THE ACUTE TOXICITY OF

A POLYCHLORINATED BIPHENYL,‘

AROCLOR'1254, To THE EARLY
LIFE STAGES OF coHo SALMON
AND STEELHEAD TROUT .

Thesis for the Degree of M. S

MICHIGAN STATE UNIVERSITY

MARK THOMAS HALTER
1 9 7 3

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ABSTRACT
THE ACUTE TOXICITY OF A POLYCHLORINATED
BIPHENYL, AROCLOR 1254, TO THE EARLY

LIFE STAGES OF COHO SALMON
AND STEELHEAD TROUT

BY

Mark Thomas Halter

Some acute effects of Aroclor 1254 (PCB) to the early
life stages of coho salmon (Onchorhynchus kisutch (Walbaum))

and steelhead trout (Salmo gairdneri.irideus Gibbons) were

 

investigated using continuous flow bioassay techniques.

PCB did not influence the acute toxicity of DDT to 5—10
week old coho salmon of Michigan or Oregon parent stock. In
14 day bioassays, fish were exposed to 45.0 to 3.0 ug/l PCB
or 3.3 to 0.2 ug/l DDT and combinations of 45.0 + 3.3 to 3.0
+ 0.2 ug/l PCB + DDT. PCB-DDT combinations produced no
greater mortalities than could be expected from exposure to
the DDT concentrations alone. The rapidity of DDT toxicity
with respect to that of PCB was suggested as the reason for
lack of interaction. Michigan salmon were more resistant to
DDT than Oregon salmon. Symptoms of PCB and DDT poisoning
were described.

Coho salmon exposed to 56.40 to 4.35 ug/l Aroclor 1254

;from the last two weeks of egg stage until four weeks beyond

Mark Thomas Halter

hatching were adversely affected at all exposure levels.
Salmonexposed to the same PCB levels during the last two
weeks of the egg stage only were adversely affected at levels
above 15 09/1. Egg hatchability, mean incubation time, fry
survival, and fry growth were measured. All egg groups ex-
posed to PCB hatched prematurely.

Two month old steelhead trout accumulated PCB at 0.189,
1.044, 1.832, and 3.106 pg/g per hour when exposed to 3.25,
10.45, 27.80, and 51.30 ug/l Aroclor 1254 for 24 days.
Uptake rates could be predicted given one uptake rate from a
known exposure level. The fish concentrated PCB 32,000 to
38,000 times over the exposure concentration but plateaus
were not reached. Bioconcentration factors in fish at 100
hour time intervals through the test were found to be in-
versely related to the exposure concentration. Fish brains
accumulated PCB at a slower rate than the whole body beyond
200 hours exposure or after accumulating about 400 pg/g PCB.

In a thirty day toxicity test the median survival times
of 100 day old steelhead trout exposed to 39.40 and 20.40
ug/l Aroclor 1254 were 293 and 400 hours, respectively. The

growth of fish exposed to more than 4.70 ug/l PCB was reduced.

THE ACUTE TOXICITY OF A POLYCHLORINATED
BIPHENYL, AROCLOR 1254, TO THE EARLY
LIFE STAGES OF COHO SALMON

AND STEELHEAD TROUT

BY

Mark Thomas Halter

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Fisheries and Wildlife

1973

ACKNOWLEDGEMENTS

I would like to thank Dr. Howard E. Johnson, my major
professor, for the advice, criticism, and encouragement he
has given me during the research and writing of this thesis.
He is a good person and one of those few who has truly cap-
tured the essence of the absent-minded professor. I also
appreciate the fine reviews of the thesis manuscript made by
Drs. Thomas G. Bahr and Norman C. Leeling.

I wish to acknowledge the Department of Fisheries and
Wildlife and the Pesticide Research Center for the cooperation
I received in the use of facilities and equipment.

For his good ideas and suggestions along the way, I
should thank David R. Rosenberger, fellow graduate student,
but I had to listen to so many bad ideas between the good ones
that some hesitancy is in order--but thank you, Dave.

Financial support for this work was provided by: The
William R. Angell Foundation and the Sports Foundation Inc.
in cooperation with the Sport Fishery Research Foundation;
the Environmental Protection Agency, Office of Water Programs,

training grant 5P3-WP-264; and the Michigan State University

-Agricultural Experiment Station. A portion of the work upon

which this work is based was performed pursuant to contract

ii

no. FDA 71-285 with the Public Health Service, Food and Drug
Administration, Department of Health, Education and Welfare.
Finally, I wish to thank my wife Susan for her support
in all ways over the last two and a half years. Her typing
ability hastened progress in the writing of this thesis con-
siderably. I further want to wish her good luck in her

future studies.

iii

TABLE OF CONTENTS

Page
INTRODUCTION 0 O O O O O O I O I O O O O O O O O O O O 1
GENERAL METHODS AND MATERIALS . . . . . . . . . . . . 5
PCB-DDT COMBINATION BIOASSAYS . . . . . . . . . . . . 13
Methods and Materials. . . . . . . . . . . . . . 13
Resu1ts O O O O O O O O O 0 O O O I O O O O O O O 18
DiscuSSion O O O O O O O I O O O O O O O O O O O 3 3
COHO SALMON EGG-'ALEVIN EXPOSURE o o o o o o o o o o o 3 5
Methods and Materials. . . . . . . . . . . . . . 35
Results 0 O O O O O O O O I O O O O O O O O O O O 36
Discussion . . . . . . . . . . . . . . . . . . . 39
PCB UPTAKE BY STEELHEAD TROUT . . . . . . . . . . . . 41
Methods and Materials. . . . . . . . . . . . . . 41
Results 0 O O O C O O O O O O O O O O O O O O O O 4 3
DiscuSSion O O O O O O O O O O O O O O O O O O O 5 3
STEELHEAD TROUT TOXICITY BIOASSAY . . . . . . . . . . 57
MethOdS and Materials 0 O O O O O O O O O O O O O 5 7
Results I O O O O O O O O O O O O O O O O O O O O 59
Discussion . . . . . . . . . . . . . . . . . . . 59
CONCLUS ION.‘ O I O O O O O O O O O O O O O O O O O O O 6 4
LITEMTURE CITED 0 O O O O O O O O O O O O I O O O O O 67
APPENDIX--BREAKDOWN OF LOSSES OF EGG, SAC-FRY AND
ALEVINS EXPOSED TO VARIOUS CONCENTRATIONS
OF AROCLOR 1254 O O O O O O O O O O O O O O 7 2

iv

LIST OF TABLES

TABLE

1.

2.

Chemical characteristics of the water used in
all bioassays. . . . . . . . . . . . . . . . . .

The toxicant concentrations, in ug/l, present in
the test tanks of the PCB-DDT bioassays per-
formed with Michigan coho salmon . . . . . . . .

The toxicant concentrations, in ug/l, present in
the test tanks of the PCB-DDT bioassays per-
formed with Oregon coho salmon . . . . . . . . .

Summary of toxicity data for Michigan coho
salmon exposed to various concentrations of PCB,
DDT or PCB-DDT combinations. . . . . . . . . . .

Summary of toxicity data for Oregon coho salmon
exposed to various concentrations of PCB, DDT or
PCB-DDT combinations 0 O O O O O O O I O I O O 0

Summary of test in which eyed coho salmon eggs
were incubated, hatched, and the resulting
alevins allowed to develop for four weeks in
various concentrations of Aroclor 1254 . . . . .

Summary of test in which eyed coho salmon eggs
were incubated for two weeks in various concen-
trations of Aroclor 1254, then transferred to
an uncontaminated system for hatching and de-
velopment. . . . . . . . . . . . . . . . . . . .

The Aroclor 1254 concentrations, in ug/l,
present in the test tanks during the PCB uptake
study as determined by gas chromatography. . . .

Actual magnification factors calculated by sub-

stitution of the appropriate number of hours
into the regression equations describing PCB
uptake 0 O O I C I O O C O O O O O O O I O O O O

Page

16

17

19

20

37

38

42

52

LIST OF TABLES--Continued

TABLE Page

10.

ll.

12.

Magnification factors calculated by substitu-

tion of the appropriate number of hours into the
regression equations, excluding the "Y" inter-

cept value, describing PCB uptake. . . . . . . . 52

The Aroclor 1254 concentrations, in ug/l,

present in the test tanks during the thirty day
toxicity test as determined by gas chromatog-

raphy. . . . . . . . . . . . . . . . . . . . . . 58

The length and weight of steelhead trout before

and after exposure to various concentrations of
ArOCJ-Or 1254 O O O O O O O O O O O O O O O O O O 62

vi

FIGURE

10.

11.

LIST OF FIGURES

The toxicant introduction system of the flow-
through test apparatus used in the present
studies. 0 O O O O O O O O O I O O O O O O O 0

Gas chromatogram of Aroclor 1254 . . . . . . .

The toxicity of various concentrations of PCB
and PCB-DDT combinations to young Michigan.
coho salmon. . . . . . . . . . . . . . . . . .

The toxicity of various concentrations of DDT
and PCB-DDT combinations to young Michigan
COho salmon O O O O O O O O O O O O I O O O O O

The toxicity of various concentrations of DDT
and PCB-DDT combinations to young Oregon coho
salmon O O O O O O O O O O O O O O O O O O O O

The toxicity of various concentrations of DDT
and PCB-DDT combinations to young Oregon coho
salmon O O O O O O O O O O O O O O O O O I O O

The toxicity of various concentrations of PCB
and PCB-DDT combinations to young Oregon coho
salmon O O O O O O O O O O O O O O O O O O O O

A comparison of standard and tissue samples
chromatograms of Aroclor 1254. . . . . . . . .

The uptake of Aroclor 1254 from water by young
steelhead trout. O O O O O O O O O O O I O O O

Aroclor 1254 concentrations in the bodies and
brains of young steelhead trout killed by expo-
sure to 51.30 ug/l Aroclor 1254. . . . . . . ,

The results of a thirty day toxicity test in

which young steelhead trout were exposed to
five concentrations of Aroclor 1254. . . . . .

vii

Page

12

22

24

26

28

30

45

47

50

61

THE ACUTE TOXICITY OF A POLYCHLORINATED
BIPHENYL, AROCLOR 1254, TO THE EARLY
LIFE STAGES OF COHO SALMON

AND STEELHEAD TROUT

INTRODUCTION

Monitoring studies have revealed that residues of two
chlorinated hydrocarbon compounds, DDT and polychlorinated
biphenyls (PCB), are prevalent in the Lake Michigan ecosystem
(Lake Michigan Interstate Pesticide Committee, 1972; Johnson
and Ball, 1972; Reinert, 1970; Hickey et al., 1966). Concen—
trations of DDT and PCB in off-shore waters of the lake are
commonly estimated at l-lO and 1—20 ng/l (pptr), respectively,
while inshore and tributary waters near agricultural, urban,
or industrial centers may be somewhat more contaminated
(Inter4Departmental Task Force on PCBs, 1972; Lake Michigan
Interstate Pesticide Committee, 1972). The bulk of residues
entering the lake are thought to be sorbed onto bottom sedi-
ments (Nisbet and Sarofim, 1972) and may be available for con-
tinuous equilibria exchange with the ambient water (Hamelink
et al., 1971) or slow microbial degradation (Matsumura et al.,
1971).

Various fish species in Lake Michigan have whole body
DDT residues of 2-20 ug/gand PCB levels are often about twice
this amount (Lake Michigan Interstate Pesticide Committee,
1972; Reinert, 1970; Henderson et al., 1970). Residues in

salmon and trout are frequently near the upper limits of these

ranges, apparently due to their high fat content and top
position in the food chain (Reinert, 1970). DDT and PCB
residues in excess of 5 ug/g have been the basis for actions
or warnings by the Food and Drug Administration in 1968 and
1971 against Lake Michigan commercial fishery products and
food fish taken by sports fishermen. The Lake Michigan
Interstate Pesticide Committee (1972) estimated that under
presently enforced FDA guidelines about 80% of the total
annual catch from the lake (which in 1967 amounted to 59
million pounds (Lyles, 1967) is nonmarketable in interstate
commerce.

The reproductive potential of coho salmon in Lake Michi-
gan may be impaired by chlorinated hydrocarbons transferred
from adult fish to their eggs. Johnson and Pecor (1969)
described a mortality syndrome associated with significant
losses among coho salmon sac-fry reared in Michigan hatch-
eries. The losses typically occurred during the seventh week
after hatching at the time of final yolk sac absorption.
These authors correlated the appearance of symptoms in af-
fected fry with DDT residues in the body and gut tissues.
However, the correlations have not always been consistent
among different fry groups or from year to year. Johnson
(1972) suggested that PCB residues present in the fry may in-
fluence overall survival.

Toxic interactions between DDT and PCB were demonstrated

with houseflies by Lichtenstein et al., (1969) who found that

topical applications of 500 ug/g of several PCB formulations
in combination with 15 ug/g of DDT produced markedly higher
mortalities than either compound applied alone. These re:
sults have also been repeated with pesticides other than DDT
(Fuhremann and Lichtenstein, 1972). Lichenstein (1969,1972)
warned that biological interactions between PCBs and other
synthetic chemicals present in biological systems are possi-
ble and should not be disregarded.

Although PCBs have been manufactured since 1929, their
presence in Lake Michigan and in other ecosystems was not
anticipated or forewarned as was the case with DDT. Conse-
quently, they remained undiscovered until 1966. Therefore,
information regarding the effects of PCB on aquatic life was
unavailable until only recently (Interdepartment Task Force
on PCBs, 1972; National Institute of Environmental Health
Sciences at al., 1972).

The present study was conducted to provide basic toxi-
cological information about the acute effects of PCB on the
early life stages of coho salmon (Onchorhyncus kisutch
(walbaumm)) and steelhead trout (Salmo gairdneri irideus
Gibbons). Specifically, the test objectives were: 1) to
compare the acute toxicities of PCB, DDT and PCB-DDT combina-
tions to young coho salmon; 2) to determine the effect of PCB
-exposure on coho eggs and alevins; 3) to determine the rate
of uptake of PCB by young trout; and 4) to determine levels

of PCB toxic to young trout over a thirty day period.

A single PCB formulation, Aroclor 1254 (Monsanto Company,
St. Louis, Missouri), was used exclusively in these experi-
ments and will be referred to as PCB in this thesis. Aroclor
1254 is the trade name for a mixture of biphenyl rings aver-
aging 54% chlorine substituents by weight. This PCB was se-
lected for testing because its gas chromatographic tracing
most closely resembles that of the residues extracted from

water and fish of Lake Michigan (Veith, 1972; Armour and

Burke, 1970).

GENERAL METHODS AND MATERIALS

Steelhead trout and coho salmon were reared in the lab-
oratory from eggs collected in April and October of 1971
respectively, at weir sites operated by the Michigan Depart-
ment of Natural Resources on the Platte river, a tributary of
Lake Michigan located in Benzie County, Michigan. Eyed coho
salmon eggs were also received in November, 1971, via air
shipment, from the Oregon Fish Commission. In each lot, the
eggs of several females were fertilized with the sperm of
more than one male.

Young fish were held in tanks supplied by the same water
source as used in all tests. Michigan fish were maintained
on Ewos starter diet, a Swedish pelleted dry food. Oregon
salmon were fed Oregon Moist diet.

All exposures were conducted using proportional diluters
(Mount and Brungs, 1967) delivering five toxicant concentra-
tions plus a control at a rate of 200 ml/min. Modified ver-
sions (Figure 1) of the toxicant introduction system of
McAllister et a1. (1972) were calibrated to deliver either
0.25 or 1.0 ml of stock solution to the mixing chambers of
the diluters with each diluter cycle. The delivery tubes

from the diluters were randomly distributed to convenient

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arrangements of screen-covered glass aquaria which had
stand-pipe controlled water volumes of 14 liters. Water
replacement time (90%) in the test tanks was approximately
2% hours. The rate of water flow per gram of fish in these
bioassays was never less than 4 l/g per day.

Toxicant stock solutions were made up in four liters
of reagent grade acetone with appropriate amounts of Aroclor
1254 and transferred directly to the Mariotte bottle of the
toxicant introduction system. After dilution the nominal
test concentrations of Aroclor 1254 in all tests were 80,
40, 20, 10, and 5 ug/l. Nominal acetone concentrations
present when 1.0 m1 of stock solution was delivered with each
diluter cycle were 800, 400, 200, 100, and 50 mg/l; or 200,
100, 50, 25, and 12.5 mg/l if 0.25 ml of stock was delivered.

Dechlorinated Michigan State University tap water
(Table 1) was used in all tests. Routine measurements of
hardness, total alkalinity, and pH over the one year test
period revealed no significant fluctuation in these charac-
teristics. Dissolved oxygen, measured in the test tanks
during each exposure, ranged from 7.3 to 8.5 mg/l. Water
temperature was maintained at 12—14°C by a Min-O-Cool aerator
in a 100 gallon reservoir through which the test water passed
prior to use. The laboratory was continuously lighted by
overhead fluorescent lamps.

Actual test concentrations of Aroclor 1254 were deter-

mined by gas-liquid chromatography (GLC) using a Micro-Tek

Table 1. Chemical characteristics of the water used in all

bioassays.

 

 

Parameter

Conc. (mg/1)

 

Hardness as CaCO3

Total Alkalinity as CaCO3
Ammonia Nitrogen
Chloride

Cu, Fe, Pb, Zn

Nitrate

Phosphorus

Sulfate

pH

Conductivity as umho/cm2

336

 

10

220 gas chromatograph equipped with a 63Ni electron-capture
detector and a % inch by 6 foot glass column packed with

3 percent SE-30 on 60-80 mesh Gas Chrom-Q. Inlet, column,
and detector temperatures were 205, 175, and 365°C,
respectively and the nitrogen carrier gas flow was 85 ml/min.
One liter water samples collected from individual test
chambers were prepared for analysis by successive extractions
with 100, 50 and 50 ml of redistilled hexane. The combined
extract was then dried over anhydrous sodium sulfate, reduced
to volume and a known amount injected into the gas chroma-
tograph. Standard solutions were injected after every one

to three samples.

Quantitation of Aroclor 1254 was based on the combined
heights of peaks 4 through 9 (shown in Figure 2). A compari—
son of water extract chromatograms to those of standards
indicated these specific peaks were stable and unmodified in
water solution. '

Recoveries from eight one liter water samples spiked with
25 to 50 ug/l of Aroclor 1254 averaged 83%. The measured
test concentrations given in this paper have not been cor-
rected for this extraction efficiency. The remainder of the
difference between nominal and measured concentration values
reported is primarily due to the sorption of PCB onto the

various surfaces of the test apparatus which the test solu-

tions contacted.

11

Figure 2. Gas chromatogram of Aroclor 1254. The combined
heights of peaks 4 through 9 were used for quan-

titation of PCB concentrations in water and fish
tissue samples.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MMMMMMM

PCB-DDT COMBINATION BIOASSAYS

Methods and Materials

Four pairs of 14 day bioassays, and one pair of 7 day
bioassays were conducted using two diluters equipped with
0.25 ml toxicant introduction devices. One system delivered
PCB-DDT mixtures while the other delivered concentrations of
PCB or DDT alone. A "dipping bird" apparatus (Mount and
Brungs, 1967) was used to maintain 200 mg/l acetone in the
control tank of each system. The first two bioassays tested
Michigan salmon and the last three tested Oregon salmon.

DDT (100% p,p' 1,1,1, trichloro 2,2 bis (-p~chloro—
phenyl) ethane) stock solutions were made up in the same
manner described in the general methods for PCB stock solu-
tions. The nominal DDT test concentrations in all bioassays
were 5, 2.5, 1.25, 0.63, and 0.31 ug/l. When combinations
*were tested, PCB and DDT were mixed in the same stock bottle.
to give nominal test concentrations of 80+5, 40+2.5, and so
on, ug/l PCB-DDT.

In each test the diluters were calibrated and run normal-
ly for 12-15 hours (overnight). Twenty-five fry were then
randomly assigned to each of the twelve test chambers. Each

day for two weeks the fish were observed at least four times

13

14

between 9 a.m. and 10 p.m. Dead fish were removed as ob-
served and the times of mortality were recorded. The fish
were fed a measured amount of food three times daily. The
food ration was reduced but not discontinued for fish groups
no longer accepting food. Uneaten food and feces were
siphoned from each tank daily. At the end of the test, sur-
viving fish were sacrificed, measured and weighed.

Between tests the diluters, delivery tubes, and aquaria
were washed with hot water and detergent and rinsed twice
with acetone.

Starting January 25, 1972, Michigan salmon about 35 days
old were treated with PCB alone and PCB-DDT combinations and
this test was followed with a bioassay of DDT alone and the
PCB-DDT combinations. Oregon salmon bioassays, begun on
February 27, first tested DDT alone and the PCB-DDT combina-
tions. This two week test was then repeated with a one week
exposure. Finally, Oregon salmon were tested with PCB alone
and the PCB-DDT combinations.

During the Michigan salmon bioassays, water samples for
toxicant determination were taken from half of the test tanks
ofieach exposure system at the beginning, middle, and end of
each test. This sampling scheme was increased to sampling
all tanks every three days during the Oregon salmon bioassays
to more adequately define actual toxicant exposure concentra-

tions.

15

The water samples were analyzed (Tables 2 and 3) by
gas-liquid chromatography as described in the general methods
section. Quantitation of PCB alone was based on the heights
of peaks 4 through 9 (Figure 2) while that of DDT was based
on its single peak height. In water samples of PCB-DDT
combinations, both compounds were quantified without separa-
tion. Under the described gas chromatographic conditions,
the retention time of DDT was identical to that of the iso-
mer(s) causing peak 11 on the chromatogram of Aroclor 1254.
The normal height of peak 11 relative to that of adjacent
peak 9 was known from experience with many chromatograms of
Aroclor 1254 extracted from water. Therefore when the en-
larged peak 11 of a PCB-DDT chromatogram was measured, peak
9 on the same chromatogram was used to estimate the portion
of peak 11 due to PCB and the remainder was taken as the peak
height of the DDT in the sample. The latter was then used
for quantitation of DDT by comparison to a standard plot of
DDT peak height vs. nanograms injected, obtained by injecting
known amounts of DDT and PCB mixtures into the gas chroma—
tograph. PCB concentrations in the combination samples could
be determined in the usual manner since DDT did not interfere
with peaks 4 through 9.

The average recoveries from twenty-three samples of water
'spiked with 5 to 50 ug/l of PCB and/or DDT were 82-84% for

each compound.

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18

Mean toxicant concentrations from individual test tanks
within each test system were compared for significant differ-
ences by the Student t-test at p = 0.05. Median survival
times (MST) with 95% confidence limits were calculated for
fish in each concentration in each bioassay by the method of

Litchfield (1949).

Results

DDT was more toxic than Aroclor 1254 to the young coho
salmon (Tables 4 and 5, Figures 3 to 7). Median survival
times of fish exposed to more than 1 ug/l DDT were always
less than 336 hours while Aroclor 1254 concentrations below
about 30 ug/l failed to kill half of the test fish within 336
hours. Median survival times of fish groups exposed to PCB-
DDT combinations was always less than 336 hours if the DDT
level was greater than 1 ug/l. In no case, however, was the
median survival time of the fish in PCB-DDT combinations
less than that of fish in the next highest DDT concentration
within the same test.

Comparison of the tests in which Michigan and Oregon
salmon of similar body weight were exposed to similar toxi-
cant concentrations show that the Michigan fish had longer
median survival times.

During the final stages of yolk-sac absorption, a general
mortality occurred among all Michigan coho salmon, producing

a 40% loss. The symptoms were similar to those described by

19

.oousmmofi uoc mcoflumuucoocoo Hmouofi

.mGOHumuucmoooo ucmoflxow mumeflxoummdm

 

 

 

 

 

 

IIII mmm A mm.o II
373 m: 3.4.50.2 004 3.0 EEESoEM
mmlmm om om N+om we we ov Aeevoumcma M
mmlam em oa.m In N
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HOHInn mm m>.m+oo.av H
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mocmoflmcou wmm Hw>H>uom :meomz .ocou puma
.mGOHumcHQEoo Beaumom no son .mum mo mc0flumuu
Icmocoo mooflum> ou oomomxo COEHmm onoo cmmflsowz Mom mono hufioflxou mo mumfifism .v mHQMB

20

.mo. u e um
ucmHTMMHo >HOGMOHMHcmflm you one moEHu Hm>fl>uom amaoofi no mQOAOMHusoosoo “mop omooex

 

 

 

 

IIII wmm om.mH

III: omm mw.o+om.m

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IIII moa A Nh.o II

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mocmoflmcoo «mm Hm>H>H5m smaomz .ocoo umme

 

 

.maoeumcaneoo aaoumoe no son .mom mo mace»
Imuucoocoo m50aum> ou oomomxo coEHmm oooo commuo How memo wuwowxou mo mHmEEom .m oHomB

21

Figure 3. The toxicity of various concentrations of PCB
and PCB-DDT combinations to young Michigan coho
salmon. Broken lines represent PCB-DDT toxicity,
solid lines PCB toxicity. Confidence interval

(95%) of the median survival times are indicated
by horizontal bars.

 

°/o MORTALITY

98-

90

70

50

30

Michigan

 

22

lJlLJ

I 1

n I 1

 

l-Lns.

>

"I C! C) 0!

4° 60 80 100

TIME (hrs)

200 300

MEDIAN SURVIVAL TIMES

Cone. ( ppb)
PCB DDT

41 .00 + 2.75
20.00 + 1 .50
10.20 + .65
47.00 —

28.25

Figure 3

MST

88
1 20

200
1 06

> 336

23

Figure 4. The toxicity of various concentrations of DDT
and PCB—DDT combinations to young Michigan coho
salmon. Broken lines represent PCB-DDT toxicity,
solid lines DDT toxicity. Confidence interval
(95%) of the median survival times are indicated
by horizontal bars.

% MORTALITY

98

9C)

70

NO

24

" Michigan 2

 

 

 

 

I
C
L f /
/
F‘ O
/
0
’ /
_ /
D
O
' /
——- F——4 I——J£hr—u———* 93‘
.A
.. o/ ’
0 o
" / / A
L I, l' .A
l’ O .A
_ 1 / /
I / /
.1.I.I.JJIIII [Ll
20 40 60 80100 200 300
TIME (hrs)
MEDIAN SURVIVAL TIMES
Conc.(ppb)
Line PCB DDT MST
A — 3.10 34.5
B 44.50 + 2.80 80.0
C 24.00 + 1.50 115.0
0 - .95 >330

Figure 4

25

Figure 5. The toxicity of various concentrations of DDT
and PCB-DDT combinations to young Oregon coho
salmon. Broken lines represent PCB-DDT toxicity,
solid lines DDT toxicity. Confidence interval

(95%) of the median survival times are indicated
by horizontal bars.

MORTALITY

%

26

 

 

- A B c
I
90 A 90/ I}
_ I/ /
o” A I 0
7T)h- I I I'F
I 7
0 0 °A
- A I A c; o/A
I ' ”—T-II-—#l
50H- I———4
. A [/0 A / OOIA
I I
_ / l 0’ A
I°A 9’ / A
I P / A
b 0!. I 6" 1/
I I l I I I L I II 1 I. L I
20 40 80 80 100 200 300
TIME (hrs)

MEDIAN SURVIVAL TIMES

Conc.(ppb)
1192 3.9.9. 9.2!.
A — 2.571320)
3 40.00 + 2.72
c — 1.40
0 20.75 + 1.25
E 11.70 + .50
F — .32

Figure 5

M51.
25

_ 42
55
70

108
125

27

Figure 6. The toxicity of various concentrations of DDT
and PCB-DDT combinations to young Oregon coho
salmon. Broken lines represent PCB-DDT toxicity,
solid lines DDT toxicity. Confidence interval
(95%) of the median survival times are indicated
by horizontal bars.

% MORTALITY

90

70

50‘

30

1O

 

28

Oregon 3.5
A 8,0 C 0,0
I . ,fi
’ /
A. ;! I;?
0A I

 

to I A
I Z W .11"
I
A? 0/ :7
I A; 4;
A I ° lo
/ A I I /
/ I o/
1 I 1 I 1 I 1 I 1| 1 I 1 J
20 40 50 80 100 200 300
TIME (hrs)

MEDIAN SURVIVAL TIM ES

Cone. (ppb)
1.10.2. 22.3. £1.
A — 3.29
B 44.00 + 2.15
C — 1.90
D 24.15 + 1.15
E 12.80 + .80
F — .72

Figure 6

Mil
25
54
‘55
99
>188
>188

29

Figure 7. The toxicity of various concentrations of PCB
and PCB—DDT combinations to young Oregon coho
salmon. Broken lines represent PCB-DDT toxicity,
solid lines DDT toxicity. Confidence interval
(95%) of the median survival times are indicated
by horizontal bars.

MORTALITY

96

98

90

70

30

(- Oregon 4

b

 

30

 

 

I" A
oI
- 10
I
__ O
’0
- / 8
"' F—7L94 od/'
I— 0/0
‘} o°/
O
' / °/ Jig?
" // /° “)7 A
o.
/ / / /
P o ./o I/f>
1 I 1 I 1 I 1 I 1| 1 I 1 I 1
20 40 60 80 100 200 300
T IME ( hrs)
MEDIAN SURVIVAL TIMES
Cone. ( 000)
Line PCB DDT MST
A 33.00 + 2.00 172
B 15.25 + .80 > 330
c 32.20 - > 336
D 9.80 + .45 > 330
E 10.50 .- > 330

Figure 7

31

Johnson and Pecor (1969). This mortality was not unexpected
and the timing and symptoms were identical to that observed
among Michigan salmon in previous years. The mortality
occurred while the first bioassay with Michigan salmon was

in progress and some losses due to this mortality occurred
among the test fish. However the symptoms associated with
these losses were distinct from those induced by the toxi-
cants in the bioassays, and were only evident during the last
100 hours of the test. In the PCB exposure system 9, 5, and
2 fish died in the control and two lowest PCB concentrations,
respectively, while the same tanks of the PCB-DDT combina-
tion system had 4, 5, and 4 killed, respectively. These mor-
talities occurred after 200 hours into the test and are not
shown in the graphs of the test results. The stock tank
mortality had ceased before the second test was initiated and
no losses occurred in the controls or two lowest test concen—
trations during this test. The median survival times for
fish in the higher PCB-DDT mixtures of the second test compare
favorably with those of the first test.

There was a marked difference in symptoms between PCB
and DDT poisoned salmon. Fish exposed to 40 ug/l Aroclor
1254 became hypoactive and ceased feeding after about three
days. They oriented near the bottom of the tank, finning only
enough to maintain their position in the water. Darkening in
color occurred in some fish but not in all. The fish did

not readily respond to a pencil tap on the aquarium frame,

32

whereas control fish reacted by darting around the tank.
About a day before death, the fish swam listlessly near the
surface of the water and did not react to a prod with a
glass rod. Death was preceded by a several hour period of
slow, deep operculating by the fish on the bottom of the
aquarium.

No external lesions appeared on fish exposed to PCB.
In other studies, Hansen et a1. (1970) and Morgan (1972)
have reported the development of external lesions on Spot

(Leiostomus xanthurus) and guppies (Poecilia reticulata),

 

 

respectively, exposed to Aroclors 1254 and 1242, respectively.

Fish exposed to about 2 ug/l DDT became hyperactive
within a day after beginning exposure. A pencil tap on the
aquarium frame sent fish darting in all directions with more
intensity and for longer duration than did a similar tap on
the control tank. Moribund fish lost equilibrium, convulsed
periodically, and died, often with mouth open and back
arched. The time of death after appearance of acute symptoms
was typically less than a day, always less than two days.

Fish killed in the PCB-DDT combinations showed the symptoms
of DDT poisoning.

The general mortality among the Michigan coho which
occurred at the final yolk—sac absorption stage had some of
the features of both PCB and DDT toxicity. Darkening occurred
in some of the fish. Swimming patterns were erratic and con-

vulsive for a time, but the fish then sank and remained on

33

the.bottom for four to five days before dying. There was no
evidence that dying fish had initiated feeding behavior.
Comparison of the length and weight of control fish
with fish tested in the two lowest toxicant concentrations
in each of the bioassays indicated no consistent effect of
PCB or DDT on growth over the two week periods. Growth
measurements were not obtained for fish in the higher test

concentrations because of partial or complete mortality.

Discussion

The bioassay results indicate that the PCBfDDT concen-
trations caused no greater mortality than could be expected
from exposure to the DDT concentration alone. We conclude
that PCB does not increase the toxicity of DDT to young coho
salmon at the acute toxicity level, although both compounds
are quite toxic in themselves. The lack of interaction be-
tween PCB and DDT may be explained by the difference in their
modes of action to fish. DDT kills rapidly whereas PCB
causes a more chronic toxicity. DDT is most likely a direct
neurotoxin (O'Brien, 1967) while the mode of action of PCB
is not yet clear. Recent studies have shown that both DDT and
PCB inhibit the ATP-ase enzyme system in fish (Cutkomp et al.,
1971; Davis and Wedemeyer, 1971; Koch et al., 1972). In our
experiments high DDT concentrations were always mixed with

high PCB concentrations and the faster-acting DDT always

34

killed the test fish before any toxicity due to PCB could
be expressed. To more completely evaluate PCB-DDT inter-

actions, tests of high PCB-low DDT should be conducted.

COHO SALMON EGG-ALEVIN EXPOSURE

Methods and Materials

The purpose of this test was to determine the effect of
Aroclor 1254 exposure on the embryo and alevin stages of coho
salmon. A proportional diluter equipped with a 1.0 ml toxi-
cant introduction system delivered a water control and five
concentrations of Aroclor 1254. A second identical system
delivered a water control and five concentrations of acetone
in the same amounts as the first system. Acetone was not
delivered to the control tanks.

On November 24, 1971, about 100 eyed Lake Michigan coho
salmon eggs were placed onto the bottom of each of the PCB
exposure chambers. Two weeks later, and about two days prior
to hatching, half of each egg group was transferred to the
appropriate chamber of the uncontaminated system. Egg hatch-
ing success and alevin survival were then monitored in both
systems until January 15, 1972, four weeks beyond hatching.
The test chambers were darkened by black plastic curtains
until the fry began to swim-up, then laboratory light was
admitted into portions of the tanks. At the end of the test,
surviving fry were sacrificed and a subsample of twenty fish,

if available, from each test chamber were weighed and

35

36

measured. Mean lengths and weights were compared by the
Student t-test at p = 0.05.

One liter water samples were taken from each tank of
the PCB exposure system every five days during the test for

actual toxicant concentration determination.

Results

The hatchability of salmon eggs continuously exposed to
56.40 ug/l Aroclor 1254 (Table 6 and Appendix A) was reduced
by 30% compared to the control eggs. Variable hatching
success was observed at the lower PCB levels. At hatching,
about half of the losses occurred as the alevins emerged from
the egg and the remainder died within the intact egg. In
the four weeks following hatching, alevin survival was in-
versely related to the PCB concentration, with no group sur-
viving as well as the control. Fry exposed to greater than
15 ug/l PCB absorbed their yolk sacs and grew slower than the
control fry.

Salmon eggs removed from PCB exposure prior to hatching
(Table 7 and Appendix A) showed good hatchability (90-97%),
except those which had been exposed to 56.40 ug/l. The lat-
ter eggs sustained a 27% reduction compared to the controls.

Alevins from egg groups which had been exposed to greater

37

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.1

.mmmo mo huwamuuoa o>Hmmooxo oomsmo HmGSM Ho maumuommo

.omm Mao» mpmamfido u o .mmwum mslcogusn n o .mlo mo maoom o co omummm

 

 

m m .ee.He~. .mm.onm.mm m.e Nee a.me m~.a.H oe.em em
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e om mo.Hem. ae.euma.mm o.Ha Hem m.ma e o
MCOHH.
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38

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k.

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M

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ucoonom .ooocH cmoz usoouom

 

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ooumcflfimucooss cm on oouuommcmuu coop .wmma HOHOOHd mo odomumuucoocoo osoflum>

..h oHQmB

39

than 15 ug/l PCB had significantly reduced survival, yolk sac
utilization and growth, but generally did better than those
fry held under continuous exposure to these concentrations

in the other system. The alevins from eggs which had been
exposed to less than 8 ug/l PCB did as well as the controls,
except as discussed below.

Eggs in both exposure systems started hatching two to
five days before the control eggs so that mean incubation
times were reduced (Tables 6 and 7). This early hatching was
preceded by an alteration of the egg surface which appeared
to be due to a breakdown or coagulation of the outer surface
of the chorion. The eggs continuously exposed to greater than
25 ug/l PCB hatched most prematurely, about 50 degree-days

or four days before the controls.

Discussion

Under continuous exposure to greater than 15 09/1
Aroclor 1254, coho salmon eggs and alevins showed marked re-
ductions in survival and growth. This was true even when
exposure was removed prior to egg hatching, although removal
did somewhat alleviate these effects. In the latter case,
the detrimental effects were probably due to the toxic action
of PCB absorbed by the embryo through the egg choriOn during
exposure. Toxicity may have been enhanced by stresses acting
upon prematurely hatched alevins. Acetone was also present

in the test system, but is not believed to be an important

40

factor in the results. Separate tests with coho eggs and
fry in our laboratory (unpublished data) showed that acetone
concentrations of 800 mg/l had no effect on the survival of
fry. Still it may be argued that acetone could serve as a
predisposing or facilitating agent for the action of known
toxicants, such as PCB.

Eggs and alevins continuously exposed to even the lowest
PCB level tested here (4.35 ug/l) had significant reductions
in at least three of the parameters measured. On the other
hand, the alevins from eggs removed from less than 8 ug/l
showed no overall effects of the PCB treatment, except for
premature hatching. The significance of these findings with
respect to successful long-term fish production can only be
evaluated on the basis of longer tests.

Jensen et al. (1970) have correlated a mortality of
Swedish salmon embryos (eggs) with PCB residues in the eggs
of 7.7-34.0 ug/g (fat basis). The severity of losses was
directly related to the amount of residues in the egg lots.
Eggs taken from adult fish reared in freshwater had higher
PCB residues and mortality than eggs taken from adults reared
in the sea. Our results in part indicate that PCB is capable
of killing salmon embryos within the egg and thus support

Jensen's data implicating PCB in egg losses.

PCB UPTAKE BY STEELHEAD TROUT

Methods and Materials

This investigation measured the rates of PCB accumula-
tion in young steelhead trout exposed for 24 days to five
concentrations of Aroclor 1254 (3.25-51.30 ug/l). The test
concentrations were delivered by a proportional diluter
equipped with a 1 m1 toxicant introduction system. Acetone
was not delivered to the control tank.

On July 13, 1971, fifty fish (avg. weight 0.35 9, avg.
length 36 mm, 60 days old) were randomly assigned to each
test chamber. The daily test routine was as described earl-
ier for the PCB-DDT combination bioassays. As fish died,
brains (and the brain case) were excised and placed in indi-
vidual aluminum foil envelopes. The bodies were placed in
similar packets and both were then frozen. On days 5, 10,
and 16 of the test, four live fish, apparently in good condi-
tion, were removed from each test tank, sacrificed, and
processed in the same manner. One liter water samples were
taken from each tank every five days for determination of
actual PCB exposure concentrations (Table 8).

Fish brain and body tissues were extracted by maceration

in a tissue grinder with several portions of hexane which had

41

42

Table 8. The Aroclor 1254 concentrations, in ug/l, present
in the test tanks during the PCB uptake study as
determined by gas chromatography.

..........

 

 

 

No. of
Nominal Measured:SD SE .Range . ...analyses
0 --- --- --- 6
5 3.25:0.16 0.080 3.01- 3.41 5
10 6.15:0.32 "0.161 5.22- 7.80 5
20 10.45:3.39* 1.517 6.00-14.12 6
40 27.80:4.11 1.840 21.94-31.96 6
80 51.30:6.66 2.980 41.79—60.64 6

 

*

Diluter delivered only about half of the desired amount of
toxicant to this test chamber on days 7 through 11 of this
test.

43

been heated to 60°C. The extraction efficiency for this
method was not determined, but repeated extractions beyond
the normal end point in more than 10 trials yielded no addi-
tional residues. Extracts were concentrated under a gentle
stream of nitrogen or by rotary evaporator and transferred

to a graduated centrifuge tube before gas chromatographic
analysis. The GLC conditions were the same as those described
for water analysis in the general methods. Clean-up pro-
cedures were found to be unnecessary. The heights of peaks

4 through 9 of Aroclor 1254 (Figure 2) were again used for
quantitation although some alteration of the relative heights
of peaks 4, 8, and 9 occurred in these tissue samples

(Figure 8).

The stock fish contained 1-2 ug/g chlorinated hydro-
carbon residues (Johnson, l972), but these amounts were insig-
nificant compared to the levels of PCB accumulated during the
exposure period. A

The PCB concentrations in live and dead fish were pooled
to calculate linear regression lines describing the uptake
rates of PCB, since examination of the raw data points indi—

cated no difference in their residue concentration.

Results

The uptake of Aroclor 1254 by steelhead trout based on
whole body residues (Figure 9) was dependent on time of
exposure and exposure concentration. Uptake was linear over

the time period monitored, from 100 to 576 hours.

44

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mo mEmHmoumEouno oHQEmm osmmflo Ucm Unmocmmm mo GOmflHmQEOo d

 

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45

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46

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oomomxo nmem unomonmou muoo onm moamnmfluu como .onfla noflmmoumou
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.m ouomflm

47

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48

The rate of PCB uptake in ug/g per hour from each test
concentration is given by the slope of the regression lines
in Figure 10. The rates were 3.106, 1.832, 1.044 and 0.189
pg/g per hour from the highest to lowest exposure concentra-
tion, respectively. To determine whether uptake rates could
be predicted if an uptake rate from a specific PCB concentra-

tion was known, the simple equation:

test concentration "X"

_-.n_._.. I! II =
known concentration "Y" X known uptake rate from Y

 

predicted uptake rate from "X"

was used to predict uptake rates from the experimental concen-
trations. The data of the highest PCB level (51.30 pg/l,
3.106 ug/g per hour) was selected as the "known" information

and the following values were then calculated:

 

"x" UPTAKE VALUES (pg/g per hour)
(Hg/l) Predicted Actual
27.80 1.680 1.832
10.45 0.633 1.044
3.25 0.195 0.189

Within experimental limits the predicted uptake rates are good.
A diluter malfunction in the delivery of toxicant to the test
chamber of 10.45 ug/l is responsible for the discrepancy be-
tween those values (see Table 8).

The degree to which Aroclor 1254 was concentrated in the

fish tissues over the exposure concentration at 100 hour

 

49

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masom mo mcamun ocm mwflmon map ca mCOflumuucmocoo vmma Hoaooum

.oa mudem

50

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51

intervals was calculated using the regression equation of
each line. Whole body residue concentrations derived by
substituting the appropriate number of hours into each equa—
tion were divided by the exposure concentration to obtain
magnification factors (Table 9). The factors increase with
time because uptake and exposure were continuous over the
test period. The reason for the inverse relationship between
exposure concentration and the calculated magnification
factors is not immediately clear however since uptake rate
declined in direct proportion to exposure concentration, as
shown above. In fact, when concentration factors are calcu-
lated from the regression equations excluding the Y—intercept
values, then the resultant factors (Table 10) show that con-
centration occurs fairly uniformly over all test levels at
about 6000X increments per 100 hours. The magnitude of the
actual concentration factors is therefore related to uptake
phenomena occurring before 100 hours in the exposure period
when uptake has not yet become linear. During this time it
appears that fish exposed to low PCB concentrations take up
relatively more PCB than do the fish in the higher PCB concen-
trations. This process stops sometime before 100 hours of
exposure but the effect on bioconcentration is still evident
in this test at 576 hours, when exposure ended.

Residue concentrations in the bodies and brains of fish
killed by exposure to 51.30 ug/l Aroclor 1254 are shown in

Figure 10. These data suggest that after 200 hours exposure,

52

Table 9. Actual magnification factors calculated by substi—
tution of the appropriate number of hours into the
regression equations describing PCB uptake.

 

 

Time (hours)
100 200 300 400 500

 

Exposure 51.30 I 7,600 13,700 19,700 25,800 31,800
Concentration 27.80 I 10,700 17,300 23,900 30,500 37,100

(Mg/1) 3.25 I 15,200 21,000 26,800 32,000 38,500

 

Table 10. Magnification factors calculated by substitution
of the appropriate number of hours into the re-
gression equations, excluding the "Y" intercept
value, describing PCB uptake.

Time (hours)
100 200. 300 400 500

 

Exposure 51.30 I 6,050 12,100 18,150 24,200 30,250
Concentration 27.80 I 6,600 13,200 19,800 26,350 32,950

(pg/1) 3.25 I 5,850 11,700 17,500 23,400 29,200

 

53

or after accumulating about 400 ug/g, the further accumula-

tion of PCB in the brain lags behind whole body uptake.

Discussion

 

The PCB exposure concentrations of this study were se-'
lected to insure that fish in the higher levels would die
during the course of the experiment, since the original pur-
pose of this study was to compare the brain concentrations
of PCB in live and dead fish from the same exposure concentra-
tion, such as was done with dieldrin and bluegills by Hogan
and Roelofs (1971). During the progress of the work however,
the brains of the sacrificed fish were improperly extracted
and brain data was obtained from dead fish only. The bodies
of the test fish were therefore analyzed and are presented
here as an uptake experiment. But when the purpose of the
test shifted, the exposure concentrations became too high to
present an uptake picture that is environmentally significant.
Nonetheless, the data do provide a quantitative demonstration
of the accumulation of a hydrophobic chlorinated hydrocarbon
from water by fish. The apparent greater efficiency of up-
take early in the test in the lower concentrations evident
here has appeared in the work of others, was mentioned by
Chadwick and Shumway (1969), but has not been discussed.
Concentration factors calculated from the PCB data of Duke
et a1. (1970, Table II, p. 179), the dieldrin data of Chadwick

and Shumway (1969, Figure 4, p. 93) or Chadwick and Brocksen

54

(1969, Figure 2, p. 696), and the parathion-blood data of
Mount and Boyle (1967, Figure 2, p. 1185) are highest in fish
exposed to the lowest concentrations.

A definite explanation for this phenomenon is, to our
knowledge, lacking. However, a hypothesis may be formulated
based on the work of P. O. Fromm, Michigan State University.
First it is assumed that the primary site of uptake of toxi-
cants into fish is at the gills (Holden, 1962; Premdas and
Anderson, 1963; Fromm and Hunter, 1969). Fromm et a1. (1971)
have shown that the rate of fluid flow through isolated-
perfused rainbow trout gills is decreased when 1 mg/l di—
eldrin, or several other chemicals, is added to the perfusion
medium. Earlier these authors (Richards and Fromm, 1969)
found that the addition of epinephrine to the perfusion fluid
caused an increase in the overall flow rate through the gills.
It is possible then that fish exposed to various concentra-
tions of toxicants in uptake studies have gill perfusion rates
decreased in direct proportion to the concentration of toxi-
cant in the exposure system. This would allow the fish ex-
posed to lower concentrations to accumulate relatively more
chemical than the fish in the higher concentrations. Perhaps
then, after a time, the fish in the higher concentrations make
a physiological adaptation in the form of an increased hormone
secretion and the gill perfusion rate returns to a more normal

level.

55

Alternatively, it may be argued that the reduction in
gill perfusion rates occurs and persists in fish exposed at
all PCB levels for as long as the exposure is continued.

The effect would occur most rapidly in those fish in the
highest PCB concentration and slowest in the lowest concen-
tration. But all fish would eventually show the same degree
of gill perfusion reduction. For a time however the fish

in the lower PCB concentrations would accumulate PCB at a
relatively higher rate than the fish in the higher concentra-
tions. Either version of this hypothesis would account for
the observed effects in uptake studies, but both are obvi-
ously only speculation.

Our overall results are similar to the findings of
Chadwick and Brocksen (1969) who studied accumulation of di-
eldrin from water by sculpin. The chief difference between
uptake of dieldrin in sculpin and PCB in steelhead appears
to be in magnitude, with PCB being more readily concentrated.

The highest bioconcentration factors measured in this
study were about 32,000-38,000 and these were still increasing
at the conclusion of the exposure. Hansen et a1. (1971) found

that spot (Leiostomus xanthurus) accumulated Aroclor 1254

 

37,000 times over the 1 ug/l exposure concentration in 28 days
and that this was about the plateau of the concentration
factors. Preliminary results reported by Stallings and Mayer
(1972) indicate that bluegills concentrated Aroclor 1254

26,000-71,000 times from water concentrations of 2-10 ug/l in

56

an 11 week exposure period but the authors did not state
whether these were plateau levels. They also reported that
PCB is not rapidly taken up from contaminated food by coho
salmon which suggests that water contamination may be the

major source of PCB residues in fish.

STEELHEAD TROUT TOXICITY BIOASSAY

Methods and Materials

The same PCB exposure system as described for the uptake
experiment was used to conduct a thirty day toxicity bioassay
with young steelhead trout. At the start of the test, 240
fish (avg. length 47.6 mm, avg. weight 1.07 gm, 100 days old)
were anesthetized in a 50 ml/l solution of MS-222, weighed,
measured, and allowed to recover in fresh water for one hour
before introduction into the test chambers. Forty fish per
concentration were tested. The daily bioassay routine was as
described for the PCB-DDT combination tests. After thirty
days of exposure, surviving fish were sacrificed and re-
measured.

Water samples were taken from each test chamber every
five days for actual toxicant concentration determination
(Table 11).

Median survival times (MST) and their 95% confidence

limits were calculated according to Litchfield (1949) and mean

lengths were compared by the Student t-test (p 0.05). The
test was not replicated but the sample size (n = 40) tended
to minimize the relative error of the calculated MST values

(Jensen, 1972).

57

58

Table 11. The Aroclor 1254 concentrations, in ug/l, present
in the test tanks during the thirty day toxicity
test as determined by gas chromatography.

 

 

No. of
Nominal Measured:SD SE Range analyses
0 O --- --- 6
5 2.55:0.18 0.081 1.93- 3.47 6
10 4.75:1.57 0.702 2.52— 6.66 6
20 10.15:2.09 0.935 8.21-13.33 6
40 20.40:3.34 1.492 16.05-26.12 6

80 39.40:5.42 2.422 32.63-47.61 6

 

59

Results

Cumulative percentages of dead fish plotted on prob—
ability scale against time to death (Figure 11) show that
39.40 and 20.40 ug/l Aroclor 1254 killed half of the test
fish in 293 and 400 hours (12.2 and 16.6 days), respectively.
Only six fish died in 10.15 ug/l PCB and none died in.the
two lower concentrations or the control. Dying fish dis:
played the symptoms of PCB poisoning described in the PCB-DDT
bioassay section which include loss of appetite, listlessness,
and slow death.

Fish growth as measured by length was significantly
reduced by exposure to greater than 4.70 ug/l PCB (Table 12).
Differences in the weight of the fish could not be statis-
tically tested since fish were initially weighed in small
groups to minimize handling stress and standard error esti-
mates were thus not available. However, the average weight
of fish exposed to PCB showed obvious declines as the test

concentrations increased.

Discussion

The chronic nature of PCB toxicity is evident from these
bioassay results. The thirty day lethal threshold concentra-
tion of Aroclor 1254 to steelhead trout lies between 10.15
and 20.40 ug/l. A threshold concentration of 17 09/1 was
determined by plotting a toxicity curve with five LC50 esti-

mates made from the bioassay data according to the method

 

Figure 11.

60

The results of a thirty day toxicity test in
which young steelhead trout were exposed to
five concentrations of Aroclor 1254. .Mortal-
ity was incomplete in 10.15 ug/l as indicated
by the broken line (C). Fish growth was re-
duced at concentrations not acutely toxic.

 

MORTALITY

%

90

70

50

3O

10

 

61

STEELHEAD TOXICITY TEST

/
/

m

/

A

 

(“m—c
// /°/
/° I
l 1 I 1 l 1 L1 1 1
200 400 600 1000
TIME (hrs)
MEDIAN SURVIVAL TIMES
Llne PCB (ppb) MST c. l.
' A 39.40 293 256-335
B 20.40 400 337 - 475
c 10.15 — —

Figure 11

62

.mo. n m um anew Houucoo Eoum ucmHmMMHm manomofiwficmflm

.mo. 0 m .ucmummmflo kHDQMOHMHGmHm you who mmsouw

.mmsoum Ham cw ow

¥

o
u o HofluHoHo

 

 

m I- mo.H .NH.oHom.mm no.8Hoe.nv os.mm

m ms.a mo.H oo.vumm.om om.mumo.ns oe.om

em mv.H mo.H «Hm.nuoo.vm mv.munv.ov mH.oH

om om.a mo.H rmm.ouom.om mm.euem.ne mn.v

o8 mn.a mo.H oH.mHmn.om mv.wumo.oe mm.m

Am mm.H mo.H m~.musm.om ne.eHmv.ns o

a nmflcflm canopm nmflcflm amuumum Aa\mzv .ocoo
Hmoflm Amy anonmz A251 numawq musnomxm mom

 

 

ou Guzmomxm Hmumm can mnommn usouu ommnammum mo pnmflm3 paw sumcma one

ovaH HOHOOH¢ MO m¢OflU6HHGOUGOO mSOHHM>

.OH mHQMB

63

recommended by Standard Methods (American Public Health
Association et al., 1971). That the growth of the test fish
was affected at levels below 16 ug/l PCB indicates the need
for longer tests in evaluating the effects of PCB.

The acute toxicity of Aroclor 1254 to fish has been in-
vestigated by others. Mayer (1972) determined a 25 day LC50
of 27 ug/l Aroclor 1254 for rainbow trout (water temperature
17°C, alkalinity 159 mg/l) but stated that toxicity had not
yet become independent of exposure time. Stalling (1970)
estimated a median lethal time of 200 hours (8.3 days) for
rainbow trout in a 50 ug/l solution of Aroclor 1254 at 20°C.

Two species of marine fish were exposed to 5 ug/l
Aroclor 1254 by Hansen et a1. (1971) for various periods of
time up to 56 days. Pinfish (Lagodon rhomboides) showed 66%:
mortality after 14 days while 62% of spot (Leiostomus

xanthurus) died in 45 days.

 

CONCLUSION

Young coho salmon and steelhead trout exposed to Aroclor
1254 concentrations above 15 ug/l will be killed if the ex-
posure period is long enough. Aroclor 1254 must therefore be
classified as a compound very toxic to fish. It is unlikely
however, that fish kills attributed to PCB poisoning in
natural waters will be reported because 1) we know of no
lakes or rivers presently suitable for aquatic life that have
been found to have PCB levels above 15 ug/l for sustained
periods; 2) high PCB concentrations in natural waters are
likely to be complexed with suspended organic and inorganic
material in the water and thus possibly be unavailable for
direct uptake by fish; 3) the behavior of fish, through either
natural movement patterns or avoidance reactions (Sprague and
Drury, 1969; Hansen, 1969) is such that lingering in polluted
water for extended periods of time would be unlikely.

Our results show that 5 09/1 Aroclor 1254 affected the
growth of salmon and trout after less than 30 days exposure.
Longer tests, if the experiences of other investigators with
other toxicants are applicable to this case, will show that
much lower levels of PCB are detrimental to fish growth and

reproduction. Such tests are presently being conducted at

64

65

the United States National Water Quality Laboratory, Duluth,
Minnesota.

Biologists attempting long tenm-bioassays with chlori-
nated hydrocarbons like PCB are presented with difficult
problems. For example, how does one go about determining
whether the observed reductions in Lake Michigan coho salmon
reproduction are related to PCB (or DDT) residues in the
eggs? To run a chronic test, exposure concentrations would
have to be 1 to 30 ng/l (PPtr), levels hard to detect ana-
lytically. The fish food, if not natural, would have to be
fortified with semi-"natural" levels of PCB residues. The
exposure system would have to be large and the exposure
period long--a minimum of one year and possibly three.
Furthermore, a duplicate experiment would probably have to
be conducted in which both PCB and DDT were incorporated into
the water and food. What guarantee would there be that when
the experiment was nearing completion that the residues in
the fish eggs would approach the levels found in natural
waters? Costs for such tests would be very high and probably
not justifiable unless the results could definitely contribute
to legal proceedings in matters related to PCBs.

PCB pollution, as with other types of contamination, is
most easily remedied by prevention. Fortunately, measures
can and have been taken with respect to the PCB problem.
Monsanto, the sole producer of PCB in this country, has modi-

fied the formulation of its "Aroclor" series and placed

66

restrictions upon many of their uses. States have initiated
monitoring programs to locate the point sources for PCB con-
tamination. The United States Environmental Protection

Agency (EPA) has suggested an interim maximum permissible
concentration of 10 ng/l (pptr) PCBs in natural waters. If
this standard is enforced, industrial hygiene will have to

be upgraded. If the PCB levels in receiving waters near
industrial centers can be limited to 10 ng/l, the concentra-
tions in dilutent bodies of water such as Lake Michigan should
fall to undectable, and hopefully biologically safe, levels

within several years.

LITERATURE C ITED

LITERATURE CITED

American Public Health Association, American Water Works
Association, and Water Pollution Control Federation,
1971. Standard methods for the examination of water
and wastewater, 13th Ed. New York, N. Y. 874 pp.

Armour, J. A. and J. A. Burke. 1970. Method for separating
polychlorinated biphenyls from DDT and its analogues.
J. Assoc. of Agric. Chem. 53: 761-768.

Chadwick, G. G. and R. W. Brocksen. 1969. Accumulations of
dieldrin by fish and selected fish-food organisms.
J. Wildlife Mgmnt. 33(3): 693-700.

Chadwick, G. G. and D. L. Shumway. 1970. Effects of dieldrin
on the growth and development of steelhead trout. IN
The biological impact of pesticides in the environment.
Ed. J. W. Gillett. Environment Health Series No. 1.
Oregon State University, Corvallis, Oregon. 210 pp.

Cutkomp, L. H., H. H. Yap, E. Y. Cheng, and R. B. Koch.
1971. ATP—ase activity in fish tissue homogenates and
inhibitory effects of DDT and related compounds. Chem-
Biol. Interactions 3(6): 439-447.

Davis, P. W. and G. A. Wedemeyer. 1971. Na+, K+-activated-
ATP-ase inhibition in rainbow trout: a site of organo-
chlorine pesticide toxicity. Comp. Biochem. Physiol.
40(3B): 823-827.

Duke, T. W., J. I. Lowe, and A. J. Wilson, Jr. 1970. A poly-
chlorinated biphenyl (Aroclor 1254) in the water sediment,
and biota of Columbia Bay, Fla. Bull. Environ. Contam.
Tox. 5(2): 171-180.

Fromm, P. O. and R. C. Hunter. 1969. Uptake of dieldrin by
isolated perfused gills of rainbow trout. J. Fish. Res.
Bd. Canada 26(7): 1939-1942. -

Fromm, P. 0., B. D. Richards, and R. C. Hunter. 1971. Effects

of some insecticides and MS-222 on isolated-perfused
gills of trout. Prog. Fish. Cult. 33(3): 138-140.

67

68

Fuhremann, T. W., and E. P. Lichtenstein. 1972. Increase
in the toxicity of organophosphorus insecticides to
houseflies due to PCB compounds. Tox. Appl. Pharm.
22: 628-640.

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. Amer. Fish. Soc. 100(2):
207-214.

Hansen, D. J. 1969. Avoidance of pesticides by untrained
sheepshead minnows. Trans. Amer. Fish Soc. 98(3):
426-429.

Hansen, D. J., P. R. Parrish, J. I. Lowe, A. J. Wilson Jr.
and P. D. Wilson. 1971. Chronic toxicity, uptake, and
retention of Aroclor 1254 in two estuarine fishes.
Bull. Environ. Contam. Tox. 6(2): 113-119.

Henderson, C., A. Inglis and W. L. Johnson. 1971. Organo—
chlorine insecticide residues in fish--fall 1969
National Pesticides Monitoring Program. Pestic. Monit.
J. 5(1): l-ll.

Hickey, J. J., J. A. Keith and F. S. Coon. 1966. An ex-
ploration of pesticides in a Lake Michigan ecosystem.
J. Appl. Ecol. 3 (Suppl.): 141-154.

Hogan, R. L. and E. W. Roelofs. 1971. Concentrations of
dieldrin in the blood and brain of the green sunfish,
Lepomis cyanellus, at death. J. Fish. Res. Bd. Can.
28(4): 610-612.

 

Holden, A. V. 1962. A study of the absorption of 14C-

labelled DDT from water by fish. Ann. Appl. Biol.
50: 467-477.

Interdepartmental Task Force on PCBs. 1972. Polychlorinated
biphenyls and the environment. Report No. ITF-PCB—72-1.
Com-72-104l9. National Technical Information Service.
U. S. Dept. of Commerce. Springfield, Virginia.

Jensen, A. L. 1972. -Standard error of LC50 and sample size
in fish bioassays. Wat. Res. 6: 85-89. ‘

Jensen, 8., N. Johannsson, and M. Olsson. 1970. PCB-indi-
cations of effects on salmon. PCB conference, Stockhom.
Sept. 29, 1970. Swedish Salmon Research Institute--
Report LFI MEOD 7/1970.

69

Johnson, H. E. and C. Pecor. 1969. Coho salmon mortality
and DDT in Lake Michigan. Trans. Thirty-Fourth N. Amer.
Wldl. and Nat. Resources Conf.: 159-166.

Johnson, H. B., and R. C. Ball. 1972. Organic pesticide
pollution in an aquatic environment. Advances in chem-
istry series, Number 111, "Fate of organic pesticides
in the ag. environment," American Chemical Society.

Johnson, J. E. 1972. The influence of chlorinated hydrocar-
bon residues and rearing temperature on survival and
growth of coho salmon and rainbow trout sac fry. M. S.
thesis. Michigan State University. 89 pp.

Koch, R. B., D. Desaiah, H. H. Yap, and L. K. Cutkomp. 1972.
Polychlorinated biphenyls: effect of long-term expo-
sure on ATP—ase activity in fish, Pimephales promelas.
Bull. Environ. Contam. Tox. 7(213): 87-92.

 

Lake Michigan Interstate Pesticides Committee. 1972. An
evaluation of DDT and dieldrin in Lake Michigan.
(Ecological Research Series EPA-R3-72-003) Offices of
Research and Monitoring. U. S. Environmental Proection
Agency, Washington, D. C.

Lichtenstein, E. P., K. R. Schulz, T. W. Fuhremann, and T. T.
Land. 1969. Biological interactions between plasti-
cizers and insecticides. J. Econ. Entomol. 62(4):
761-765.

Lichtenstein, E. P. 1972. PCBs and interactions with insec-
ticides. Environmental Health Perspectives. 1: 151-153.

Litchfield, J. T. Jr. 1949. A method for rapid graphic
solution of time-percent effect curves. J. Pharmac.
Exp. Ther. 97: 399-408.

Lyles, C. H. 1967. Fishing statistics of the United States.
Statistical Digest 61. U. S. Government Printing Office.
490 pp.

Matsumura, F., K. C. Patil and G. M. Boush. 1971. DDT metab—
olized by microorganisms from Lake Michigan. Nature.
230: 325-326.

Mayer, F. L. 1972. PCB Newsletter (4th) U. S. Environ-
mental Protection Agency, Office of Research and Moni-
toring, National Water Quality Laboratory, Duluth,
Minnesota. Mimeo.

70

McAllister, W. A. Jr., W. L. Mauck and F. L. Mayer, Jr.
1972. A simplified device for metering chemicals in
intermittent-flow bioassays. Trans. Amer. Fish. Soc.
101(3): 555-557.

Morgan, J. R. 1972. Effects of Aroclor 1242 (a polychlori-
nated biphenyl) and DDT on cultures of an alga, proto-
zoan, daphnid, ostracod, and guppy. Bull. Environ.
Contam. Tox. 8(3): 129-137.

Mount, D. I. and W. A. Brungs. 1967. A simplified Dosing
apparatus for fish toxicology studies. Water Res.
1: 21-29.

Mount, D. I. and H. W. Boyle. 1969. Parathion--use of blood
concentrations to diagnose mortality of fish. Env. Sci.
Tech. 3(11): 1183-1185.

National Institute of Environmental Health Sciences, National
Institute of Health, Department of Health, Education and
Welfare. 1972. Environmental health perspectives.
Experimental issue No. 1, April, 1972.

Nisbet, I. C. T. and A. F. Sarofim. 1972. Rates and routes
of transport of PCBs in the environment. Environmental
Health Perspectives. 1: 21-38.

O'Brien, R. D. 1967. Insecticides: action and metabolism.
Academic Press, New York. 332 pp.

Premdas, F. H. and J. M. Anderson. 1963. The uptake and
detoxification of C14-labelled DDT in Atlantic salmon,
Salmo salar. J. Fish. Res. Bd. Canada 20(3): 827-837.

 

Reinert, R. E. 1970. Pesticide concentrations in Great Lakes
fish. Pestic. Monit. J. 3(4): 233-240.

Richards, B. D. and P. O. Fromm. 1969. Patterns of blood
flow through filament and lamellae of isolate-perfused
rainbow trout (Salmo gairdneri) Gills. Comp. Biochem.
Physiol. 29: 1063-1070.

 

Sprague, J. B. and D. E. Drury. 1969. Avoidance reaction of
salmonid fish to representative pollutants. Adv. in
Wateeroll. Res. Proc. 4th Int. Conf. pp. 169*179.

Stalling, D. L. 1970. PCB Newsletter (4th). U. S. Environ-
mental Protection Agency, Office of Research and Moni-
toring, National Water Quality Laboratory, Duluth,
Minnesota. Mimeo.

71

Stalling, D. L. and F. L. Mayer, Jr. 1972. Toxicities of
PCBs to fish and environmental residues. Environmental
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mental Health Perspectives. 1: 51-54.

APPENDIX

BREAKDOWN OF LOSSES OF EGG, SAC-FRY AND ALEVINS
EXPOSED TO VARIOUS CONCENTRATIONS
TO AROCLOR 1254

72

73

EGG-FRY EXPOSURE TEST

0 ug/l PCB with

0 mg/l acetone

 

 

 

 

 

 

 

 

 

 

 

 

 

 

11-29-71 ................ 92_ eggs
‘7
‘r———- 10 dead eggs
12-13-71 ....... . ........ 89 eggs
0 ug/l PCB w_ 0 mg/l acetone 0 mg/l acetone
_45 eggs _44_ eggs
~————+~ 1 dead eggs —————+- 0 dead eggs
,____,. l hatched dead r————+- 3 hatched dead
V)
12-22—71. 43_ fry hatched 41 fry hatched
j
1_———+ 2 dead fry dead fry
1-15-72.. 41 fry
43 er—hatChed = 95.6% hatched 41 er—hatChed = 93.2% hatched
45 eggs 44 eggs
41 fry _ . 41_fry = .
43 fry hatched — 95.3% surv1va1 4l.fry hatched 100.0% surV1va1
41 fryfi = 91.1% survival 41 fry = 93.2% survival
45 eggs 44 eggs

74
EGG-FRY EXPOSURE TEST

4.35 ug/l PCB with ‘50

mg/l.acetone

 

11-29-71.. ........ . ..... 1 8 eggs
_1___.
——————>— 13 dead eggs
V
12-13-71....... ........ . 95 eggs

 

 

 

I

4-35 ug/l PCB w ég'mg/l acetone

 

49 eggs
—T——

4

r—-> 2 hatched dead

-————9- dead eggs

 

 

 

 

 

12-22-71. %3_ fry hatched
._____9. 6 dead fry
I
V
1-15-72.._;_ fry
43 frxhatched = 87 8% hatched
49 eggs .
37 fr); = ‘
43 fry hatched 86.0% surv1val
37 fry __ = 75.5% survival
49 eggs

1

_vag/l acetone

 

.19. eggs

._____,. 0

dead eggs

~—————+- 2 hatched dead

V
44

._71_

5 _12 dead fry

V

 

fry hatched

 

 

 

 

32 fry
44 fryrhatched = 95.7% hatched
46 eggs
32 fry _ '
44 fry hatched'— 72.7% surV1va1
32 fry = 69.6% survival
46 eggs

75
EGG-FRY EXPOSURE TEST

'7.75 ug/l PCB with 100 mg/l acetone

 

11—29-71................ 110' eggs

--——->- 4 dead eggs

 

Y
12-13“7l ..... 0000.00.00.
. 106 eggs

i l

7.75 ug/l PCB w_100 mg/l acetone '100 mg/l acetone

 

 

 

 

 

 

 

 

 

 

 

 

56 eggs :6 eggs
———e* 6 dead eggs ————>- 1 dead eggs
‘b—e- 6 hatched dead ‘———>- 4 hatched dead
V \Y
12-22-71...44 fry hatched 45 fry hatched
8 dead fry 1 dead fry
1-15-72....36 fry 44_fry
44 fry hatChEd = 78.6% hatched 45 fry hatChed = 90.0% hatched
56 eggs 50 eggs
36 fry = . 44 fry = .
44 fry hatched 81.8% surv1va1 45 fry hatched 197.8% surv1va1
36 fr
44 eggs = 64.3% survival :3 2358 = 88.0% survival

11-29-71 ................

12-

12-

76

EGG-FRY EXPOSURE TEST

15.35 ug/l PCB with 200 mg/l acetone

 

 

V
13-71 ..... . ..........

193 eggs
7

98 eggs

-——> 5 dead eggs

 

V

15.35 ug/l PCB w 200 mg/l acetone

 

50 e 5
fi_ 99

————>’15 dead eggs

 

22-71..19 fry hatched

_1 dead fry

1-15-72...12 fry

 

 

 

l9 fry hatched = 38.0% hatched*
50 eggs

12 fry - _

19 fry hatched — 63.2% surVival
l2 fry = 24.0% survival
50 eggs

————)'l6 hatched dead*

200 mg/l acetone

 

22 eggs

———4> 3 dead eggs

I——-—+o l hatched dead

I

44 fry hatched

 

_4_dead fry

40 fry

 

 

 

44 fry hatchEd = 91.7% hatched
48 eggs

40 fry _ '

44 fry hatched — 90.9% surv1val
40 fry = 83.3% survival
48 eggs

*Bacteria or fungi caused excessive egg mortality in these
chickens.

77
EGG-FRY EXPOSURE TEST

25.90 ug/l PCB with 400 mg/l acetone

 

11-29-71............. 109 eggs

-———+- 7 dead eggs

 

V
102 eggs

25.90 Hg/l PCB w 400 mg/l acetone 400 mg/l acetone

 

 

 

 

 

 

 

 

 

59 eggs 4; eggs

-———%> 1 dead eggs ~———5— 1 dead eggs

~———4> l hatched dead c———€> 0 hatched dead

V I)
12-22—72..57 fry hatched 42 fry hatched

3 dead fry 10 dead fry

1-15-72..._4 fry 32 fry
57 fry hatChed = 96.6% hatched 42 £33 hatChed = 97 7 hatched
59 eggs 43 eggs '
4 fry = - 32 fry, = -
57 fry hatched 7.0% surv1va1 42 fry hatched 76.2% surv1va1
4 fry = 6.8% survival 32 fry = 74.4% survival

 

 

59 eggs 43 eggs

78
EGG-FRY EXPOSURE TEST

56.40 Ug/l PCB with 800 mg/l acetone

 

 

 

11-29-71. ...... 00000... 2?- eggs
-———€>16_dead eggs
{V

12—13-7100000 000000000 84 eggs

 

1 e
I I

66.40 ug/l PCB y 8 0 mg/l acetone 800 mg/l acetone

 

 

46 eggs 36 eggs

————+rlg dead eggs ————+-_g_dead eggs

'———_T’_§ hatched dead

II

—-€>‘_6 hatched dead
Y

 

 

12-22-71..gg fry hatched

66 dead fry

1—15-72..._; fry

 

 

25 fry hatched

16 dead fry

1.5. fry

 

 

29 fry hatChed = 63.0% hatched 25 fry hatChed = 65.8% hatched
46 eggs 38 eggs

3 fry 2 . 15 fry = -

jg fry hatched 10.3% surv1va1 25 fry hatched 60.0% surv1va1

3 fry

= 6.5% survival
46 eggs

 

15

fry = 39.5% survival

 

38

eggs

MICHIGAN STATE UNIV. LIBRARIES

31293011096132