beginning test: Begin exposure no later than April 1 by distributing 12
acclimated yearling brooff trout per duplicate using a stratified random
assignment (see D.3). This allows about a four month exposure to the
toxicant before the onset of secondary or rapid growth phase of
the gonads.
Extra test animals may be added at the beginning so that fish can
be removed periodically for special examinations (see B.12),
or for residue analysis (see C.U).
3. Food: Use a good frozen trout food (e.g., Oregon Moist). Fish should
be fed the largest pellet they will take a minimum of two times
daily. The amount should be "based on a reliable hatchery feeding
schedule. Alevins and early Juveniles should be fed trout starter
a minimum of five times daily. Each batch of prepared food should be
checked for pesticides (including DDT, TDE, dieldrin, endrin, aldrin,
24
-------
BHC, chlordane, toxaphene, 2.U-D, and PCBs), and the kinds and amounts
should be reported to the project officer or recorded.
U. Disease: Handle disease outbreaks according to their nature,
with all tanks receiving the same treatment whether there seems
to be sick fish in all of them or not. The frequency of treatment should
be held to a minimum.
5. Measuring fish: Record mortalities daily, and measure fish
directly at initiation of test, after three months and at
thinning (see B.6) (total length and weight). Fish should not
be fed 2U hours before weighing and lightly anesthetized with
MS-222 to facilitate measuring (100 m£ MS-222/liter water).
6. Thinning: When secondary sexual characteristics are well
developed (approximately two weeks prior to expected spawning),
separate males, females and undeveloped fish in each duplicate
and randomly reduce sexually mature fish (see D.M to the desired
number of 2 males and U females, and discard undeveloped fish
after examination. Place two spawning substrates (described
earlier) in each duplicate. Record the number of mature, immature,
deformed and injured males and females in each tank and the number
from each category discarded. Measure total length and weight of all
fish in each category before any are discarded and note which ones
were discarded (see C.U).
7. Removing eggs; Remove eggs from the redd at a fixed time
each day (preferably after 1:00 p.m. Evansville time, so the
fish are not disturbed during the morning).
Egg incubation and viability: Impartially select 50 eggs from
the first eight spawnings of 50 eggs or more in each duplicate
and place them in an egg incubator cup for hatch. The remaining
eggs from the first eight spawnings (>50 eggs) and all subsequent
eggs from spawnings should be counted and placed in separate egg
incubator cups for determining viability (formation of neural keel
after 11-12 days at 9° C). The number of dead eggs from each spawn
removed from the nest should be recorded and discarded. Never place
more than 250 eggs in one egg incubator cup. All eggs incubated
for viability are discarded after 12 days. Discarded eggs can be
used for residue analysis and physiological measurements of toxicant
related effects.
Progeny transfer: Additional important information on hatchability
and alevin survival can be gained by transferring control eggs
immediately after spawning to concentrations where spawning is
reduced or absent, or to where an affect is seen on survival of
eggs or alevin, and by transferring eggs from these concentrations
to the control tanks. Two growth chambers for each duplicate
spawning tank should always be reserved for eggs produced in that
tank.
25
-------
10. Hatch and, alevin thinning: Remove dead eggs daily "rom the
hatchability cups described in Step 8 above. When hatching
commences, record the number hatched daily in each cup. Upon
completion of hatch in any cup, randomly (see D.4) select 25 alevins
from that cup. Dead or deformed alevins must not be included
in the random selectio'n but should be counted as being dead or
deformed upon hatch. Measure total lengths of the 25 selected
and discarded alevins. Total lengths are measured by the
photographic method used by McKim and Benoit (1971). The fish
are transferred to a glass box containing 1 inch of test water.
They should be moved to, and from this box in a water filled
container, rather than by netting them. The glass box is
placed on a translucent millimeter grid over a fluorescent light
box which provides background illumination. Photos are then
taken of the fish over the millimeter grid and are enlarged into
8 X 10 inch prints. The length of each fish is subsequnetly de-
termined by comparing it to the grid. Keep lengths of discarded
alevins separate from those which are kept. Place the 25 selected
alevins back into the incubator cup and preserve the discarded
ones for initial weights.
11. Alevin-juvenile exposure: Randomly (see D.4) select from the incubation
cups two groups of 25 alevins each per duplicate for 90-day
growth and survival exposures in the growth chambers. Hatching
from one spawn may be spread out over a 3 to 6 day period;
therefore, the median-hatch date should be used to establish
the 90-day growth and survival period for each of the two
groups of alevin. If it is determined that the median-hatch
dates for the five groups per duplicate will be more than three
weeks apart, then the two groups of 25 alevin must be selected
from those which are less than three weeks old. The remaining
groups in the duplicate which do not hatch during the three
week period are used only for hatchability results and then
photographed for lengths and preserved for initial weights. In
order to equalize the effects of the incubation cups on growth,
all groups selected for the 90-day exposure must remain in the
incubation cups three weeks before they are released into the
growth chambers. Each of the two groups selected per duplicate
must be kept separate during the 90-day period. Record
mortalities daily, along with total lengths 30 and 60 days
post-hatch and total length and weight at 90 days post-hatch.
Alevins and early juveniles should not be fed 24 hours before
weighing. Total lengths are measured by transferring the growth
chambers described earlier to a translucent millimeter grid
over a fluorescent light box for photographing as described in
Step 10 above. Survival and growth studies should be terminated
after three months. Terminated fish can be used for tissue
residue analysis and physiological measurements of toxicant
related effects.
26
-------
12. Parental termination: All parental fish should be terminated
when a three week period passes in which no spawning occurs
in any of the spawning tanks. Record mortality and weigh
and measure total length of parental fish, check sex and
condition of gonads (e.g., reabsorption, degree of maturation,
spent ovaries, etc.) (see C.4).
13. Special examinations; Fish and eggs obtained from the
test should be considered for physiological, biochemical, and
histological investigations which may indicate certain toxicant
related effects.
14. Necessary data; Data that must be reported for each tank of
a chronic test are:
a. Number and individual weights and total lengths of
normal, deformed, and injured mature and immature
males and females at initiation of test, three months
after test commences, at thinning and at the end of
test.
b. Mortality during the test.
c. Number of spawns and eggs. A mean incubation time
should be calculated using date of spawning and the
median hatch dates.
d. Hatchability.
e. Fry survival, growth and deformities.
C. Chemical system
1. Preparing a stock solution; If a toxicant cannot be introduced
into the test water as is, a stock solution should be prepared
by dissolving the toxicant in water or an organic solvent. Acetone
has been the most widely used solvent, but dimethylformanide (DMF)
and triethylene gly_col may be preferred in many cases. If none
of these solvents are acceptable, other water-miscible solvents
such as methanol, ethanol, isopropanol, acetonitrile, dimethy1-
acetamide (DMAC), 2-ethoxyethanol, glyme (dimethylether of
ethylene glycol, diglyme (dimethyl ether of diethylene glycol)
and propylene glycol should be considered. However, dimethyl
sulfoxide (DMSO) should not be used if at all possible because
of its biological properties.
Problems of rate of solubilization or solubility limit should be
solved by mechanical means if at all possible. Solvents, or as
a last resort, surfactants, can be used for this purpose, only
after they have been proven to be necessary in the actual test
27
-------
system. The suggested surfactant is p-tert-octylphenoxynonaethoxy-
ethanol (p-1, 1, 3, 3-tetramethylbutylphenoxynonaethoxyethanol,
OPEin) (Triton X-100, a product of the Rohm and Haas Company,
or equivalent).
The use of solvents, surfactants, or other additives should be
avoided whenever possible. If an additive is necessary, reagent
grade or better should be used. The amount of an additive used
should be kept to a minimum, but the calculated concentration
of a solvent to which any test organisms are exposed must never
exceed one one-thousandth of the 96-hr. TL50 for test species
under the test conditions and must never exceed one gram per liter
of water. The calculated concentration of surfactant or other
additive to which any test organisms are exposed must never
exceed one-twentieth of the concentration of the toxicant and
must never exceed one-tenth gram per liter of water. If any
additive is used, two sets of controls must be used, one exposed
to no additives and one exposed to the highest level of additives
to which any other organisms in the test are exposed.
2. Measurement of toxicant concentrat ion: As a minimum the con-
centration of toxicant must be measured in one tank at each
toxicant concentration every week for each set of duplicate tanks,
alternating tanks at each concentration from week to week. Water
samples should be taken about midway between the top and bottom
and the sides of the tank and should not include any surface scum
or material stirred up from the bottom or sides of the tank.
Equivolume daily grab samples can be composited for a week if it
has been shown that the results of the analysis are not affected
by storage of the sample.
Enough grouped grab samples should be analyzed periodically
throughout the test to determine whether or not the concentation
of toxicant is reasonably constant from day to day in one tank
and from one tank to its duplicate. If not, enough samples must
be analyzed weekly throughout the test to show the variability
of the toxicant concentration.
3. Measurement of_ other variables; Temperature must be recorded
continuously (see A.11).
Dissolved oxygen must be measured in the tanks daily at least five
days a week on an alternating basis, so that each tank is analyzed
once each week. However, if the toxicant or an additive causes
a depression in dissolved oxygen, the toxicant concentration with
the lowest dissolved oxygen concentration must be analyzed daily in
addition to the above requirement.
A control and one test concentration must be analyzed weekly for
pH, alkalinity, hardness, acidity, and conductance or more often,
28
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if necessary, to show the variability in the test water. However,
if any of these characteristics are affected by the toxicant,
the tanks must be analyzed for that characteristic daily, at
least five days a week, on an alternating basis, so that each
tank is analyzed once every other week.
At a minimum, the test water must be analyzed at the beginning
and near the middle of the chronic test for calcium, magnesium,
sodium, potassium, chloride, sulfate, conductance, total solid,
and total dissolved solids.
4. Residue analysis: When possible and deemed necessary, mature
fish, and possibly eggs, larvae, and juveniles, obtained from
the test, should be analyzed for toxicant residues. For fish,
muscle should be analyzed, and gill, blood, brain, liver, bone
kidney, GI tract, gonad, and skin should be considered for
analysis. Analyses of whole organisms may be done in addition
to, but should not be done in place of, analyses of individual
tissues, especially muscle.
5. Methods: When they will provide the desired information with
acceptable precision and accuracy, methods described in Methods
for Chemical Analysis of Water and Wastes (EPA, 1971) should be
used unless there is another method which requires much less
time and can provide the desired information with the same
or better precision and accuracy. At a minimum, accuracy should
be measured using the method of known additions for all analytical
methods for toxicants. If available, reference samples should be
analyzed periodically for each analytical method.
D. Statistics
1. Duplicates; Use true duplicates for each level of the toxic
agent, i.e., no water connections between duplicate tanks.
2. Distribution of tanks: The tanks should be assigned to locations
by stratified random assignment (random assignment of one tank
for each level of the toxic agent in a row followed by random
assignment of the second tank for each level of the toxic agent
in another or an extension of the same row).
3. Distribution of test organisms; The test organisms should be
assigned to tanks by stratified random assignment (random assign-
ment of one test organism to each tank, random assignment of a
second test organism to each tank, etc.).
4. Selection and tninning test organisms: At time of selection or
thinning of test organisms the choice must be random (random, as
defined statistically).
29
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E. Miscellaneous
1. Additional information; All routine bioassay flow through
methods not covered in this procedure (e.g., physical and
chemical determinations, handling of fish) should be followed
as described in Standard Methods for the Examination of Water
and Wastewater (American Public Health Association, 1971).
2. Acknowledgments: These procedures for the brook trout were
compiled by J. M. McKim and D. A. Benoit for the Committee on
Aquatic Bioassays. The participating members of this committee
are: Robert Andrew, John Arthur, Duane Benoit, Gerald Bouck,
William Brungs, Gary Chapman, John Eaton, John Hale, Kenneth
Hokanson, James McKim, Quentin Pickering, Wesley Smith, Charles
Stephan, and James Tucker.
3. References; For additional information concerning flow through
bioassay tests with brook trout, the following references are
listed:
Allison, L. N. 1951. Delay of spawning in eastern brook trout
by means of artificially prolonged light intervals. Progressive
Fish-Culturist, 13: 111-116.
American Public Health Association. 1971. Standard methods
for the examination of water and wastewater. 13th ed. APHA,
New York.
Carson, B. W. 1955. Four years progress in the use of
artificially controlled light to induce early spawning of brook
trout. Progressive Fish-Culturist, 17: 99-102.
Drummond, Robert A., and Walter F. Dawson. 1970. An inexpensive
method for simulating Diel patterns of lighting in the laboratory.
Trans. Amer. Fish. Soc., 99(2): 434-435.
Environmental Protection Agency. 1971. Methods for Chemical
Analysis of Water and Wastes. Analytical Quality Control Laboratory,
Cincinnati, Ohio.
Fabricius, E. 1953. Aquarium observations on the spawning
behavior of the char, Salmo alpinus. Rep. Inst. Freshwater Res.
Drottingholm, 34: 14-48.
Hale, J. G. 1968. Observations on brook trout, Salvelinus
fontinalis spawning in 10-gallon aquaria. Trans. Amer. Fish.
Soc.. 97: 299-301.
30
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Henderson, N. E. 1962. The annual cycle in the testis of
the eastern brook trout, Salvelinus fontinalis (Mitchill)
Canadian Jour. Zool., 40: 631-645.
Henderson, N. E. 1963. Influence of light and temperature
on the reproductive cycle of the eastern brook trout
Salvelinus jontinalis (Mitchill). J. Fish. Res. Bd. Canada,
20(4) : 859-897.
Hoover, E. E., and H. E. Hubbard. 1937. Modification of
the sexual cycle in trout by control of light. Copeia,
4: 206-210.
MacFadden, J. 1961. A population study of the brook trout
Salvelinus fontinalis (Mitchill). Wildlife Soc. Pub. No. 7.
McKim, J.'M., and D. A. Benoit. 1971. Effect of long-term
exposures to copper on survival, reproduction, and growth
of brook trout Salvelinus fontinalis (Mitchill). J. Fish. Res,
Bd. Canada, 28: 655-662.
Mount, Donald I. 1968. Chronic toxicity of copper to fathead
minnows (Pimephales promelas, Rafinesque). Water Research,
2: 215-223.
Mount, Donald I., and William Brungs. 1967. A simplified
dosing apparatus for fish toxicology studies. Water Research,
1: 21-29.
Pyle, E. A. 1969. The effect of constant light or constant
darkness on the growth and sexual maturity of brook trout.
Fish. Res. Bull. No. 31. The nutrition of trout, Cortland
Hatchery Report No. 36, pages 13-19.
Wydoski, R. S., and E. L. Cooper. 1966. Maturation and
fecundity of brook trout from infertile streams. J. Fish.
Res. Bd. Canada, 23(5): 623-649.
Approved by the Committee on
Aquatic Bioassays, NWQL
Approved by the Director, NWQL
-------
Appendix A
Test (Evansville, Indiana) Photoperiod
For Brook Trout Partial Chronic
Dawn to Dusk
Time Date Day-length (hour and minute)
6:00 - 6:15) MAR. 1 12:15)
6:00 - 7:00) 15 13:00)
6:00 - 7:30) APR. 1 13:30)
6:00 - 8:15) 15 14:15)
)
6:00 - 8:45) MAY 1 14:45)
6:00 - 9:15) 15 15:15)
)
6:00 - 9:30) JUNE 1 15:30) Juvenile-
6:00 - 9:45) 15 15:45) adult exposure
6:00 - 9:45) .JULY 1 15:45)
6:00 - 9:30) 15 15:30)
6:00 - 9:00) AUG. 1 15:00)
6:00 - 8:30) 15 14:30)
6:00 - 8:00) SEPT. 1 14:00)
6:00 - 7:30) 15 13:30)
6:00 - 6:45) OCT. 1 12:45)
6:00 - 6:15) 15 12:15)
) Spawning and
6:00 - 5:30) NOV. 1 11:30) egg incubation
6:00 - 5:00 15 11:00)
6:00 - 4:45) DEC. 1 10:45)
6:00 - 4:30) 15 10:30)
6:00 - 4:30) JAN. 1 10:30) Alevin-juvenile
6:00 - 4:45) 15 10:45) exposure
6:00 - 5:15) FEB. 1 11:15)
6:00 - 5:45) 15 11:45)
32
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Appendix B
Months
Mar.
Apr.
May
June
July
Aug.
Sept.
Oct.
Nov.
Dec.
Jan.
Feb.
Mar.
Temperature Regime for Brook Trout Pa
Temperature ° C
Juvenile-
adult
exposure
Spawning
and
egg incubation
Alevin-
juvenile
exposure
9
12
14
15
15
15
12
9
9
~~9
9
9
9
A constant temperature
must be established just
prior to spawning and egg
incubation, and maintained
throughout the 3-month
alevin-juvenile exposure.
33
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TECHNICAL REPORT DATA
sc read InsuiiLtions on the rrrcrrc ttciw conipk ring;
1. REPORT NO. 2.
EPA-600/3-76-112
4. TITLE AND SUBTITLE
EFFECTS OF AROCLOR& 1254 ON BROOK TROUT, SALVELINUS
FONTINALIS
3. RECIPIENT'S ACCESSION- NO.
5. REPORT DATE
December 1976
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Virginia M. Snarski and Frank A. Puglisi
8. PERFORMING ORGANIZATION REPORT NO
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Environmental Research Laboratory - Duluth, MN
Office of Research and Development
U.S. Environmental Protection Agency
Duluth, Minnesota 55804
10. PROGRAM ELEMENT NO.
1BA608
11. CONTRACT/GRANT NO.
None (in-house)
12. SPONSORING AGENCY NAME AND ADDRESS
Same as above
13. TYPE OF REPORT AND PERIOD COVERED
Final 1Q77-1Q74
14. SPONSORING AGENCY CODE
EPA/600/03
15. SUPPLEMENTARY NOTES
16. ABSTRACT
No adverse effects were observed on survival, growth, and reproduction of brook
trout exposed for 71 weeks to 0.94 yg/1. and lower concentrations of the poly-
chlorinated biphenyl Aroclor*' 1254 (P = 0.05). Survival and growth to 90 days of
alevin-juveniles from exposed parents were also unaffected ( P = 0.05).
Polychlorinated biphenyl concentrations in the brook trout were directly
proportional to the water exposure concentrations (P = 0.05). The PCB tissue
concentrations appeared to have reached a steady state by the first sampling
after 14 weeks of exposure. The PCB residues (wet-tissue basis) in chronically
exposed fish were approximately 2 yg/g in the fillet and 9 yg/g in the "whole body"
(entire fish minus one fillet and the gonads) at the highest water concentration,
0.94 yg/1. The higher residue in the whole body compared to the corresponding
fillet was due to the higher fat content of the former.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
Bioassay
Trout
Survival
Growth
Reproduction
Freshwater
Tissues
Chlorohydrocarbon
3 DISTRIBUTION STATEMEN"
RELEASE TO PUBLIC
b.IDENTIFIERS/OPEN ENDEDTERMS
Aroclor® 1254
Polychlorinated bipheny!. 06/F/T
biphenyl
Bioaccumulation
\v SECURITY CLASS (ThisKeportl
UNCLASSIFIED
I 20. SECURITY CLASS / This page]
UNCLASSIFIED
EPA Form 2220-1 (9-73)
c. COSATI 1'icld/Gioup
21. NO. OF
42
22 PRiCE
o/
S. GOVERNMENT PRINTING OFFICE 1977-757-056/5552 Region No. 5- I I
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