to aerate water in the test tanks. Activated carbon filters were
installed in the air lines which eliminated that source of con-
tamination. Another source of contamination by PCBs and PAEs
was avoided by using redistilled acetone in preparing stock
solutions of toxaphene. Well water used in the study was checked
for the presence of PCB and PAEs, but none were detected at a
sensitivity of 10 ng/l.

Physical reduction by grinding followed by extraction of the fish
tissues were accomplished by the procedures of Benville and
Tindle56 and Hesselberg and Johnson57, Initial sample cleanup
was automated gel permeation chromatography>8 followed by modified
silicic acid chromatography-9, Quantitation of toxaphene residues
was done by gas liquid chromatography (GLC) with 63Ni-electron
capture detection. A 2.1 m long x 2 mm i.d. coiled glass column
packed with 3% (w/w) OV-7 on chromosorb W-hp was utilized with

a nitrogen flow rate of 40 ml/min. A column temperature of 200 C
was used for total toxaphene residues to reduce the long retention
time, and a column temperature of 180 C was optimum for toxaphene
isomer resolution. The minimum detection limit of toxaphene in
fish tissue was 0.05 mg/g, but below 0.1 pg/g was difficult to
quantitate. Complete details on the toxaphene residue technology
used in this study are described by Stalling and Huckins®9,

Recovery of toxaphene from spiked tissue samples was 97% & 100%,

from water spiked at 100 ng/1 was 97% & 104%, at 50 ng/1 was
75.6% & 84.7% and at 25 ng/1 was 44% & 56%. , 8

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10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

Hemphill, J. E. Toxaphene as a Fish Toxin. Progr. Fish-Cult.
16:41-42, 1954.

Henegar, D. L. Minimum Lethal Levels of Toxaphene as a
Piscicide in North Dakota Lakes. Bureau of Sport Fisheries
and Wildlife, U.S.D.I. Wash., D.C. Resource Pub. 7. 1966.
16 v.

Hooper, F. F. and A. R. Grzenda. The Use of Toxaphene as a
Fish Poison. Trans. Amer. Fish. Soc, 85:180-190, 1957.

Johnson, W. C. Toxaphene Treatment of Big Bear Lake, California.

Calif. Fish and Game. 52(3):173-179, 1966.

Johnson, W. D., G. F. Lee, and D. Spyridakis. Persistence of
Toxaphene in Treated Lakes. Air Water Pollut, 10:555-560,
1966.

Kallman, B. J., 0. B. Cope, and R. J. Navarre. Distribution
and Detoxification of Toxaphene in Clayton Lake, New Mexico.
Trans, Amer, Fish, Soc, 91(1):14-22, 1962.

Rose, E. T. Further Notes on Toxaphene in Fish Population
Control. Quart. Biol. Rep. 10:5-7, 1958.

Royer, L. M. Bioassay Method for the Determination of

Toxaphene in Lake Water. J. Fish. Res. Bd. Can, 23(5) :723-
727, 1966.

Stringer, G. E. and R. G. McMynn. Three Years Use of Toxaphene
as a Fish Toxicant in British Columbia. Can, Fish Cult.
28:37-44, 1960.

Tanner, H. A. and M. L. Hayes. Evaluation of Toxaphene as a
Fish Poison. Colorado Coop. Fish. Res. Unit, Quart. Rep.
(3-4:31-39, 1955.

Terriere, L. C., U. Kiigemagi, A. R. Gerlach, and R. L.
Borovicka. The Persistence of Toxaphene in Lake Water and

its Uptake by Aquatic Plants and Animals. J. Agr. Food
Chem. 14(1) :66-69, 1966.

Cushing, C. E., Jr. and J. R. Olive. Effects of Toxaphene and

Rotenone uvon the Macroscopic Bottom Fauna of Two Northern

Colorado Reservoirs. Trans, Amer. Fish. Soc, 86:294-301, 1956.

37


SECTION IV
MATERIALS AND METHODS

GROWTH, REPRODUCTION, AND MORTALITY

This portion of the study basically was conducted according to
the recommended procedures for partial chronic tests with brook
trout by EPA~’. Yearling brook trout were obtained from the
Manchester National Fish Hatchery, Iowa, on vil 3, 1972. The
fish were treated with 2 mg/l of Hyamine 35004 continuously for
one hour daily for three consecutive days. On April 10, 26 fish,
12 for growth and 14 for residue studies, were placed in each of
12 stainless steel tanks measuring 51 cm deep x 137 cm long x 36 cm
wide and having a water depth of 30 cm. Well water (Table 1) was
delivered to the tanks at a rate of 800 ml/min/tank through a
proportional diluter system modeled after Mount and Brungs*>.

Each tank was aerated with filtered air to maintain oxygen concen-
trations above 70% of saturation. The adult fish were fed the
Modified Oregon Test Diet 4t ad libitum throughout the study. The
young fry were fed a commercial trout starter (EWOS).

All fish were weighed and measured (total length) on April 22,

and 12 fish from each tank were tagged with surgical wound

clips attached to the anterior base of the dorsal fin to follow
growth on the same group of fish throughout the study. Growth

was determined on the twelve tagged fish on August 11 and again

on October 5. After the last growth determination, the fish were
thinned to two males and four females per tank and two spawning
substrates were placed in each tank. Fifty eggs from each spawning
were placed in incubator cups for hatchability and subsequent
growth determinations on the surviving fry. After spawning ceased,
four adults from each tank were preserved for pathological evalua-
tion. Most of the remaining eggs from each spawning (#250) were
placed in separate incubator cups for determining viability
(formation of neural keel after 11-12 days). All eggs died in the
eyed stage and the mortality appeared to be caused by a covering
of a clear slime bacteria. Additional eggs were obtained from
White Sulphur Springs National Fish Hatchery, West Virginia on
January 12, 1973. One hundred eggs were placed in each of six
incubator cups per duplicate tank. The eggs were exposed to
toxaphene for 22 days before the median hatch date. When hatching


TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)

1 REPORT NO 2. 3. RECIPIENT'S ACCESSION NO,
EPA-600/3-75-013
4. TITLE AND SUBTITLE 5. REPORT DATE
November 1975 (Issuing Date)
TOXAPHENE EFFECTS ON REPRODUCTION, GROWTH, AND ls. PERFORMING ORGANIZATION CODE
MORTALITY OF BROOK TROUT
7, AUTHOR(S) 8. PERFORMING ORGANIZATION REPORT NO.

Foster L. Mayer, Jr., Paul M. Mehrle, Jr., and
William P. Dwyer

9. PERFORMING ORGANIZATION NAME AND ADDRESS 10. PROGRAM ELEMENT NO,
Fish-Pesticide Research Laboratory 1BA021 (ROAP/Task 16AAK
Fish and Wildlife Service 71. CONTRACT /BRANX NO.
United States Department of the Interior
Columbia, Missouri 65201 EPA-TAG-0153(D)

12. SPONSORING AGENCY NAME AND ADDRESS 13. TYPE OF REPORT AND PERIOD COVERED
Environmental Research Laboratory Final (4/72-3/74)
Office of Research and Development 14. SPONSORING AGENCY CODE

U.S. Environmental Protection Agency
Duluth, Minnesota 55804
15. SUPPLEMENTARY NOTES

EPA-ORD

16. ABSTRACT

Yearling brook trout (Salvelinus fontinalis) were continuously exposed to toxaphene
(0, 39, 68, 139, 288, and 502 ng/1) in a flow-through diluter system. Day length
and water temperature were altered monthly to correspond to natural conditions.
Adult growth was reduced in the 288 and 502 ng/1 toxaphene exposures, and the added
stress of spawning activities caused extensive mortalities in these concentrations.
The numbers of eggs spawned and percent viability were inversely related to
increasing toxaphene concentrations. All groups of fry exposed to toxaphene had
reduced rates of growth and survival. Biochemical investigations on fry backbones
demonstrated that bone collagen may be a sensitive indicator of normal and abnormal
growth and development prior to being observed in the whole fish. Toxaphene was
accumulated by brook trout 5,000 to 76,000 times that in the water and the more
chlorinated isomers of toxaphene were preferentially stored.

ike KEY WORDS AND DOCUMENT ANALYSIS
ja. DESCRIPTORS fb. IDENTIFIERS/OPEN ENDED TERMS |c. COSATI Field/Group
Growth Brook trout 6C
Collagens Toxaphene 6F
Reproduction (biology) Chronic effects 6T
Mortality Continuous exposure
Pesticides Residue dynamics
Trout Salmonids
Partial life cycle
18, DISTRIBUTION STATEMENT 19, SECURITY CLASS (This Report) 21, NO, OF PAGES
UNCLASSIFIED 51
20. SECURITY CLASS (This page) 22. PRICE
RELEASE TO PUBLIC UNCLASSIFIED

EPA Form 2220-1 (9-73) 43


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ABSTRACT

Yearling brook trout (Salvelinus fontinalis) were continuously
exposed to toxaphene (0, 39, 68, 139, 288, and 502 ng/1l) in a
flow-through diluter system. Day length and water temperature
were altered monthly to correspond to natural conditions. Adult
growth was reduced in the 288 and 502 ng/1 toxaphene exposures,
and the added stress of spawning activities caused extensive
mortalities in these concentrations. The numbers of eggs
spawned and percent viability were inversely related to
increasing toxaphene concentrations. All groups of fry

exposed to toxaphene had reduced rates of growth and survival.
Biochemical investigations on fry backbones demonstrated that
bone collagen may be a sensitive indicator of normal and abnormal
growth and development prior to being observed in the whole fish.
Toxaphene was accumulated by brook trout 5,000 to 76,000 times
that in the water and the more chlorinated isomers of toxaphene
were preferentially stored.

This report was submitted in fulfillment of Contract Number
EPA-IAG-0153 (D) by the Fish-Pesticide Research Laboratory,
Fish and Wildlife Service (USDI) under the sponsorship of the
Environmental Protection Agency. Work was completed March 1974.

iii


The data presented in our study suggested that toxaphene decreased
collagen synthesis which resulted in whole-body collagen and
backbone collagen concentrations being decreased. Concomitantly,
the rate of mineralization appeared to proceed normally and was
not affected by toxaphene. The increase in calcium and phosphorous
in the backbone of fry exposed to 68 and 139 ng/1 of toxaphene
after 90 days of exposure, was hypothesizedto be due to the
decrease in collagen content. The resulting effect of toxaphene
on bone composition was an increase in the ratio of minerals:
collagen which is depicted in Fig. 2. The implication of this
type of bone composition on trout fry development beyond 90

days remains to be elucidated, as does the mode of action of
toxaphene causing this condition.

RESIDUE DYNAMICS

Toxaphene in water was concentrated by yearling brook trout from
5,000 times in the low concentration to 16,000 times in the high
concentration (Table 10). Equilibria between water concentrations
of toxaphene and residues in brook trout were reached after
approximately 140 days of continuous exposure. After 161 days,
whole body residues of toxaphene in fish from the 288 and 502 ng/1
concentrations were 2.4 and 8.0 yg/g, respectively. Toxaphene
concentrations in adult brook trout of this magnitude would be
considered detrimental since extensive mortality occurred in the
288 and 502 ng/1 toxaphene exposures just prior to and during
spawning activities (Fig. 1). Kallman, Cope, and Navarre also
reported symptoms of poisoning associated with toxaphene concent-
trations of 8 to 15 ag/g in bullheads (Ictalurus natalis and I.
melas). Fish from all toxaphene exposures were analyzed for the
distribution of toxaphene residues between the fillet and the
remaining tissue (offal) after 161 days. Toxaphene residues in
the offal were 2.8 to 4.2 times that found in the fillet (Table
11). The highest residue detected in the fillets was 4.9 pg/g,
whereas, residues in offal were 13 mg/g. All toxaphene residues
in fillets were below the allowable FDA limit of 5 ug/g for
toxaphene in foods consumed by man°”.

Residues of toxaphene in the eggs immediately after spawning were
0, 0.4, 0.9, 1.8, 2.9, and 5.2 mg/g for the 0, 39, 68, 139, 288,
and 502 ng/1 exposure concentrations, respectively. Egg viability
was significantly reduced (P<0.05) when egg concentrations of
toxaphene were equal to or greater than 0.9 ug/g (Table 5). Since
the original eggs died in the eyed stage, no relationship between
toxaphene residues in eggs and egg hatchability could be determined.

26


SECTION V
RESULTS AND DISCUSSION
GROWTH, REPRODUCTION, AND MORTALITY

After three months of exposure to toxaphene, the yearling brook
trout were easily excitable and of lighter coloration in the 288
and 502 ng/1 concentrations. The presence or absence of fin
erosion appeared to be an index to the excitability of the fish.
While conducting the three month growth measurements, examination
of the fish revealed an incidence of fin erosion of 3, 7, 3, 10,
31, and 50% in the 0, 39, 68, 139, 288, and 502 ng/1 concentrations,
respectively. By late August, the nervousness had subsided and
most of the eroded fins had healed. Warner, Peterson, and

Bor gman’ 1 also reported an increased responsiveness to external
stimuli in goldfish (Carassius auratus) continuously exposed to
1.8 g/l of toxaphene for 96 hours.

The last measurements to determine the effect of toxaphene on
growth were completed October 5. The growth of the fish was
significantly decreased (P<0.05) in the 288 and 502 ng/1 toxaphene
concentrations only after six months of continuous exposure (Table 3).
All fish visually appeared to be in good health and no mortality had
occurred. However, just prior to spawning, the additional stress
of physiological changes occurring before and during spawning
caused a 50 and 100% mortality in the 288 and 502 ng/1 toxaphene
concentrations, respectively (Fig. 1). Eight percent of the fish
died in each of the 39, 68, and 139 ng/l concentrations, but no
fish died in the controls. Histopathological analyses of selected
tissues indicated atrophy of liver cells, degeneration of pancreatic
acinar tissue, and proliferation of interrenal tissues of the kidney
in all fish exposed to toxaphene.

The 96-hr LC50 of toxaphene for 16-month-old brook trout was
10.8 pg/1 (Table 4) and was approximately 38 times that amount of
toxaphene killing 50% of the fish in the chronic study.

Egg viability was reduced at a much lower concentration of toxaphene
than was parental growth (Table 5). Concentrations of toxaphene

of 68 ng/l and higher significantly reduced viability (P<0.05).

No relationship between the number of females spawning or numbers

of spawns and toxaphene exposure was evident except in the highest
concentration. The reduction in the number of females spawning

and numbers of spawns in the high concentration was due to the
extensive mortality prior to spawning. The number of eggs per
spawn tended to be less in the higher toxaphene concentrations,

but the difference was not statistically significant.

12


Due to the loss of the eggs in the first part of the study,
additional eyed eggs were obtained and exposed to toxaphene
for 22 days before hatching. No effect on hatching was
observed. The resulting fry were continuously exposed to
toxaphene for an additional 90 days for growth determination.

Growth of the fry as measured by length, was significantly
affected (P<0.05) after 30 days of toxaphene exposure in the
139 and 288 ng/1 concentrations (Table 6). All fry in the

502 ng/1l treatments were dead by the end of the 30 day period.
After 60 days exposure, growth was significantly reduced in all
groups of fish exposed to toxaphene (39-502 ng/l). The effects
on the weight of fry were the same as on length at 90 days.

Mortality of the brook trout fry in all toxaphene concentrations
was higher than that in the control fish at 30 days and thereafter
(Table 7). The mortality rate of fry in the controls was also
high during the first 60 days of exposure and all fish fed poorly.
However, no disease organisms or other problems could be found.
The rate of mortality in the control fish decreased after 60

days of exposure and the fish began feeding normally.

In our 12 month study, the no-effect concentration of toxaphene
to brook trout under continuous exposure conditions was below
39 ng/1 based on fry growth and mortality. Nicholson, et al.
reported toxaphene concentrations ranging from 7-410 ng/1 in
water from Flint Creek, Alabama. Concomitant studies by Grzenda,
et al.62 were conducted on zooplankton, bottom fauna, and fish
populations of Flint Creek. There was no convincing evidence
that continuous toxaphene contamination resulted in any gross
damage to the aquatic organisms. The fish that Grzenda, et al.
studied were warm water fish and may have been more tolerant to
toxaphene. Also, the fish in Flint Creek may have developed
resistance to toxaphene as described by Ferguson, et a1.63 in
Mississippi.

Mount and Stephan64 proposed that an application factor, calculated
by dividing the maximum acceptable toxicant concentration (MATC),
the highest continuous toxicant concentration that has no adverse
effect on growth, reproduction, and survival, by the 96-hr LC50
value, be used to determine safe concentrations of toxic pollutants.
However, Eaton’? states that the lethal threshold concentration
might provide better acute values for calculating application
factors than 96-hr LC50 values. Further, Mount and

Stephan suggest that the application factor for a given toxicant,

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samples of backbones from approximately 20 fry at each of the
30, 60, and 90 day sampling periods were assayed for pure
collagen according to the method of Flanagan and Nichols°>.

The concentration of HyP in pure collagen was measured and used
to derive a factor for converting the concentration of HyP in
backbone hydrolysates to concentration of collagen in the
backbone. The following formula was used to estimate the amount
of collagen in the backbone:

1
g HyP x g HyP = g collagen
g bone g collagen g bone

The percent of HyP in the pure collagen extracts was 6.51, 6.56,
and 6.61 in the 30, 60, and 90 day samples, respectively. The
average of the three values, 6.56%, was used to derive the con-
version factor for each group. The data were analyzed by analysis
of variance and the least significant difference multiple means
comparison test.

RESIDUE DYNAMICS

The sampling schedule for yearling brook trout during the 161 day
uptake phase of the study is presented in Table 2. The fish sampled
from the 39, 139, and 502 ng/1 toxpahene exposures were reserved

to determine which toxaphene isomers are accumulated or eliminated
by brook trout. However, total toxaphene residues were determined
on all fish analyzed. Toxaphene residues were determined on both
fillet and offal of four adult fish from each toxaphene concentra-
tion after 161 days of exposure. Upon completion of the uptake
phase, twelve adult fish from each concentration were placed in
uncontaminated water at 9 C to determine the elimination rate of
toxaphene. Three fish were sampled from each exposure after 7,

14, 28, and 56 days in the fresh water. Yearling and adult fish
were analyzed individually. Thirty, 25, 20, 15, and 10 brook trout
fry were sampled from each duplicate tank after 7, 15, 30, 60, and
90 days of exposure, respectively. The fry sampled from each duplicate
were pooled for residue analysis, since at least two grams of tissue
were required for analysis. All fish were frozen immediately after

sampling.

Various sources of polychlorinated biphenyls (PCBs) and phthalic
acid ester (PAE) contaminants were major obstacles in the measure-
ment of low concentrations of toxaphene in water and fish. A
major source of PCB contamination was found to be in the air used


SECTION I
CONCLUSIONS

Growth of yearling brook trout is reduced through continuous
exposure to 288 and 502 ng/1 of toxaphene for six months.

Increased stress due to spawning activities in the presence
of toxaphene results in high rates of mortality at toxaphene
concentrations of 288 and 502 ng/1.

Toxaphene concentrations of 68 ng/1 and higher reduce egg
viability.

Toxaphene increases the ratio of minerals to organic content
in the vertebral column of brook trout fry during a 90-day
exposure. The no-effect concentration of toxaphene is less
than 39 ng/1l.

The effects of chronic toxaphene exposure on brook trout fry
growth and development are predictable by collagen measure-
ments on fry after only seven days.

The no-effect concentration of toxaphene on growth of brook
trout fry is below 39 ng/1 after 90 days of exposure.

Toxaphene was accumulated by brook trout 5,000 to 76,000
times that in water.

The more chlorinated toxaphene isomers are preferentially
stored by brook trout while the less chlorinated ones are
more rapidly eliminated.

The maximum acceptable toxicant concentration of toxaphene
in water is below 39 ng/l for brook trout.


Table 9.

BACKBONE COMPOSITION OF BROOK TROUT FRY AS AFFECTED

BY TOXAPHENE
= — —
Days Backbone Toxaphene concentration, ng/1
hatch|{ constituent 0 39 68 139 288 502
30 Hydroxyproline, | 51 34> | 26> | 20° 25> -c
mg/g
% collagen b b b b
(estimated)* 78 51 40 30 38 -
% phosphorous 5.7 7.3 9.9 8.8 11 -
% calcium 1.2 1.0 1.3 1.0 1.4 -
b
60 Hydroxyproline,} 20 14> 14> 11 - -
ng/g
b b b
% collagen 29 20 20 16 - -
(estimated)
b
% phosphorous 12 11 9.9 7.7 - -
% calcium 2.6 2.8 2.5 1.3 - -
90 Hydroxyproline,} 19 16” | 16° | i6? - -
mg/g
b b b
% collagen 30 25 25 25 - -
(estimated)
b b
% phosphorous 11 16 20 20 - -
b b
% calcium 10 15 21 21 - -

a
Estimated collagen based upon pure collagen containing 6
hydroxyproline.

b Significantly different from controls (P<0.05), n = 6 to

© all ft

sh were dead.

24

-56%


experimentally determined for one species of fish in one type

of water, might be applicable to other waters and other aquatic
species. The MATC of a given toxicant could be estimated there-
after for other species, by determining the lethal threshold
concentration for the species of concern in the appropriate water
quality and multiplying by the previously determined application
factor for the toxicant.

The MATC of toxaphene for brook trout was not established in

this study. However, the effects of toxaphene on fry growth

were supported by physiological and biochemical determinations
(see PHYSIOLOGY AND BIOCHEMISTRY) and a MATC of toxaphene for
brook trout would have to be below 39 ng/1. An application factor
of 0.0095 was derived by using the 39 ng/1 concentration from the
chronic study and the lethal threshold concentration of 4.1 pg/1
(Table 4). The application factor closely approximates the 0.01
factor recommended in establishing water quality criteria for
organochlorine insecticides44,

PHYSIOLOGY AND BIOCHEMISTRY

Development of trout fry is usually evaluated by making length
and weight measurements. In the present study we evaluated the
effects of toxaphene on differentiation and development of brook
trout fry by the conventional method as well as by biochemical
techniques to ascertain if a method for evaluating toxicant effects
on development prior to discernible effects on weight and length
gain could be established. The biochemical technique applied
was measuring the synthesis of the amino acid hydroxyproline and
the protein collagen. The use of collagen as a representative
"differentiated" protein for study of embryonic development has
been reported in amphibian embryological investigations-2»

During the first cleavage stages of frog embryos (Xenopus laevis)
collagen synthesis is repressed, but during gastrulation collagen
synthesis begins and increases 500-fold through neurulation,
hatching, and posthatching stages.

Collagen is the major fibrous protein of all vertebrates and most
of the invertebrate phyla®5, its most important function in
vertebrates is serving as the major component of the organic matrix
of connective tissues and bones. It is around and within the
collagen fibrils that calcification and mineralization takes place
and as development proceeds, more calcium and phosphate salts are
deposited resulting in mature bone.

21


LIST OF FIGURES

PAGE
Relationship between mortality and spawning 13
activity of adult brook trout exposed to 288 and
502 ng/1 of toxaphene. Spawning activity was
based on the number of spawns in time of all
groups of fish expressed as a percentage.
Effect of toxaphene on backbone composition of 27

brook trout fry after 30, 60, and 90 days of
exposure.


CONTENTS

ABSTRACT

LIST OF FIGURES

LIST OF TABLES

ACKNOWLEDGMENTS

Sections

I CONCLUSIONS

Il RECOMMENDATIONS

TIL INTRODUCTION

Iv MATERIALS AND METHODS

VI

VII

GROWTH, REPRODUCTION, AND MORTALITY
PHYSIOLOGY AND BIOCHEMISTRY
RESIDUE DYNAMICS

RESULTS AND DISCUSSION
GROWIH, REPRODUCTION, AND MORTALITY
PHYSIOLOGY AND BIOCHEMISTRY
RESIDUE DYNAMICS

REFERENCES

LIST OF PUBLICATIONS

PAGE

iii
vi

vii

viii


Table 2. SAMPLING SCHEDULE FOR TOXAPHENE RESIDUES IN YEARLING
BROOK TROUT
(number/concentration)

Days of Water concentration, ng/1
Exposure 0 39 68 139 288 502
1 4 4 4
3 4 4 4
7 4 4 4
10 4 4 4
18 4 4 4
24 4 4 4
60 6 4 6 4 6 4
140 6 4 6 4 6 4
161 8 4 8 4 8 4

10


ACKNOWLEDGMENTS

Assistance in the diluter construction and maintenance by
W. A. McAllister, D. D. Holz, and L. McLain was greatly
appreciated. We thank Ms. M. E. DeClue, 0. Yarbrough, and
S. Cummins for their technical assistance in performing the
collagen, calcium, and phosphorous assays. Also, special
thanks to P. L. Crutcher, Pathologist, for conducting the
histopathological examinations. Toxaphene residue analyses
were conducted by J. N. Huckins with assistance from

J. L. Johnson. Portions of the study were funded by the
Fish and Wildlife Service, United States Department of the
Interior.

viii


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33.

34,

35.

36.

37.

38.

39.

40.

41.

42,

43.

44,

Sanders, H. 0. Pesticide Toxicities to Tadpoles of the
Western Chorus Frog Pseudacris triseriata and Fowler's
Toad Bufo woodhousii fowleri. Copeia 2:246-251, 1970.

Sanders, H. 0. Toxicity of Some Insecticides to Four Species
of Malacostracan Crustaceans. Bureau of Sport Fisheries and
Wildlife, U.S.D.1I. Technical Paper 66. 1972. 19 p.

Sanders, H. O. and 0. B. Cope. Toxicities of Several
Pesticides to Two Species of Cladocerans. Trans. Amer. Fish,
Soc. 95(2):165-169, 1966.

Sanders, H. 0. and O. B. Cope. The Relative Toxicities of
Several Pesticides to Naiads of Three Species of Stoneflies.
Limnol. and Oceanogr. 13(1):112-117, 1968.

Schoettger, R. A. and J. R. Olive. Accumulation of Toxaphene

by Fish Food Organisms. Limnol, and Oceanogr. 6(2):216~219, 1961.

Workman, G. W. and J. M. Neuhold. Lethal Concentrations of
Toxaphene for Goldfish, Mosquitofish, and Rainbow Trout with
Notes on Detoxification. Progr. Fish-Cult. 25:23-30, 1963.

U. S. Environmental Protection Agency. Recommended Bioassay
Procedures for Brook Trout Salvelinus fontinalis (Mitchill)
Partial Chronic Tests. National Water Quality Laboratory.
Duluth, Minnesota. January 1972. 12 p.

Mount, D. I. and W. A. Brungs. A Simplified Dosing Apparatus
for Fish Toxicology Studies. Water Res. 1:21-29, 1967.

National Academy of Sciences. Nutrient Requirements of
Domestic Animals. No. 11. Nutrient Requirements of Trout,
Salmon, and Catfish. Nat. Acad. Sci. Wash. D.C. 1973. 57 p.

McKim, J. M. and D. A. Benoit. Effect of Long-term Exposures
to Copper on Survival, Reproduction, and Growth of Brook

Trout Salvelinus fontinalis. J, Fish, Res, Bd. Can. 28:655-
662, 1971.

McAllister, W. A., Jr., W. L. Mauck, and F. L. Mayer, Jr.
A Simplified Device for Metering Chemicals in Intermittent-
flow Bioassays, Ixans, Amer, Fish, Soc, 101:555-557, 1972.

U. S. Environmental Protection Agency. Proposed Criteria

for Water Quality. Vol. I. U. S. Environmental Protection
Agency. Wash., D. C. October 1973. 425 p.

39


SECTION III
INTRODUCTION

The extensive use and persistence of organochlorine insecticides
have resulted in their widespread occurrence in the environment.
Their presence in aquatic habitats as well as their adverse
effects on aquatic organisms have been reportedl,2, Although
much of the use of organochlorine insecticides has been reduced
in recent years, some are still extensively used; 30 to 40
million pounds of toxaphene are applied annually on crops and
livestock in the United States3. Toxaphene is the technical
grade of chlorinated camphene, containing 67-69% chlorine. The
empirical formula is CjQHj9Clg. Its primary use is as an
insecticide on cotton, but it is also registered for use on
certain grains, alfalfa, fruit, and vegetables. Since the use
of DDT was restricted in 1969, toxaphene has often been used to
replace it, both by itself and in combination with other insecti-
cides*.

Previously, there have been few reports of toxaphene residues in
fish, water, or food products. However, toxaphene, hgs been
identified in analysis of water and tissue samples””’. Due to the
numerous toxaphene isomers (=175)°, it is unlikely that most
routine gas chromatographic analyses would be sensitive to less
than 1-5 wg/1 of toxaphene in water or 0.5 ug/g in whole fish.

The presence of polychlorinated biphenyls, other organochlorine
insecticides, and phthalic acid esters in environmental samples
further complicate analytical results unless special separation
and cleanup or other special procedures are used. For example,
Nicholson, et al.9 measured the annual cycle of toxaphene residues
in a river and found them to range from 7 to 410 ng/1. However,
this level of sensitivity was possible only by collection of
toxaphene from 19 m> of water on carbon.

The persistence of toxaphene in aquatic environments was demon-
strated in past years when it was evaluated as a piscicidel0-20,
Detoxification times for lakes that had been treated with toxaphene
ranged from four weeks to over six years based on fish mortality

and residue studies. The time required for detoxification of
lentic waters was dependent on physical and chemical characteristics
of the water. Detoxification rates of toxaphene were apparently
greater in more eutrophic situations.


SECTION II
RECOMMENDATIONS

Additional research on other aquatic organisms is recommended
to determine more accurately the no-effect concentration of
toxaphene.

Consideration should be given to using hydroxyproline and
collagen measurements as early biochemical indicators of
growth and developmental changes in fishes.

Utilizing the data in this study, water concentrations of
toxaphene should be below 39 ng/1 to protect aquatic life.


different toxaphene isomers in brook trout were not due to any
alterations in the toxaphene that the fish were exposed to.
Water samples from the 502 ng/1_ concentration were analyzed
using difference chromatography Oand the resulting curves
demonstrated that no major changes in early or late eluting
toxaphene isomers occurred in water prior to uptake by brook
trout.

35


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23


45,

46.

47.

48.

49.

50.

51.

52,

53.

54.

55.

56.

57.

58.

Benoit, D. A. and F. A. Puglisi. A Simplified Flow-splitting
Chamber and Siphon for Proportional Diluters. Water Res. 7:
1915-1916, 1973.

Drummond, R. A. and W. F. Dawson. An Inexpensive Method for
Simulating Diel Patterns of Lighting in the Laboratory. Trans.
Amer. Fish, Soc. 99(2):434-435, 1970.

Eaton, J. G. Chronic Malathion Toxicity to the Bluegill
Lepomis macrochirus Rafinesque). Water Res. 4:673-684, 1970.

Snedecor, G. W. Statistical Methods. Ames, Iowa, Iowa State
Univ. Press, 1965. 534 p.

Cochran, W. G. and G. M. Cox. Experimental Designs. New York,
N.Y., John Wiley & Sons, Inc., 1968. 617 p.

Litchfield, J. T., Jr. and F. Wilcoxon. A Simplified Method
of Evaluating Dose-effect Experiments. J, Pharmacol. Therap.
96:99-113, 1949,

Woessner, J. F. The Determination of Hydroxyproline in Tissue
and Protein Samples Containing Small Proportions of this
Amino Acid. Arch. Biochem. Biophys. 93:440-447, 1961.

Rollins, J. W. and R. A. Flickner. Collagen Synthesis in
Xenopus Oocytes after Injection of Nuclear RNA of Frog
Embryos. Science 178:1204-1205, 1972.

Green, H. B., B. Goldberg, M. Schwartz, and D. D. Brown.
The Synthesis of Collagen During Development of Xenopus
laevis. Developmental Biol. 18:391-400, 1968.

Fiske, C. H. and Y. Subbarow. The Colorimetric Determination
of Phosphorous. J. Biol. Chem. 63:373-400, 1925.

Flanagan, B. and G. Nichols. Metabolic Studies of Bone in
vitro. J. Biol. Chem. 237:3686-3692, 1962.

Benville, P. E. and R. C. Tindle. Dry Ice Homogenization
Procedure for Fish Samples in Pesticide Residue Analysis.

J. Agr. Food Chem. 18:948-949, 1970

Hesselberg, R. J. and J. L. Johnson. Column Extraction of
Pesticides from Fish, Fish Food, and Mud. Bull. Environ.
Contam. & Toxicol. 7:115-120, 1972.

Tindle, R. C. and D. L. Stalling. Apparatus for Automated
Gel Permeation Cleanup for Pesticide Residue Analysis.
Analytical Chemistry 44(11):1768-1773, 1972.

40


Hydroxyproline and hydroxylysine in the collagen molecule are
derived from the hydroxylation of their respective precursors,
proline and lysine, after their incorporation into the
polypeptide (protocollagen). The enzyme collagen hydroxylase
or peptidyl proline hydroxylase, which commences gts activity
during gastrulation, catalyzes the hydroxylation® . Ascorbic
acid, ketoglutarate, and ferrous iron are cofactors for the
enzyme.

Toxaphene significantly decreased growth in brook trout fry after
30, 60, and 90 days of exposure as determined by both length-
weight measurements and biochemical analyses. Only biochemical
analyses were performed on whole fry after the 7 and 15 day
samplings, since the fry were too small to be handled for length-
weight measurements; however, significant decreases in growth,

as determined biochemically, were observed. In the 7 and 15

day samplings, toxaphene concentrations of 68, 139, and 288 ng/1
significantly decreased (P<0.05) the concentration of hydroxy-
proline in the sac fry (Table 8). The no-effect toxaphene
concentration was between 39 and 68 ng/l. These results suggest
that toxaphene significantly decreased collagen synthesis and
consequently decreased the precursor molecules of the organic
framework of skin, scale, and bone. The use of whole-fry
hydroxyproline analyses as an indicator or predictor of future
growth and development appears to be quite adequate based upon
length-weight and biochemical analyses performed on fry from

the 30, 60, and 90 day samplings. Instead of whole-fry analyses
from these latter sampling periods, we performed collagen, as well
as mineral analyses on the backbones.

All toxaphene concentrations significantly altered (P<0.05)

the composition of backbones after 30, 60, and 90 days of exposure
(Table 9). More pronounced effects were observed after 90 days
of exposure than after 30 and 60 days of exposure. These
results demonstrate changes similar to those thought to be in-
dicated by the hydroxyproline measurements from the 7 and 15

day samplings, i.e., collagen content was significantly de-
creased by toxaphene, and the effect of toxaphene on collagen
synthesis prior to the 30 day sample had a significant impact

on bone composition and development. Concomitantly, toxaphene
decreased the growth of the trout fry as determined by length
measurements (Table 6). However, only the 139 and 288 ng/l
exposures decreased growth during the first 30 days, whereas all
toxaphene concentrations decreased growth as determined biochem-
ically. These results illustrate that the actual growth (length)

22


10

11

12

13

14

15

LIST OF TABLES
CHEMICAL CHARACTERISTICS OF WELL WATER AT THE FISH-
PESTICIDE RESEARCH LABORATORY

SAMPLING SCHEDULE FOR TOXAPHENE RESIDUES IN YEARLING
BROOK TROUT

PAGE

10

THE EFFECT OF TOXAPHENE ON YEARLING BROOK TROUT GROWTH 14-15

LC50's (AND 95% CONFIDENCE LIMITS) OF TOXAPHENE TO
YEARLING BROOK TROUT DETERMINED DATLY

THE EFFECT OF TOXAPHENE ON BROOK TROUT REPRODUCTION
BROOK TROUT FRY GROWTH AS AFFECTED BY TOXAPHENE

MORTALITY IN BROOK TROUT FRY CONTINUOUSLY EXPOSED TO
TOXAPHENE

COLLAGEN SYNTHESIS IN BROOK TROUT SAC FRY AS AFFECTED
BY TOXAPHENE

BACKBONE COMPOSITION OF BROOK TROUT FRY AS AFFECTED
BY TOXAPHENE

WHOLE-BODY RESIDUES OF TOXAPHENE IN YEARLING BROOK
TROUT CONTINUOUSLY EXPOSED TO TOXAPHENE

TOXAPHENE RESIDUES IN ADULT BROOK TROUT FILLET AND
OFFAL AFTER 161 DAYS OF EXPOSURE

WHOLE-BODY RESIDUES OF TOXAPHENE IN BROOK TROUT FRY
CONTINUOUSLY EXPOSED TO TOXAPHENE

TOXAPHENE RESIDUES IN ADULT BROOK TROUT DURING
ELIMINATION STUDY

MEAN PERCENT CHANGE OF SELECTED GLC PEAKS FROM TOXAPHENE
RESIDUES IN ADULT BROOK TROUT AFTER CESSATION OF 502
ng/1 TOXAPHENE EXPOSURE

MEAN PERCENT CHANGE OF SELECTED GLC PEAKS FROM RESIDUES

IN YEARLING BROOK TROUT CONTINUOUSLY EXPOSED TO
TOXAPHENE

vii

16

17
19

20

23

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29

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32

33

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Brook trout fry from the second batch of eggs (adults not exposed)
concentrated toxaphene up to 76,000 times that in the exposure
water in only 15 days (Table 12). Uptake in all toxaphene exposures
passed through a maximum after 15 days, declined through 60 days,
and then tended to increase again between 60 and 90 days of exposure,
Accumulation factors of toxaphene in the fry exposed for 90 days
was similar to that found in the adults. The reason for the
greater uptake of toxaphene during the first 15 days may have
been due to anatomical differences present during this stage of
life. Gills are generally not functional until the yolk sac

is absorbed and most respiration occurs through a vascular
network enveloping the yolk69, Also, the yolk contains glyceride
fat droplets in which toxaphene would be readily stored and these
nutrients are utilized towards the end of the yolk sac phase

of development®?. Shortly after the yolk sac is absorbed and
just prior to the time that the fry begin to feed (30-34 days
post-hatch), fat content is probably lowest and therefore a

lower equilibrium between toxaphene in the water and that in

the fish occurs. As the fry begin to feed actively (35-36

days post-hatch) and lipid content increases, the accumulation

of toxaphene again increases.

The decline in whole body residues in adult brook trout 56 days
after transfer to uncontaminated water varied with toxaphene
concentration and ranged from 0 to 54% (Table 13). Toxaphene
residues in fish from the 68 and 139 ng/l exposures did not
decline. However, residues in fish from the 288 and 502 ng/1
exposures were reduced by 32 and 51%, respectively. The elin-
ination of toxaphene was reflected by a marked decrease in
toxaphene isomers with GLC retention times less than 1.48
relative to p,p'-DDE (Table 14). These isomers are generally
less chlorinated, more hydrophilic, and therefore can be more
easily degraded and/or excreted than higher chlorinated compo-
nents. However, the abrupt change that occurred in the
elimination of toxaphene isomers up to a relative retention time
of 1.48 and the accumulation of later eluting ones is not fully
understood. Differences of isomer storage in toxaphene residues
from fish during the uptake portion of the study were not as
dramatic as those changes observed in the elimination study
(Table 15). However, with few exceptions the more chlorinated
toxaphene isomers were stored to a greater extent by brook trout
than were the less chlorinated isomers. Preferential storage of
the more chlorinated isomers was probably due to their being
more lipophilic and less easily degraded than the less chlor-
inated isomers. Differences in storage and elimination of

30


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19


DISCLAIMER

This report has been reviewed by the Environmental Research
Laboratory, U.S. Environmental Protection Agency, and approved
for publication. Approval does not signify that the contents
necessarily reflect the views and policies of the U.S. Environ-
mental Protection Agency, nor does mention of trade names or
commercial products constitute endorsement or recommendation
for use.

di


22.

23.

24,

25.

26.

27.

28.

29,

39.

31.

32.

Meehan, W. R. and W. L. Sheridan. Effects of Toxaphene on
Fishes and Bottom Fauna of Big Kitoi Creek, Afognak Island,
Alaska. Bureau of Sport Fisheries and Wildlife, U.S.D.I.
Resource Pub. 12. 1966. 9 pn.

Needham, R. G. Effects of Toxaphene on Plankton and Aquatic
Invertebrates in North Dakota Lakes. Bureau of Sport Fisheries
and Wildlife, U.S.D.I. Resource Pub. 8. 1966. 16 p.

Cohen, J. M., L. J. Kamphake, A. E. Lemke, C. Henderson, and
R. L. Woodward. Effect of Fish Poisons on Water Supplies.

Part 1. Removal of Toxic Materials. J, Amer. Water Works
Ass. 52(12):1551-1566, 1960.

Douderoff, P., M. Katz, and C. M. Tarzwell. Toxicity of Some
Organic Insecticides to Fish. Sewage Ind. Wastes. 25(7):
840-844, 1953,

Ginsburg, J. M. Tests with New Toxicants, in Comparison with
DDT, on Mosquito Larvae and Fish. New Jersey Mosquito
Extermination Ass, 34:132-135, 1947.

Henderson, C., Q. H. Pickering, and C. M. Tarzwell. Relative
Toxicity of Ten Chlorinated Hydrocarbon Insecticides to Four
Species of Fish. Trans. Amer. Fish. Soc. 88(1):23-32, 1959.

Katz, M. Acute Toxicity of Some Organic Insecticides to Three
Species of Salmonids and to the Threespine Stickleback. Trans.

Amer, Fish. Soc. 90(3):264-268, 1961.

Macek, K. J., C. Hutchinson, and 0. B. Cope. The Effects of
Temperature on the Suscentibility of Bluegills and Rainbow
Trout to Selected Pesticides. Bull. Environ. Contam, Toxicol.
4(3) 174-183, 1969.

Macek, K. J. and W. A. McAllister. Insecticide Susceptibility
of Some Common Fish Family Representatives. Trans. Amer, Fish,
Soc. 99(1):20-27, 1970.

Mahdi, M, A. Mortalitv of Some Species of Fish to Toxaphene
at Three Temperatures. Bureau of Sport Fisheries and Wildlife,
U.S.D.I. Resource Pub. 10. 1966. 10 p.

Sanders, H. 0. Toxicity of Pesticides to the Crustacean

Gammarus lacustris. Bureau of Sport Fisheries and Wildlife,
U.S.D.I. Technical Paver 25. 1969. 18 p.

38


SECTION VI
REFERENCES

Johnson, D. YW. Pesticides and Fishes-A Review of Selected
Literature. Trans. Amer, Fish. Soc, 97(4) :398-424, 1968.

Katz, M., F. L. Pederson, M. Yoshinaka, and D. Sjolseth.

Effects of Pollution on Fish Life. J. Water Pollution Control
Fed, 41(6):994-1016, 1969.

Hercules Incorporated. Toxaphene:Use Patterns and Environmental
Asvects. Hercules Inc. Wilmington, Del. November 1970. 172 p.

Courtenay, W. R., Jr. and M. H. Roberts, Jr. Environmental
Effects on Toxaphene Toxicity to Selected Fishes and Crustaceans.
U.S. Environmental Protection Agency. Wash., D.C. April 1973.

74 ve.

Barthel, W. F., J. C. Hawthorne, J. H. Ford, G. C. Bolton,

L. L. McDowell, E. H. Grissinger, and D. A. Parsons. Pesticides
in Water-Pesticide Residues in Sediments of the Lower Mississippi
River and its Tributaries. Pesticides Monitoring J. 3(1):8-66,
1969.

Bugg, J. C., Jvr., J. E. Higgins, and E. A. Robertson, Jr.
Residues in Fish, Wildlife, and Estuaries-Chlorinated Pesticide
Levels in the Eastern Oyster (Crassostrea virginica) from
Selected Areas of the South Atlantic and Gulf of Mexico.

Pesticides Monitoring J. 1(3):9-12, 1967.

Schafer, M. L., J. T. Peeler, W. S. Gardner, and J. E. Campbell.
Pesticides in Drinking Water-Waters from the Mississippi and
Missouri Rivers. Environ. Sci. Technol. 3(12):1261-1269, 1969.

Casida, J. E., R. L. Holmstead, S. Khalifa, J. R. Know, T.
Ohsawa, K. J. Palmer, and R. Y. Wong. Toxaphene Insecticide:
A Complex Biodegradable Mixture. Science 183(4124) :520-521,
1974,

Nicholson, H. P., A. R. Grzenda, G. J. Lauer, W. S. Cox, and

J. I. Teasley. Water Pollution by Insecticides in an Agricultural
River Basin. I. Occurrence of Insecticides in River and Treated
Municipal Water. Limnol. and Oceanogr. 9:310-317, 1964.

36


Table 1. CHEMICAL CHARACTERISTICS OF WELL WATER AT THE FISH-
PESTICIDE RESEARCH LABORATORY

Svecified sensitivity Concentration,
Analyte limits, mg/1 mg/1
Ca 0.1 70
Mg 0.1 27
K 0.5 3.9
S0, 0.01 4.4
ND3 0.05 <0.05
NOy 0.05 < 0.036
NAy /y 0.01 0.066
Phenol 0.001 <0.001
Cly 0.001 <0.001
cL 0.01 29
F 0.01 0.34
cN 0.005 0.006
Fe 0.01 0.014
Cu 0.001 0.0045
an 0.001 <0.001
Cd 0.001 < 0.0005
Cr 0.01 <0.01
Pb 0.001 0.0015
Alkalinity 1.0 237
Hardness (EDTA) 1.0 272
pH 0.1 7.4
Temperature to0.5c 16 ¢



multiple means comparison test (least significant difference)

was used to compare treatments. The effects of toxaphene

on egg viability were determined by conducting an analysis of
variance on the arcsin transformation for proportions (angle =
arcsin percentage) 48 followed by a least significant difference
test. Mortalities occurring in adults and fry were analyzed by

the binomial chi-square analysis for data arranged in two classes49,
LC50s for the acute toxicity test were calculated by the method 9f
Litchfield and Wilcoxon,

PHYSIOLOGY AND BIOCHEMISTRY

Eight brook trout fry from each concentration were sampled at 7,

15, 30, 60 and 90 days after the median hatch date. The fry

from the 7 and 15 day samples were blotted to remove excess water,

then frozen on dry ice. Individual fry were weighed, homogenized

in 2 ml 10% trichloroacetic acid, and centrifuged for 5 minutes

(1500 rpm). The supernatant was discarded, and the protein

precipitate was washed twice with distilled water. Hydrolysis of

the protein precipitate was performed at 120 C in 3 ml of 6N HCL

for 24 hours. The hydrolysate was diluted to 25 ml with distilled
water. Hydroxyproline (HyP) was determined in a 2 ml aliquot according
to the method of Woessner 1. Detection of HyP in the protein
hydrolysate was used as the indicator for collagen synthesis,
because HyP is restricted to collagen or elastin in animal tissues.
Since the total amount of elastin is very small in comparison with
that of collagen, and since the hydroxyproline content is only
about one-tenth as high in elastin, its contribution to the total
hydroxyproline content is negligible compared with that of collagen.
Fry from the 30, 60, and 90 day samples were blotted and placed on
dry ice. After the fry were frozen, the backbone was removed. The
backbone of each fry was dried at 110 C in a forced air oven for

2 hr, then weighed. The dried bone was subjected to hydrolysis

at 120 C in 3 ml 6N HClovernight. The hydrolysate was diluted to
10 ml with distilled water. HyP was determined in the hydrolysate
as previously mentioned. Phosphorous was dgyermined on the hydrolysate
by a modification of the Fiske and Subbarow”” spectrophotometric
method. Calcium was assayed in the hydrolysate using atomic

absorption spectrophotometry. The percision of each of the methods
varied by less than 3%, and the recovery from spiked samples was

95-99%.

53

The amount of HyP found in the bone hydrolysates was used as a
direct measurement for collagen in the bone. The percent collagen
in the backbone was also estimated by an indirect method. Pooled


EPA-600/3~-75-013
November 1975

TOXAPHENE EFFECTS ON REPRODUCTION, GROWTH, AND

MORTALITY OF BROOK TROUT

by

Foster L. Mayer, Jr.
Paul M. Mehrle, Jr.
William P. Dwyer

Fish-Pesticide Research Laboratory
Fish & Wildlife Service
United States Department of the Interior
Columbia, Missouri 65201

Contract No. EPA-IAG-0153 (D)

Project Officer

Leonard H. Mueller
Environmental Research Laboratory
Duluth, Minnesota 55804

U.S. ENVIRONMENTAL PROTECTION AGENCY

OFFICE OF RESEARCH AND DEVELOPMENT

ENVIRONMENTAL RESEARCH LABORATORY
DULUTH, MINNESOTA 55804


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17


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 categories were established to
facilitate further development and application of environmental
technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in
related fields. The five series are:

Environmental Health Effects Research
Environmental Protection Technology
Ecological Research

Environmental Monitoring
Socioeconomic Environmental Studies

UPWNE

This report has been assigned to the ECOLOGICAL RESEARCH series.
This series describes research on the effects of pollution on
humans, plant and animal species, and materials. Problems are
assessed for their long- and short-term influences. Investi-
gations include formation, transport, and pathway studies to
determine the fate of pollutants and their effects. This work
provides the technical basis for setting standards to minimize
undesirable changes in living organisms in the aquatic, terres-
trial and atmospheric environments.

This document is available to the public through the National
Technical Information Service, Springfield, Virginia 22161.


59,

60.

61.

62.

63.

64,

65.

66.

67.

68.

69.

Huckins, J. N., D. L. Stalling, and J. L. Johnson. Silicic
Acid Contaminants and Modification of the Armour and Burke
Method for PCB-pesticide Separations. J. Ass, Offic. Anal.
Chem. (In press).

Stalling, D. L. and J. N. Huckins. Analysis and Gas
Chromatography-Mass Spectrometry Characterization of Toxaphene
in Fish and Water. U. S. Environmental Protection Agency.
Washington, D. C. (In press).

Warner, R. E., K. K. Peterson, and L. Borgman. Behavioral
Pathology in Fish: A Quantitative Study of Sublethal Pesticide
Toxication. J. Appl. Ecol. 3(Supp1):223-247, 1966.

Grzenda, A. R., G. L. Lauer, and H. P. Nicholson. Water
Pollution by Insecticides in an Agricultural River Basin.
II. The Zooplankton, Bottom Fauna, and Fish. Limnol. and
Qceanogr. 9:318-323, 1964,

Ferguson, D. E., D. D. Culley, W. D. Cotton, and R. 0. Dodds.
Resistance to Chlorinated Hydrocarbon Insecticides in Three
Species of Freshwater Fish. BioScience 14(11):43-44, 1964,

Mount, D. I. and C. E. Stephan. A Method for Establishing
Acceptable Toxicant Limits for Fish-Malathion and the
Butoxyethanol Ester of 2,4-D. Trans. Amer. Fish, Soc. 21:
185-193, 1967.

Piez, K. A. and R. C. Likins. The Nature of Collagen. II.

Vertebrate Collagens. In:Calcification of Biological Systems.
Wash., D. C. Ass. Advance. Sci., Publ. No. 64, 1958. 420 p.

Mussini, E., J. J. Hutton, and S. Udenfriend. Collagen Proline
Hydroxylase in Wound Healing, Granuloma Formation, Scurvy,
and Growth. Science 157:927-929, 1967.

Nusagens, B., A. Chantraine, and C. M. Lapiere. The Protein
in the Matrix of Bone. Clin. Opth. Rel. Res. 88:252-274, 1972.

U. S. Environmental Protection Agency. Toxic Pollutants:
Environmental and Health Criteria for Toxaphene. Office of
Water Programs, U.S. Environmental Protection Agency, Wash.,
D. C. 1973. 53 p.

Brown, M. E. The Physiology of Fishes. Vol. I. Metabolism.
New York, N. Y., Academic Press Inc., 1957. 477 p.

41


of the fry was altered by a toxaphene concentration of 139 ng/1
or greater, but the development or composition of bone which
will ultimately reflect the structure and size of the fry is
altered by a concentration less than 39 ng/1.

After 60 days of exposure, toxaphene had a similar biochemical
effect as after 30 days of exposure (Table 9). The collagen
concentration was significantly decreased (P<0.05) by toxaphene,
the phosphorous concentration in the bone from the 139 ng/1
group was significantly decreased (P<0.05), and the calcium
concentration was unchanged by all toxaphene concentrations.

The significance of the phosphorous decrease was not completely
understood. The main point to note, however, was the decrease
in the percentage of collagen between the 30 and 60 day sampling
periods. This decrease was apparently due to the increase in
calcium and phosphorous salts which occurred as the backbone
matured. It appeared that the process of mineralization began
most extensively between the 60 and 90 day period, as judged by
the increase in phosphorous and calcium concentrations. The
mineralization process did not appear to be affected by toxaphene
as was the collagen content. The length measurements in all
groups exposed to toxaphene were significantly decreased (Table 6),
which support the collagen analyses. Thus, the biochemical
analyses and length measurements reflected the same effects
after 60 days of toxaphene exposure.

Similar results as in the 30 and 60 day samples were observed
after 90 days of toxaphene exposure, except that the mineral
contents were significantly altered (P<0.05). The length and
weight measurements were also decreased by all toxaphene exposures
which reflect the same effects as the biochemical analyses. The
no-effect concentration of toxaphene on growth and development

as determined by both length-weight and biochemical measurements
was below 39 ng/l. However, evaluation of collagen and mineral
concentrations after 90 days of exposure gave an indication of
not only status of growth and development, but also the quality
of bone. The mineralization of bone is accomplished by a complex
mechanism which involves the immature bone tissue accumulating
phosphorous salts and then calcium salts67, ‘The data presented
in Table 9 supports this fact. Also, the mineralization process
can take place independently of the collagen matrix, i.e., the
organic substrate is not believed to be necessary for initiation
of mineralization§7.

25


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34


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20


Several investigators have observed acute effects of toxaphene

on aquatic organisms*» 1-38. However, little data are available
from which the chronic effects of toxaphene on fish and aquatic
invertebrates or its biological significance in the aquatic
environment can be assessed. We therefore undertook the following
study to assist the U.S. Environmental Protection Agency in
establishing water quality criteria and standards for toxaphene.
The objectives of the study were: 1) to determine the effect of
continuous exposures of toxaphene on brook trout (Salvelinus
fontinalis) growth, reproduction, and mortality; 2) to investigate
potential physiological and biochemical "predicators" of abnormal
growth and development; 3) to determine the accumulation of
toxaphene in several life stages of brook trout; 4) to determine
the residual isomer changes and elimination rates of toxaphene.


SECTION VIT
LIST OF PUBLICATIONS
Mayer, F. L., Jr., P. M. Mehrle, and W. P. Dwyer. Brook Trout

Development as Affected by Toxaphene. 35th Midwest Fish & Wildlife
Conference Abstracts. St. Louis, Missouri. December 1973. p. 58-59.

42




was complete, four groups of 25 fry each per duplicate tank
were selected from the incubation cups and placed in growth
chambers measuring 14 cm deep x 38 cm x 15 cm wide and having

a water depth of 10 cm. The remaining fry were placed in the
adult tank for the physiological and residue dynamics portions
of the study. Total lengths of the fry were. determined by

the photographic method of McKim and Benoit 2 immediately after
completion of hatching, and at 30, 60, and 90 days thereafter.
The weight of the fry was measured at 90 days. Mortalities were
recorded daily.

A diluter system with the modification of McAllister, Mauck,

and Mayer’? and a dilution factor of 0.5 between the concentrations
was used to deliver five concentrations of toxaphene and a control
for the chronic test. The nominal toxaphene concentrations were
0, 41, 75, 125, 270, and 500 ng/l. The concentrations were
selected to include an estimated no-effect concentration (108 ng/1)
which was determined by multiplying the 96 hr LC50 value (10.8 pg/1)
by 0.01 as recommended in establishing water quality criteria44,
Toxaphene concentrations in the water of each exposure tank were
measured weekly, and the average measured concentrations were 0,
39, 68, 139, 288, and 502 ng/l with an analytical sensitivity of
10 ng/l. Acetone was used as the carrier solvent for toxaphene
and did not exceed 0.28 ml/liter. An experimental-use sample of
toxaphene (X-16189-49) was furnished by Hercules Inc. and was

used throughout the study. Flow-splitting chambers designed by
Benoit and Puglisi45 were utilized to thoroughly mix and divide
each toxaphene concentration for delivery to the duplicate adult
exposure tankg. The water temperature in the tanks was controlled
by Min-0-Cook®) refrigeration units suspended in a circulating
water bath. Artificial daylight was provided by the method of
Drummond and Dawson’ The water temperature regime and Rhoto~
period were those recommended by EPA for brook trout tests oO,

An acute toxicity test of toxaphene on yearling brook trout was
also conducted. The lethal threshold concentration (determined
when the rate of death was 10% or less of the original number of
fish in any concentration during the preceding 24 hour period) was
determined following recommendations by Eaton’ TA proportional
diluter delivering five concentrations and a control and with a
dilution factor of 0.75 between the concentrations was utilized.
The test was conducted at 10 C in the same size exposure tanks as
were used in the chronic study. Twenty fish were exposed to each
concentration, and mortalities were recorded daily.

The design of the chronic study was a randomized block design.
Growth data of both the adults and fry were analyzed by analysis
of variance to determine if significant differences existed, A