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ABSTRACT

RELATIONSHIP OF DIELDRIN TOXICITY TO
CONCENTRATIONS OF DIELDRIN IN THE BLOOD
AND BRAIN OF THE GREEN SUNFISH,
LEPOMIS CYANELLUS

 

by Roger L. Hogan

Green sunfish, Lepomis cyanellus, were exposed to

 

concentrations of dieldrin which were maintained for
periods over 124 hours. Blood and brain samples from

the fish analyzed by gas chromatography indicated threshold
toxic levels of approximately 6.0 micrograms per gram for
blood and 9.0 micrograms per gram for the brain. Surviving
fish had levels markedly lower than those experiencing
mortality. Correlations were noted between dieldrin levels
in blood and brain, and between blood and water dieldrin
concentration. Surviving fish exhibiting severe pesticide
poisoning symptoms had significantly higher blood and
brain concentrations than did those showing moderate to

minimal symptoms.

RELATIONSHIP OF DIELDRIN TOXICITY TO
CON ENTRATIONS OF DIELDRIN IN THE BLOOD
AND BRAIN OF THE GREEN SUNFISH,

LEPOMIS CYANE LUS

 

BY

Roger L.1Hogan

A THESIS

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

Department of Fisheries and Wildlife

1959

W
Q
.0.
Fifi
E
4/3
*3
(J)

I would like to take this opportunity to express
my sincere appreciation to the following peOple:
Dr. Eugene w. Roelofs for his counseling and advice, and
Dr. Howard E. Johnson and Dr. T. Wayne Porter for their
constructive comments during and after the study. This
research was funded by the Federal Water Pollution Control

Administration training grant no. 5Tl-NP-lO9.

ii

TABLE OF CONTENTS

Page
INTRODUCTION . . . . . . . . . . . . . . . . l
The problem . . . . . . . . . . . . . . 1
Related studies . . . . . . . . . . . . l, 2
METHODS. . . . . . . . . . . . . . . . . . . 3
Test apparatuS. . . . . . . . . . . . . 3
Adsorption and aeration losses. . . . . 3, 4
Study description . . . . . . . . . . . 4, 6, 8
Extraction efficiencies . . . . . . . . 8
RESULTS. . . . . . . . . . . . . . . . . . . 9
Uptake rates. . . . . . . . . 9 . . . . 9

Blood and brain threshold levels. . . . 10, ll

Dieldrin concentrations and symptoms. . 12

DISCUSSIOLQ' O O O O O O O O O O O O O O O O 0 14

Water dieldrin concentration and influence
on threshold levels . . . . . . . . . . 14

Relationships of findings to other
research. . . . . . . . . . . . . . . . 15, l6, 17

Conclusions and needed research . . . . 17, 18

LITERATURE CITED . . . . . . . . . . . . . . l9

APPEquIX O O O O O O O O O O O O O O O O O O 22

iii

TABLE

1.

LIST OF TABLES

Hourly rate of dieldrin uptake per gram
of 10 green sunfish . . . . . . . . . .
Relationship of blood and brain dieldrin
concentrations to death and to symptoms

in surviving green sunfish. . . . .

iv

PAS

11

[11

LIST OF FIGURES

FIGURE

1. Adsorptive losses of dieldrin in test
tan}: and filter a o o o o o

2. Test tank water concentrations of dieldrin

3. Regression of blood dieldrin level of
fish on water concentration at time of
death . . . . . . .

5

7

12

LIST OF APPENDICES

Relationship of length, weight,

green sunfish to mortality .

vi

and sex of

INTRODUCTION

 

With the current emphasis on the dangers of the
chlorinated hydrocarbon pesticides it becomes increasingly
important to extablish methods by which their presence and
effects on fish can be assessed. There have been several
studies done on levels of various pesticides and their
distribution within the organs of fish, but very few of
these have been of a toxicological nature. Mount, 23 a1.
(1966) established the long-term toxic level of endrin in

the blood of the channel catfish, Ictalurus punctatus

 

(Rafinesque), and thus suggested the use of blood as an
indicator tissue for endrin—caused fish kills. Blood
threshold levels of endrin were set for the gizzard shad,

Dorosoma cepedianum, by Brungs and Mount (1967). Blood

 

serves as the transporter for the chlorinated hydrocarbon
pesticides and distributes them throughout the exposed
fish.

It is generally accepted that the nervous system is
the primary site of action for chlorinated hydrocarbon
pesticides. {atsumura and O'Brien (1966a), working with
DDT and the central nervous system of the American

cockroach, Periplaneta americana (L.), found lipids in

 

the nerve cord to be the primary binding substance.

Using DDT and components of the American cockroach nerve,

Matsumura and O'Brien (1966b) suggested that the pesticide
combines with the nerves to form a charge transfer complex
which interferes with potassium ion efflux and nerve
transmittance. Narahashi and Haas (1968) came to similar
conclusions while studying the effects of DDT on components
of lobster (Homarus sp.) nerve membrane. Some investigators,
notably Dale, g3 a1. (1962) have correlated clinical signs
of DDT poisoning with concentrations of the pesticide in
the brain of the rat and also noted sharp increases in
plasma concentrations of DDT after administration. The
plasma concentrations did not vary significantly, however,
from the onset of symptoms, through convulsions, to death.
Dale, EE.§£- (1963) and Hayes and Dale (1964) found essen-
tially the same brain—DDT level—symptom correlation in
their studies with rats, and Stickel, £3.31. (1966), working
with birds, observed essentially the same phenomenon.
Using rats and the chlorinated hydrocarbon insecticide
dieldrin, Heath and Vandekar (1964) concluded that the
brain was the target organ upon which the dieldrin acted
to cause death.

This study was initiated to determine the relationship
of dieldrin toxicity to concentrations of dieldrin in the

blood and brain of the green sunfish, Lepomis cyanellus.

 

73.“,
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«IETHODS

Green sunfish with a weight range of 33.75 grams to
70.62 grams and a mean weight of 47.20 grams, were obtained
from a pond located on the Kellogg Bird Sanctuary near
Hickory Corners, Michigan. They were held a minimum of
three weeks in a SOD—gallon holding tank with a carbon-
filtered water re—cycling system. Six blood and six brain
samples taken to determine the presence of pesticide
residues, were negative. Following the holding period,

3 replications were run with 10 fish in each replication.
The fish were placed in a 70—liter test tank to which was
attached a pump to facilitate complete mixing of the water
and dieldrin, and a glass wool filter to eliminate detritus
and bacteria build-up. Water in the tank maintained a
temperature of 18 degrees C. and had been previously carbon
filtered to eliminate any traces of chlorinated hydro-
carbons. Dieldrin was added to the tank by a marriotte
bottle containing 4 liters of water and 2000 micrograms
(500 ppb) of dieldrin. The pesticide had been added to

the marriotte bottle with 5 milliliters of acetone. The
bottle could be calibrated to add from 2 to 10 milliliters
per minute with reliable long-term accuracy. Rates of
addition during the 3 replications ranged from 4.0 to 6.5

milliliters per minute. Adsorption on glass walls of the

 

 

tank accounted for a large portion of dieldrin losses in
the 70-liter test tank and filter. Three studies were
carried out to determine the amount of dieldrin losses
attributable to glass adsorption. In each study 1500
micrograms of dieldrin were added to the covered, non—
aerated tank, and 6 water samples were taken at intervals
between 1 and 24 hours. An average of 5.% of the initial
dieldrin dosage was adsorbed in 24 hours, with 45 %
occurring within the first 8 hours (Figure l). Decreases
in water concentration from the 8-hour level to the 24-
hour level, at which time the adsorption study was terminated,
occurred at the average rate of 1.19% per hour. Aeration
losses, as discussed by Mount (1962) and Bowman, g; 2;.
(1964), were computed in one of the adsorption studies
by using the 24-hour dieldrin water level as the starting
point. The water was then vigorously aerated for an
additional 24 hours after which a water sample was taken
to determine the dieldrin concentration. Allowing for
the 1.19% per hour adsorption loss determined earlier,
the 24—hour aeration loss was found to be 10.44%.
Twenty—four hours before the initiation of each
replication, 1000 micrograms of dieldrin were added to the
test tank to allow for glass adsorption losses. Water

samples were taken at the end of the 24—hour period to

5.5: oz<
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determine levels of dieldrin existing just prior to the
addition of the fish. Three replications having a mean
dieldrin concentration of 6 parts per billion were run,
the duration of each being determined by the length of
time required for 50% of the test fish to die (Figure 2).
The criteria for death was set at that point where there
was no longer Opercular action and the fish could not be
stirred to movement by its removal from the tank. All
fish were removed immediately after death and blood
samples taken by severing the caudal peduncle. Blood
aliquots were then weighed and frozen. The brain cases
were removed and fixed in a 10% formalin solution for

24 hours after which the brains were excised and weighed.
Fixation facilitated the removal of the brains from the
brain case and also provided a method of temporary storage.
The surviving fish were sacrificed using a 10% tricaine
methane-sulphonate solution; brain and blood samples

were taken in the above described manner. Length, weight,
sex, and previously noted symptoms of all specimens were
recorded.

Blood and brain samples were assayed using a modifi—
cation of the method described by Schafer, Busch, and
Campbell (1963). The samples were saponified with alcoholic
sodium hydroxide over a hot plate which allowed a slight

refluxing action. After cooling, the samples were extracted

 

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into petroleum ether and concentrated to 15 milliliters.
Measured volumes were then injected into an Aerograph 5508
electron capture gas chromatograph equipped with a column
of Chromosorb w packing coated with a % QF—l liquid
phase. Operating temperature was 175 degrees C and the
nitrogen flow, 35 milliliters per minute. Three aliquots

of each sample were injected and the resultant peaks

lb
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compared to .1 microgram per milliliter standards which
were run after each sample injection. Standards were

prepared with Shell analytical standard 99+% HEOD (dieldrin).

 

Reagent blanks and blood and brain samples from the control
fish were consistently negative. Five samples each of
blood and brain were fortified with dieldrin and inter-
spersed with the normal samples in order to determine
efficiency of extraction. Average extraction efficiencies
were noted of 94.92% with a standard deviation of 8.8%
for the blood, and 92.84% with a standard deviation of
9.7% for the brain.

An average of twenty-five loo-milliliter water samples
were taken during each of the replications. These 100-
milliliter aliquots were extracted into 20 milliliters
of petroleum ether by shaking for 4 minutes and collecting
and concentrating the supernatant. Average extraction
efficiency was determined by the method described above

and found to be 97.46% with a standard deviation of 5.2%.

RESULTS

During the experiments determinations of dieldrin
uptake rates were made during periods when water concen-
trations of the pesticide remained at the same level for
durations ranging from 5 to 8 hours, after fish had been
exposed from 70—110 hours. At least one sample was taken
from each of the 3 replications and micrograms of dieldrin
uptake/gram of fish/hour were computed. These computa-
tions were made under the assumption that all dieldrin
losses not due to glass adsorption and aeration could be
attributed to uptake by fish. These losses were determined
by comparing the amount of pesticide present at the beginning
of each time period, and the amount added by the marriotte
bottle, to the dieldrin level at the end of the time periods.
After allowance was made for aeration and adsorption losses,
values were calculated (Table 1).

Table 1. Hourly rate of dieldrin uptake per gram of
10 green sunfish.

 

 

Time 2 water Dosage Total Weight Rate of
Period concentration rate of 10 green uptake
(hours) for the ug/min. sunfish (s) (ug/g/hr)

period (ppb)

 

8 5.98 1.59 460.20 .198
8 6.28 1.59 460.20 .213
6 5.94 1.88 458.37 .253
8 6.32 1.97 497.41 .251
5 6.51 1.87 497.41 .248

 

10

As is shown by Table 2, with two exceptions in each
instance, all fish that died had blood dieldrin levels of
above 5.17 micrograms per gram, and all that survived had
levels less than this amount. Comparing the means of the
two groups of blood levels using the Mann—Whitney U test
with N2 = 16, significance was shown at the .001 level
(Siegel, 1956). The variance and standard deviation for
blood dieldrin concentrations of fish that died were
1.180 and 1.086 respectively. Concentration of dieldrin
in the water at time of death influenced the pesticide
blood level (Figure 3). Regression analysis of blood on
water gave significance at the .01 level with 13 degrees
of freedom.

As was the case with the blood, higher concentrations
of dieldrin were found in the brains of the fish that
died. With one exception, all mortalities had brain
concentrations above 8.70 micrograms per gram, and all
that survived, again with one exception, had brain levels
below this figure (see Table 2). The Mann—Whitney U test
indicated differences between the means of the two sample
groups (N2 = 16) to be significant at the .001 level.
Values for the variance and standard deviation of the
dieldrin concentrations in the brains of the dead fish
were 4.049 and 2.012 respectively. Blood to brain correla—

tion was indicated by a correlation coefficient of .95

11

Table 2. Relationship of blood and brain dieldrin concentrations
to death and to symptoms in surviving green sunfish.

 

 

Blood dieldrin Brain dieldrin

 

 

 

concentration concentration Symptoms
(ug/g) (ug/g)
3.153 12.183 Death FR
5.738 10.789 '
5.167 11.537
6.446 13.350
6.714 11.988
5.381 8.900
6.055 9.090
6.015 8.718
6.384 9.420
3.740 8.706
5.308 10.477
6.521 8.602
5.554 10.108
6.931 10.461
3.659 9.740 Hyperactive - some equilibrium loss
5.147 7.998 Hyperactive - some equilibrium loss
4.878 7.398 Hyperactive - erratic darting
4.740 7.147 Hyperactive
5.616 8.654 "
5.137 6.999 "
4.646 6.059 "
3.935 8.212 General darkening-vertical bars of
5.787 6.880 dark coloration-some hyperactivity
4.685 3.889 General darkening
4.674 3.763 " "
4.967 6.052 " "
3.526 3.811 " "
3.256 6.086 None
3.641 6.623 "
2.786 5.403 "

12

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with 29 degrees of freedom. Regression analysis of brain
on blood gave significance at the .05 level.

Symptoms of pesticide poisoning observed during the
study were similar to those described by Ferguson, 23 a1.
(1964). The first noticeable symptom was a rising toward
the surface of the water. A general darkening of body
coloration accompanied by formation of dark, vertical
bars occurred, followed by a period of hyperactivity
characterized by rapid pectoral and Opercular action,
erratic swimming or darting, a gradual loss of equilibrium,
and death. ‘Using the fish that survived, and dividing
them into two groups based on the severity of their symptoms
(described in Table 2), differences between the blood
and brain levels of the two groups were tested using the
Mann—Whitney U test. Blood concentration difference
between the two groups was significant at the .025 level
(N2 = 9) with the most severe symptoms having the highest
dieldrin concentration. Brain level difference was
significant at .001 with the same relationship of dieldrin

to symptoms noted above.

DISCUSSION

 

Data on blood levels of dieldrin at death indicate
a blood threshold level for the green sunfish of approx-
imately 6.0 micrograms per gram.

The blood concentrations appear high when compared
to the long-term work done with endrin. Mount, 23 a1.
(1966) in a 44—day study, using the channel catfish found
toxic blood levels of endrin to be .28 micrograms per gram
and above. In a 8-day study, Brungs and Mount (1967)
working with gizzard shad found the critical level to be
.10 micrograms per gram. However, Henderson (1959) and
Katz (1961) studying 96-hour TLM values for dieldrin and
endrin, found endrin toxicity to be 10-20 times as great
as that of dieldrin. This fact when combined with the
hardiness of the green sunfish and the short—term nature
of the study indicates the dieldrin blood levels to be
within expected limits.

Water concentration of dieldrin appears to exert a
slight influence on the critical levels of the pesticide
in the blood of fish experiencing mortality. This effect
should be expected in short—term studies when blood is
viewed as a tranSporter dependent upon the amount of
pesticide passed over the gill surfaces to determine

""7

amounts picked up by blood proteins (Moss and nathaway,

14

 

 

15

1964). Mount, 23.21- (1966) working with long—term endrin
blood thresholds, found them to be essentially independent
of concentrations of endrin in the water.

Brain levels for fish that died were more variable
than those for the blood; however, a clear cut off point
is evident between dieldrin concentrations in mortalities
and survivors. The data indicate this threshold to be
approximately 9.0 micrograms per gram. Blood level
correlates with brain levels and both blood and brain
concentrations showed a relationship to categories of
observed symptoms in the test fish. In the two instances
where fish died with blood levels below the established
threshold, brain residues were above their critical
concentration (Table 2). The remaining exception was a
surviving specimen which had low blood dieldrin concentra—
tion but a brain concentration over the threshold level.
This individual was also exhibiting strong pesticide
poisoning symptoms.

Gakstatter (1966) working with channel catfish and

bluegills, Lepomis macrochirus, indicated that the brain

 

did not serve a storage function but that dieldrin
residues decline rapidly following exposure. This same
phenomenon was also noted for blood. Heath and Vandekar
(1964) studying effects of dieldrin on rats, found that

the faster the pesticide was added to the test animals,

~.-*..l

six—Lu?

-‘IVWJL “_-..

 

‘Efi”'“'

the higher was their brain concentration. The investigators

felt dieldrin, when added slowly, was being carried by the
soluble blood proteins and was absorbed by body fat, but
when added rapidly the body fat was not able to take it
up rapidly enough, with the result that the pesticide
reached the central nervous system and eventually caused
death. Gakstatter (1966) found starved test fish to have
a higher blood and brain dieldrin level than normal fish,
and he suggested that the inherent protective system pro—
vided by the body fat can be overloaded by exceeding its
storage capacity. This could conceivably be accomplished
by long~term exposure to pesticides at moderate levels
and would have the same end effect as exposure to high
lethal concentrations. In each of two prior experiments
by the author, 10 bluegills exposed to 41 ppb and 28 ppb
of dieldrin, suffered a 50% mortality within 14 hours.
Dieldrin concentration in the brains of fish that died
ranged from 6.5 to 8.4 micrograms per gram with a mean
of 7.78, and those surviving, 1.03 to 6.03 micrograms per
gram with a mean of 3.27. A strong symptomatic correlation
with brain concentration was also noted.

Ferguson, e: a1. (1964) working with endrin and
dieldrin found a definite difference in 36—hour TLM limits
between resistant and non-resistant fish. Ludke, e; a1.

(1968) studying endrin and its effects on resistant golden

 

 

17

shiners, Notemigonus crysoleucas, found whole body

 

residues 82 times and blood residues 64 times as great
in resistant fish as those found in non—resistant fish
killed by the exposure. The investigators, therefore,
indicate a limitation which must be considered when using
blood and brain thresholds as a diagnostic tool in fish—
kill determinations.

Evidence indicates the brain to be a primary site

of action for dieldrin. Additional research needs to be

 

 

undertaken before being able to determine definitely
dieldrin's mode of action and the target site or sites
upon which it acts. Hayashi and Matsumura (1967) have
initiated a line of research which could productively be
applied to other organisms. Using two species of cock-
roaches, the investigators compared the effects of dieldrin
on ion movement in the nervous tissue of susceptible and
resistant specimens. Initial response to dieldrin
appeared to be much the same as was observed for DDT.
iore detailed studies patterned after work done by Ludke,
g3 a1. (1968) could prove valuable in answering many of
the questions regarding blood and its transport function,
and modes of action of the pesticide at its target site.
Blood and tissue chemistry investigations such as under-
taken by Eisler and Edmunds (1966) should be initiated

on resistant organisms to determine differences between

18

resistant and susceptible forms.

LITERATURE CITED

 

Bowman, M. C., F. Acree, Jr., C. S. Lofgren, and M. Beroza.

1964. Chlorinated hydrocarbon insecticides: fate
in aqueous suSpensions containing mosquito larva.
Sci. 146:1480-1481.

Brungs, W. A. and D. I. Mount. 1967. Lethal endrin
concentration in the blood of gizzard shad. J. Fish.
Res. Ed. Can. 24(2):429—431.

Dale, W. E., T. B. Gaines, and W. J. Hayes, Jr. 1962.
Storage and excretion of DDT in starved rats.
Toxicol. Appl. Pharmacol. 4:89-106.

Dale, W. E., T. B. Gaines, W. J. Hayes, Jr., and G. W.
Pearce. 1963. Poisoning by DDT: relation between
clinical signs and concentration in the rat brain.
Sci. 142:1474-1476.

Eisler, Ronald, and Philip H. Edmunds. 1966. Effects
of endrin on blood and tissue chemistry of a marine
fish. Trans. Amer. Fish. Soc. 95(2):153-l59.

Ferguson, Denzel E., Dudley D. Culley, William D. Cotton,
and Ross P. Dodds. 1964. Resistance to chlorinated
hydrocarbon insecticides in three species of fresh—
water fish. BioSci. 14(11):43—44.

Ferguson, D. E., J. Larry Ludke, and George C. Murphy.
1966. Dynamics of endrin uptake and release by
resistant and susceptible strains of mosquito fish.
Trans, Amer. Fish. Soc. 95(4):335-344.

Gakstatter, Jack Henry. 1966. The uptake from water by
several species of freshwater fish of P P' DDT,
dieldrin and lindane: their tissue distribution
and elimination rate. Thesis, University of North
Carolina.

Hayashi, M. and F. Matsumura. 1967. Insecticide mode
of action: effect of dieldrin on ion movements in
the nervous system of Periplaneta americapa and
Blattella germanica cockroaches. J. Agr. Food
Chem. 15:622-627.

 

 

19

Pvt-xii 1V

 

20

Hayes, Wayland J. and William E. Dale. 1964. Concentra—
tion of DDT in the brain and other tissues in relation
to symptomatology. Toxicol. Appl. Pharmoc. 6(3):349.

Heath, D. F. and M. Vandekar. 1964. Toxicity and meta—
bolism of dieldrin in rats. Brit. J. Ind. Med. 21:
269-279.

Henderson, G., Q. H. Pickering, and T. M. Tarzwell. 1959.
Relative toxicity of ten chlorinated hydrocarbon
insecticides to four species of fish. Trans. Amer.
Fish. Soc. 88(1):23-32.

Katz, Max. 1961. Acute toxicity of some organic insec—
ticides to three Species of salmonids and to the
three Spine stickleback. Trans. Amer. Fish. Soc.
90(3):264-268.

Ludke, J. Larry, Denzel E. Ferguson, and W. David Burke.
1968. Some endrin relationships in resistant and
susceptible populations of golden shiners, Notemi-
ggnus chrysoleucas. Trans. Amer. Fish. Soc. 97(3):
260—263.

 

Matsumura, F. and R. D. O'Brien. 1966a. Interactions of
DDT with components of American cockroach nerve.
J. Agr. Food Chem. 14:39-43.

. 1966b. Adsorption and binding of DDT by the
central nervous system of the American cockroach.
J. Agr. and Food Chem. 14:36-39.

 

Moss, J. A. and D. E. Hathaway. 1964. Transport of
dieldrin and telodrin in the blood. Biochem. J. 91:

Mount, D. I. 1962. Chronic effects of endrin on blunt—
nose minnows and guppies. USFWS Res. Rept. 58:38 p.

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21

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APPENDIX

Relationship of length, weight, and sex of green sunfish
to mortality.

 

 

 

Length Weight Sex
131 41.60 Male
147 60.08 Male
125 39.80 Female
115 33.39 Male
141 60.02 Male
135 47.56 Male
128 38.52 Female

Died 136 45.70 Male
131 42.90 Male
130 41.20 Male
140 45.00 Male
156 70.62 Male
126 39.22 Female
145 38.30 Male
125 33.35 Male
122 44.20 Male
134 55.85 Male
141 53.61 Male
136 48.51 Female
136 45.76 Male
136 50.07 Male

Survived 135 47.31 Male
139 54.74 Male
140 59.72 Male
134 42.30 Male
141 51.10 Male
124 36.89 Female
145 56.86 Male
140 54.50 Male
130 37.30 Female

22

 

 

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