INVESTIGATIONS IN FISH CONTROL

35. Toxicology of Thiodan 

in Several Fish 

and Aquatic Invertebrates

U

United States Department of the Interior

Fish and Wildlife Service 

Bureau off Sport Fisheries and Wildlife
INVESTIGATIONS IN FISH CONTROL

Investigations in Fish Control, published by the Bureau of Sport Fisheries and Wildlife, include reports on the results of work at the Bureau's Fish Control Laboratories at 

La Crosse, Wis., and Warm Springs, Ga., and reports of other studies related to that work. 

Though each report is regarded as a separate publication, several may be issued under a 

single cover, for economy.

Current reports in this series are 

(Reports 1 and 2 are in one cover.)

1. Laboratories and Methods for Screening Fish-Control Chemicals, by Robert E. Lennon and 

Charles R. Walker. 1964. 15 p.

2. Preliminary Observations on the Toxicity of Antimycin A to Fish and Other Aquatic Animals, by Charles R. Walker, Robert E. Lennon, and Bernard L. Berger. 1964. 18 p.

(Reports 3 through 8 are in one cover.)

3. Minimum Lethal Levels of Toxaphene as a Piscicide in North Dakota Lakes, by Dale L. 

Henegar. 1966. 16 p.

4. Effects of Toxaphene on Plankton and Aquatic Invertebrates in North Dakota Lakes, by 

Robert G. Needham. 1966. 16 p.

5. Growth Rates of Yellow Perch in Two North Dakota Lakes After Population Reduction with 

Toxaphene, by Donald C. Warnick. 1966. 9 p.

6. Mortality of Some Species of Fish to Toxaphene at Three Temperatures, by Mahmoud Ahmed 

Mahdi. 1966. 10 p.

7. Treatment of East Bay, Alger County, Michigan, with Toxaphene for Control of Sea Lampreys, by William E. Gaylord and Bernard R. Smith. 1966. 7 p.

8. Effects of Toxaphene on Fishes and Bottom Fauna of Big Kitoi Creek, Afognak Island, 

Alaska, by William R. Meehan and William L. Sheridan. 1966. 9 p.

(Reports 9 through 11 are in one cover.)

9. Relation of Chemical Structure to Fish Toxicity in Nitrosalicylanilides and Related Compounds, by Charles R. Walker, Roland J. Starkey, and Leif L. Marking. 1966. 12 p.

10. Evaluation of _p_,_p_'-DDT as a Reference Toxicant in Bioassays, by Leif L. Marking.

1966. 10 p.

11. Evaluation of an Electronic Method of Measuring Hematocrits of Fish, by Richard A. 

Schoettger and Arnold M. Julin. 1966. 11 p.

(Reports 12 through 17 are in one cover.)

12. Toxicity of MS-222 to Selected Fishes, by Leif L. Marking. 1967. 10 p.

13. Efficacy of MS-222 as an Anesthetic on Four Salmonids, by Richard A. Schoettger and 

Arnold M. Julin. 1967. 15 p.

14. Method for Determining MS-222 Residues in Fish, by Charles R. Walker and Richard A. 

Schoettger. 1967. 10 p.

15. Residues of MS-222 in Four Salmonids Following Anesthesia, by Charles R. Walker and 

Richard A. Schoettger. 1967. 11 p.

16. Annotated Bibliography on MS-222, by Richard A. Schoettger. 1967. 15 p.

17. MS-222 as an Anesthetic for Channel Catfish: Its Toxicity, Efficacy, and Muscle Residues, 

by Richard A. Schoettger, Charles R. Walker, Leif L. Marking, and Arnold M. Julin.

1967. 14 p.

(Reports 18 through 21 are in one cover.)

18. Toxicity of 22 Therapeutic Compounds to Six Fishes, by Wayne A. Willford. 1967. 10 p.

19. Toxicity of Bayer 73 to Fish, by Leif L. Marking and James W. Hogan. 1967. 13 p.

20. Toxicity of Dimethyl Sulfoxide (DMSO) to Fish, by Wayne A. Willford. 1967. 8 p.

21. Labor-Saving Devices for Bioassay Laboratories, by Robert J. Hesselberg and Ralph M. 

Burress. 1967. 8 p.

Fish Control Laboratories

Bureau of Sport Fisheries and Wildlife

U.S. Department of the Interior

P. 0. Box 862

La Crosse, Wisconsin 54602

Continued on inside back cover 
INVESTIGATIONS IN FISH CONTROL

35. Toxicology of Thiodan 

in Several Fish 

and Aquatic Invertebrates

By Richard A. Schoettger

United States Department of the Interior, Walter J. Hickel, Secretary

Leslie L. Glasgow, Assistant Secretary for

Fish and Wildlife, Parks, and Marine Resources

Fish and Wildlife Service, Charles H. Meacham, Commissioner

Bureau of Sport Fisheries and Wildlife, John S. Gottschalk, Director

Washington, B.C. January 1970
CONTENTS

Page 

Abstract ........................ 3

Materials ....................... 4

Methods ........................ 5

Toxicity ...................... 5

Solutions for testing water quality effects on

toxicity ..................... 6

Deposition and metabolism of Thiodan ......... 7

Results and discussion .................. 9

Toxicity ....................... 9

Effects of water quality on toxicity ......... 14

Deposition and metabolism of Thiodan ......... 18

Summary ......................... 27

References ........................ 28
TOXICOLOGY OF THIODAN 

IN SEVERAL FISH AND AQUATIC INVERTEBRATES

By Richard A . Schoettger, Fishery Biologist

Bureau of Sport Fisheries and Wildlife 

Fish Control Laboratory, La Crosse, Wisconsin

ABSTRACT.--This investigation provides toxicological data on Thiodan to 

meet published criteria for evaluating chemicals for fish control. Thiodan, a 

"chlorinated hydrocarbon insecticide, was tested against rainbow trout and their 

fertilized eggs, western white suckers, Daphnia magna, and damselfly naiads . 

The median tolerance limits for trout and suckers ranged between 0.3 and 8 .1 ppb 

and the fish were at least seven times more susceptible than the invertebrates. 

Toxicity was influenced by temperature, length of exposure, and alkaline pH. 

Exposures for up to 2 hours to 50 ppm of Thiodan were not toxic to fertilized trout 

eggs. The deposition and metabolism of Thiodan residues in western white 

suckers, northern creek chubs , and goldfish were traced with the aid of 

carbon-14 labeled Thiodan, and chemical analyses of Thiodan in tissues . Residues 

occurred in the skin and muscles of fish exposed to acute and to multiple subacute 

concentrations . The death of fish which were poisoned subacutely was correlated 

with size and the lipid content of muscle. The residues in the various tissues 

seemed to be associated with the method of treatment. A water-soluble metabolite 

of Thiodan in the bile of treated fish appeared to be a glucosiduronic acid conjugate of Thiodan alcohol. A possible metabolic pathway for Thiodan degradation 

is discussed. Thiodan appears to have little value as a selective piscicide 

against rough fishes such as carp or suckers, but under certain conditions it may 

be a good general fish toxicant.

Fishery biologists generally turn to the 

use of chemicals for manipulating fish populations when other methods of management such 

as seining, drawdown, draining, or biological 

controls are ineffective or too expensive. 

According to Stroud and Massman (1963) the 

principal chemicals used in fishery reclamations 

in the United States and Canada are the insecticides rotenone and toxaphene. They reported 

that another insecticide, Thiodan,!./was applied 

experimentally to four small lakes in Canada . 

The trials indicated that it is highly toxic to fish, 

detoxifies more rapidly than toxaphene, and is 

less expensive than rotenone, but may be detrimental to plankton and bottom fauna .

I/ The term Thiodan (the trade name for endosulfan) is used in this paper because it is in 

common usage.

Thiodan and toxaphene are classed 

chemically with the organochlorine or chlorinated 

camphene insecticides, which include aldrin, 

dieldrin, and endrin. It is generally recognized 

that this group of chemicals is relatively persistent in the environment. For example, 

toxaphene-treated waters may remain toxic to 

fish for 2 or 3 years (Tanner and Hayes, 1955; 

Hooper and Grzenda, 1955); residues of toxaphene may persist in fish tissues and in other 

parts of the ecosystem (Kallman, Cope, and

This paper is based on a dissertation submitted 

to the Faculty of the Zoology Department of 

Colorado State University in 1966, in partial 

fulfillment of the requirements for the degree 

of doctor of philosophy. Financial assistance 

from the National Institutes of Health is gratefully acknowledged.
Investigations in Fish Control 35: Bureau of Sport Fisheries and Wildlife

Navarre, 1962). The United States Department 

of the Interior has banned all use of this and 

other persistent chemicals in Department waters 

because of the effects on aquatic life. Some 

question remains whether Thiodan should be 

considered for fishery uses because of its 

chemical similarity to toxaphene. Recently 

Meyer (1965) reported on the organophosphorus 

insecticide Guthion as a potential fish poison 

less persistent than organochlorine insecticides. 

The rapid degradation of antimycin A (a piscicide registered under the tradename of Fintrol) 

shows promise for fish management (Walker, 

Lennon, and Berger, 1964). Perhaps a variety 

of fish control agents with well-defined chemical 

and toxicological properties will enable biologists 

to select suitable compounds for specific 

management problems.

Thiodan was patented in the United States 

as an insecticide by Frensch et al. (1957) and 

later in Germany as a fish toxicant (Frensch et 

al., 1959). Cuerrier (1960) made field observations on the efficacy of Thiodan in fish control. 

Adlung (1957) and Liidemann and Neumann (1962) 

measured its relative toxicity to several aquatic 

vertebrates and invertebrates. However, these 

data are insufficient to meet the criteria, as 

established by Lennon and Walker (1964), for 

gaging the potential of chemicals for fish control. 

The objective of my investigation was to provide 

additional information on Thiodan by determining 

its toxicity to other fish and aquatic invertebrates , the effect of pH and water quality on 

toxicity, and its uptake and metabolism in fish.

MATERIALS

Thiodan is the registered tradename of 

a chlorinated fused-ring heterocyclic compound 

developed by Farbwerke Hoechst A. G., Frankfurt, Germany, as an insecticide (F.M.C. 

Corporation, 1964). The Food Machinery and 

Chemical Corporation (F.M.C.), Niagara 

Chemical Division, Middleport, N.Y. , is 

licensed to manufacture, use, and sell Thiodan 

in the United States . The American common 

name of Thiodan is endosulfan (6,7,8,9,10,10- 

hexochloro 1 ,5,5a_, 6,9,9a_-hexahydro-6,9- 

methano -2,4,3 -benzodioxathiepin -3 -oxide).

The empirical formula is C^H^Cl^O^S and its 

structure is as follows (F.M.C. Corporation, 

1958a and 1964):

Cl

Cl

According to the manufacturer, the 

technical Thiodan used in this study is 96.4 percent pure. It contains A and B isomers which 

melt in the ranges of 108° to 109° C., and 206° 

to 208° C., respectively(Lindquist and Dahm, 

1957). The acid hydrolysis of either isomer 

yields only one isomer of Thiodan alcohol which 

has the structure:

ci

Cl

Thiodan is insoluble in water and stable 

in sunlight, but hydrolizes to sulfite and Thiodan 

alcohol in the presence of moisture, bases or 

acids. Other physical and chemical properties 

are reported by the maker (F.M.C. Corporation, 

1964).

The F.M.C. Corporation supplied 

approximately 17 milligrams of carbon-14
Richard A. Schoettger: Toxicology of Thiodan in Several Fish and Aquatic Invertebrates

labeled Thiodan for tracer investigations . It has 

an activity of 5.91 microcuries (uc) per mg. 

Carbon-14 constitutes 6.8 percent of the molecule and 25 percent of the total carbon. The 

locations of carbon-14 in the Thiodan molecule 

are shown by asterisks in the structural 

formula. The synthesis of radioactive Thiodan 

was outlined by Forman et al. (1960).

METHODS

Toxic ity

Thiodan was bioassayed against small 

rainbow trout and western white suckers and 

against Daphnia magna, damselfly naiads and 

fertilized eggs of rainbow trout (table 1). Most 

of the experiments were conducted in the 

Zoology Laboratory, Colorado State University, 

Fort Collins, Colo., but the egg trials were 

carried out at the Fish Control Laboratory, 

La Crosse, Wis. The fish were maintained in 

aerated tap water at 10° C . If deaths in a particular lot of fish exceeded 5 to 10 percent, it 

was discarded. Both species were held 10 days 

before use and were fed Colorado's standard 

pelleted diet. They were fasted for 48 hours 

and acclimated to experimental temperatures for 

24 hours before the trials. The naiads were 

acclimated to laboratory conditions for several 

days before use.

The bioassays were conducted according 

to procedures described by the American Public 

Health Association (1960), with some modifications . Trials with fish were in glass aquariums 

containing 20 liters of tap water; those with 

damselfly naiads were in 6 liters. Experiments 

with D. magna were in plastic vessels each 

holding 1 liter of reconstituted water. In the 

latter, tap water was deionized and formulated 

to contain 45 to 50 ppm of bicarbonate alkalinity 

with sodium bicarbonate, and 45 to 50 ppm of 

calcium chloride . Chemical analyses of the tap 

water showed it to contain the following: 

Oxygen 11.8 ppm (7° C .); total hardness 45 .0 

ppm as calcium carbonate; total alkalinity as 

bicarbonate 38 .0 ppm as calcium carbonate; 

calcium 12.0 ppm; magnesium 2.3 ppm; ferric 

and aluminum oxide 1.6 ppm; sulfate 1.3 ppm; 

chloride 6.9 ppm; chlorine 0.0; and pH 7 .4 .

Dssired concentrations of Thiodan were 

prepared by adding aliquots of 1 -part-per - 

thousand stock solutions of technical Thiodan 

in ethanol to the water. The concentrations 

were based on active ingredient. Ethanol was 

added to controls in an amount equal to the 

largest aliquot of stock used in the test series. 

A minimum of aeration was applied to all of the 

solutions tested against fish to maintain an oxygen concentration of not less than 5 ppm. Ten 

fish and 10 or 20 invertebrates were used to 

measure toxicity, and the trials were replicated 

1 to 4 times. The loadings of fish in test vessels 

were approximately 0.5 to 1.0 gram per liter.

The bioassays with each species were 

carried out at two temperatures: Trout, 1.5° 

and 10° C.; suckers, 10° and 19°; D. magna, 

10° and 19°; and naiads, 8° and 19° . Temperature control was achieved by manipulating 

room temperature. At 10° and below, temperature varied + 1° C ., and at 19° the fluctuation 

was + 2-3°."

Observations on the toxicity of Thiodan 

were made 24, 48, 72, 96, and 120 hours after 

the experimental animals were first exposed. 

Individuals showing no respiratory movement 

and no response to a tactile stimulus were recorded as dead and were removed.

Between experiments, test containers 

were cleansed with detergent, filled with 5- to 

10-percent potassium hydroxide, allowed to 

stand several hours, washed again, and then 

rinsed with ethanol and dried. This procedure 

removed or destroyed Thiodan which adhered 

to the glass as indicated by periodic bioassays 

of cleansed aquariums.

The median tolerance limits (TLm, the 

concentration tolerated by 50 percent of the test 

animals) of trout, suckers, naiads, and D. 

magna to Thiodan were determined for each 

temperature and observation period. A polynomial quadratic equation was derived from the 

data for the 24- and 48-hour bioassays as 

described by Goulden (1956). TLm was interpolated from the regression line. The 95-percent 

confidence limits around the TLm's were computed from the standard errors for the populations
Investigations in Fish Control 35: Bureau of Sport Fisheries and Wildlife

Table 1: Species, sizes, and sources of experimental animals.

Species

Size range 

Length Weight 

(inches)__(grams) Source

Rainbow trout, Salmo gairdneri 

Western white suckers, Catastomus 

commersoni

Do.

Northern creek chubs, Semotilus

atromaculatus

Goldfish, Carassius auratus 

Rainbow trout (fertilized eggs) 

Water fleas, Daphnia magna

Damselfly naiads, Ischura sp.

1.6-2.2 1.0-1.8 SFH!/, Bellvue, Colo.

1.8-2.6 0.9-2.5

5.3-8.2 40.1-70.0

Cache la Poudre River, 

Fort Collins, Colo.

6.7-10.9 96-337

0.25-0,50

NFH, Genoa, Wis. 

NFH, Manchester, la. 

Laboratory cultures at 

Colorado State Univ.. 

Fort Collins, Colo. 

From beneath the ice 

of a pond near Fort 

Collins, Colo.

_!/ State fish hatchery.

_2/ Mean.

_3/ National fish hatchery.

The TLm's for 72, 96, and 120 hours were 

interpolated from averages of the replicate bioassays by methods of the American Public 

Health Association (1960).

Eggs of rainbow trout were fertilized at 

the National Fish Hatchery, Manchester, Iowa, 

and transported to the laboratory. Twenty-five 

hours later they were divided into groups of 100 

each and dipped into solutions containing 10, 

100, 1,000, 10,000, or 50,000 ppb of Thiodan 

for 30 or 120 minutes . Test solutions were 

prepared with technical Thiodan dissolved in 

acetone, and control solutions with a corresponding concentration of acetone alone . After 

dipping, the eggs were placed in a Heath Incubator at 12° C. for hatching. At 24-hour intervals the eggs were observed for mortality, and 

2 or 3 specimens were collected randomly for 

microscopic examination. The samples were 

prepared for the examination, and stages of 

development were identified as described by 

Knight (1963).

Solutions for testing water quality effects on 

toxic ity

Salt solutions were made to a hardness 

of 500 ppm, as calcium carbonate, by adding 

calcium chloride, calcium sulfate, magnesium 

chloride, or magnes ium sulfate to 3 liters of tap 

water in 1 -gallon glass jars. Thiodan dissolved 

in ethanol was added to make a concentration of 

20 ppb. Controls were salt solutions without 

the toxicant, solutions with Thiodan, but no 

salts, and vessels with only tap water.

Effects of pH on toxicity were measured 

in solutions of pH 6.4, 7 A, 8.4, and 9.4 . The 

pH was adjusted by adding potassium hydroxide 

and/or acetic acid to 3 liters of tap water in

1 -gallon glass jars. A concentration of 20 ppb 

of Thiodan was added to each jar. Control 

solutions had pH values appropriate for a particular pH test, but were without Thiodan.

Following preparation of experimental 

media, the glass jars were sealed and the contents aged at 19° C. + 2° to determine degradation 

time. Salt solutions were aged up to 436 hours, 

and solutions of different pH for up to 96 hours. 

Then they were aerated and bioassayed for 24 

hours with 10 suckers with an average length of

2 inches and weight of 1.3 grams.
Richard A. Schoettger: Toxicology of Thiodan in Several Fish and Aquatic Invertebrates

The comparative differences in toxicities 

of the solutions were analyzed statistically by 

the exact test for 2 x 2 tables (Goulden, 1956).

Deposition and metabolism of Thiodan

Radioactive Thiodan--Western white 

suckers were bioassayed in 1 -gallon glass jars 

containing 1.5 or 3.0 liters of tap water at 19° C . 

One sucker was placed in each jar per 1.5 liters 

of solution. The fish had an average length of 

5,3 inches and weighed 40.1 grams. Radioactive Thiodan was dissolved in ethanol, and a 

stock was prepared as in the toxicity experiments. 

Portions of this stock were added to the jars to 

give concentrations of 20 and 80 ppb of labeled 

compound. At the time of these experiments, 

the exact purity of the labeled Thiodan was unknown. Forman et al. (1960) later reported 

that it contained 85- to 93-percent Thiodan, but 

the impurities were not radioactive. Thus, the 

amount of pure Thiodan in each jar was slightly 

less than 20 or 80 ppb.

The fish exposed to a concentration of 

80 ppb were removed for analysis at the time of 

death. Those treated with 20 ppb were killed 

with chloroform at intervals between 1 and 2 

hours of exposure. Samples of gill filament, 

heart, liver, kidney, skin, muscle, gut (with 

and without feces), brain, and blood were 

collected. Blood was obtained by cardiac 

puncture immediately after death. The samples , 

excluding blood, were rinsed briefly in distilled water, placed in tared vials, and dried at 

90° to 100° C. to constant weight. They were 

digested in 3.0 ml of 2-percent potassium hydroxide. A 0.5-ml aliquot of each digest was 

placed on a tared stainless steel planchet of 

approximately 20 cm and diluted with deionized 

water. A homogeneous distribution of the 

material on the plancnet was promoted by 

stirring in a small quantity of laboratory detergent which reduced surface tension. This also 

reduced contraction of the samples on the 

planchets while they dried to constant weight. 

The samples were then ready for measurement 

of radioactivity.

Determinations of radioactivity were 

made with an internal proportional counter and

the counts corrected for background and counter 

efficiency. Characteristics of the counter were 

reported by Nader, Hagee, and Setter (1954). 

The 0.95 counting error was computed for each 

sample according to the nomograms of Jarret 

(1946). Concentrations of Thiodan greater 

than the counting error were considered significantly different from zero. Untreated control 

fish were also counted, but none contained 

statistically significant radioactivity.

Radioactive Thiodan was used to determine whether and how it may be metabolized in 

fish. Large northern creek chubs and western 

white suckers were exposed to 40 ppb of labeled 

Thiodan at 21° C. using methods and materials 

described in the radiation studies. The fish 

were killed with chloroform after 3 or 5 hours 

of exposure.

Samples of chub liver, kidney, blood, 

and brain were dried to constant weight at 

90° C., macerated, and extracted in absolute 

ethanol. The ethanol was evaporated almost to 

dryness at room temperature, and the residues 

were washed with alternate rinses of benzene 

and deionized water into 125-ml separatory 

funnels . The mixtures were agitated and 

allowed to separate for 1 to 12 hours, depending 

on persistence of emulsions. The emulsions 

persisted with all samples , except brain. Aliquots of benzene or water fractions were placed 

on planchets and dried for measurements of 

radioactivity. Small amounts of persistent 

emulsions were included with the water fraction. 

The counts were corrected for background, 

geometry and backscatter, but not absorption 

since the amount of lipid per planchet was usually 

less than 0.2 ing.

The tissues which had been extracted 

with alcohol were prepared for analyses of 

radioactivity as described earlier for potassium 

hydroxide digests of sucker tissue.

The gallbladder bile of chubs and suckers 

was dried to constant weight at 65° C. and rehydrated in 30-percent ethanol in water. Aliquots 

of the rehydrated bile were washed with benzene 

and water into separatory funnels, separated, 

and analyzed for radioactivity like the alcoholic 

extracts of chub tissues . Additional aliquots
Investigations in Fish Control 35: Bureau of Sport Fisheries and Wildlife

were diluted to 10 ml with deionized water. 

Approximately 1 ml of 0.1M sodium ace'tate and 

3.0 ml of 0.2M_ acetic acid were used to buffer 

the bile to pH 4 .5 . The buffered mixtures were 

incubated for 24 hours at 37° C. with 0.5 ml 

(2,500 Fishman units) of beta-glucuronidase,U 

a procedure similar to that described by 

Talalay (1963). The samples were raised to 

pH 7.5, then extracted with benzene, and the 

levels of benzene- and water-soluble radioactive 

substances were determined.

After approximately a 6-month delay in 

the research, a check was made of the molecular integrity of the stock supply of radioactive 

Thiodan. The results of paper chromatographic 

analyses, methods according to Mills (1959), 

indicated that the labeled chemical had degraded 

to what appeared to be Thiodan alcohol. Experiments with radioactive Thiodan were dis - 

continued.

Nonradioactive Thiodan-- Further investigations on the deposition and metabolism of 

Thiodan in fish were conducted at the Fish Control Laboratory, La Crosse, Wis. Goldfish 

were maintained in flowing well water at 12° C . 

and fasted for 24 hours before the tests. An 

individual was placed in each of 10 glass jars 

containing 15 liters of reconstituted water at 

12° C . The water was reconstituted by adding 

various salts to deionized water, methods 

according to Lennon and Walker (1964). Stock 

solutions of technical Thiodan were prepared in 

acetone and an aliquot containing 0.105 mg. of 

toxicant was added to each test solution to give 

a concentration of 7 .0 ppb. Control fish were 

exposed to similar quantities of acetone. The 

living test and control fish were transferred 

daily into new test solutions. The solutions 

were aerated periodically to insure adequate 

oxygen.

The goldfish experiment was terminated 

after 21 days. All of the fish, except two, died 

during the tests and were removed for tissue 

analyses . The two survivors were killed by

2/ Ketodase. Warner-Chilcott, Morris 

Plains, N. J.

overanesthetization in MS-222. The gill filaments , gonads , livers , spleens, hearts, kidneys, 

guts (including feces), skin, muscle, brains, 

peritoneal fat (from two specimens), scales, 

and fins were dissected from the dead fish. 

Heparinized samples of blood were collected 

by cardiac puncture, and the gallbladder was 

removed from the liver.

According to preliminary tests using 

analytical methods which are outlined later, 

I found it necessary to combine tissues, except 

muscle, from several fish. Samples of approximately 10 to 50 grams were needed for the 

analyses. The gills from the first five fish 

which died were combined into one sample, 

those from the last three and the two survivors 

into another. Samples of the skin, liver, gut 

and content, gonad, and two composite samples, 

one of the heart, kidney, spleen, and blood and 

another of the scales and fins were prepared 

like the gill. All of the brains were pooled into 

one sample. Peritoneal fat was dissected from 

two fish and pooled. The various samples 

were weighed and combined immediately after 

dissection. They were weighed again, after 

drying to constant weight at 70° C., to determine water content. The gallbladder bile was 

measured volumetrically and set aside for other 

analyses. The dried tissues were extracted 

with petroleum ether for 5 to 6 hours in a 

soxhlet apparatus . The lipid content was determined by allowing the petroleum ether to 

evaporate and weighing the residue. They were 

then redissolved in the same solvent.

The extracts were analyzed for Thiodan 

by the colorimetric procedure described by 

Maitlen et al. (1963). He observed that captan, 

chlordan, heptachlor, and ovex caused some 

interference in the method, but 45 pesticides 

and several chlorinated solvents did not. The 

method is applicable for 5 to 50 ug of Thiodan 

and also measures Thiodan alcohol. The analyses were made on a Beckman Model DB spectrophotometer.

Before Thiodan can be analyzed colorimetrically, it must be separated (cleaned up) 

from lipids in ether extracts . Cleanup was
Richard A. Schoettger: Toxicology of Thiodan in Several Fish and Aquatic Invertebrates

accomplished by acetonitrile-petroleum ether 

partition, and by chromatography on a 60/100 

mesh, activated Florisil-carbon column 

described by Murphy and Barthel (1960).

Efficiency of the cleanup technique was 

determined by running Thiodan standards, or 

known amounts of Thiodan added to fish fat 

through the procedure. The columns were 

eluted with 8-percent diethylether in petroleum 

ether. The overall recovery averaged 56.3 

percent and varied approximately 3 percent. 

The average recovery was used to correct 

measurements of Thiodan in fish tissues . 

Maitlen et al. (1963) obtained recoveries of 

approximately 77 percent, and Moats (1963) 

indicated that only one isomer of Thiodan is 

eluted from Florisil. Thiodan alcohol was recovered completely after acetonitrile partition, 

but, as was also reported by Moats (1963), none 

could be eluted from the Florisil column.

In spite of cleanup, low levels of lipids 

persisted in Thiodan-fish fat standards and in 

the tissue samples, particularly those from 

muscle. The lipids did not appear to significantly alter absorbance or wavelength. Analyses of standards containing even greater lipid 

levels and known amounts of Thiodan supported 

the observations.

The bile samples were composed of bile 

from two fish which died on the same day, or 

successive days. For example, the bile of 

fish which died on the fourth and fifth days of 

treatment were combined into one sample, that 

from individuals dying on the seventh and eighth 

days formed another, and so forth. The samples 

were extracted with petroleum ether in a 

separatory funnel before and after incubation 

with beta-glucuronidase, as in the radiation 

experiments . The extracts were analyzed 

colorimetrically for Thiodan.

RESULTS AND DISCUSSION

Toxicity

Fish The toxic ity of Thiodan to rainbow 

trout and western white suckers is influenced by

temperature and expsoure. The 24- to 120-hour 

TLm's for trout at 1.5° and 10° C. range from 

5 .9 to 0.7 ppb and 2.1 to 0.3 ppb respectively 

(table 2). The TLm ranges for suckers at 10° 

and 19° are 8.1 to 2.5 ppb and 6.6 to 2.8 ppb 

(table 2). The results show that in general the 

toxicity of Thiodan to these species of fish increases with warmer temperatures and longer 

exposures.

The relation between toxicity and temperature and exposure is shown graphically in 

figure 1. In general, toxicity increases significantly up to approximately 72 or 96 hours and 

suggests that the toxicity is associated with 

accumulations of Thiodan in excess of the 

amounts that may be metabolized or stored by 

the fish. Relatively small changes in toxicity 

occurred with additional exposure (96 and 120 

hours), with the exception of suckers at 10° C. 

The plotted line for suckers at 10° is linear 

whereas that for trout is curvilinear. The 

latter species may be able to metabolize 

Thiodan more efficiently at 10° than the former, 

though not in equivalent amounts since suckers 

were more resistant.

The nonlinearity of several of the curves 

in figure 1 may also be related to the amount of 

toxicant available for uptake by fish in static 

tests such as these. Holden(1962) showed, for 

example, that within 10 hours 80 to 90 percent 

of the radioactive DDT in static tests was taken 

up by the fish, detritus, or on the sides of the 

containers . It is possible that constant-flow 

tests, or tests where the toxicant is periodically 

renewed would reveal greater toxicity over 

longer exposures.

Extrapolations of the 24- and 48-hour 

regressions used to determine TLm's for trout 

and suckers indicate that, with one exception, 

there is less effect of temperature and exposure 

on the concentrations necessary for 100-percent 

mortality than on the TLm's. The 100-percent 

lethal levels for trout are approximately 3 to 5 ppb 

and 8 to 10 ppb for suckers . The 24 -hour concentration of 9.5 ppb for trout at 1.5° C. is 

exceptional. Adlung (1957) observed that temperature had little effect on the "absolute lethal 

dosage" of Thiodan to goldfish.
10 Investigations in Fish Control 35: Bureau of Sport Fisheries and Wildlife

Table 2: Toxicity of Thiodan to two species of fish and aquatic invertebrates at two 

temperatures [Median tolerance limits in parts per billion; 95-perc;ent 

confidence intervals in parentheses].

Species and 

temperature

Rainbow trout:

At 1.5° C.

At 10° C.

Western white sucker:

At 10° C.

At 19° C.

Daphnia magna :

At 10° C.

At 19° C.

Damselfly naiads:

At 8° C.

At 19° C.

At 24 

hours

5.9

(5.2-6.6)

2.1

(1.5-2.7)

8.1

(7.4-8.^8)

6.6

(5.9-7.3)

178.0

(162-194)

68.0

(54-82)

235.0

(165-305)

275.0

(240-310)

At 48 

hours

2.1

(1.4-2.8)

1.1

(0.7-1.5)

6.4

(5.6-7.2)

4.3

(3.3-5.3)

132.0

(113-151)

62.0

(49-75)

120.0

(65-180)

175.0

(135-215)

At 72 

hours

1.4

 

0.4

 

4.9

 

3.1

 

87.5

 

60.5

 

84.5

 

150.0

At 96 

hours

0,8

 

0.3

 

3.5

 

3.0

 

52.9

 

56.0

 

71.8

 

107.0

At 120 

hours

0.7

 

0.3

 

2.5

 

2.8

 

47.5

 

53.5

 

62.0

 

75.0

The laboratory and field trials with 

Thiodan reported by other investigators show 

that it is highly toxic to a variety of species. 

Adlung (1957) killed goldfish with 10 ppb in 20 

hours at 19° to 22° C. Ludemann and Neumann 

(1960a) determined a 48-hour LC50 of 11 ppb for 

carp (Cyprinus carpio) at 17° to 19° . Relative 

to other organochlorine insecticides, they 

found that endrin was more toxic by a factor of 

two and toxaphene less toxic by a factor of five. 

Also, they later reported that 10 and 5 ppb 

killed all rainbow trout and northern pike (Esox 

lucius) respectively within 48 hours at 19° to 

21° (Ludemann and Neumann, 1961a).

F. M. C. Corporation (1958b) applied 

46 ppb of Thiodan to a 27-acre pond and killed 

all minnows, perch, sunfish, bullheads, and 

suckers within 7 days . Cuerrier (1960) was 

able to eliminate bluntnose minnows, golden 

shiners, common suckers, bullheads, perch, 

smallmouth bass, and sunfish from a lake with 

a concentration of 15 ppb. In both field trials, 

there was mortality of frogs and aquatic insects . Plankton declined, but later recovered.

The symptoms of Thiodan poisoning in 

trout and suckers are similar to those described by other investigators for fish exposed 

to organochlorine insecticides (Adlung, 1957; 

Henderson et al., 1959; and Ludemann and 

Neumann, 1960a and 1961a). The fish seem 

overly excitable at first and swim rapidly about 

the aquariums. Later they surface, lose 

equilibrium, and move with spasmodic jerks. 

In time, the majority sink to the bottom, and 

opercular movements become erratic . Many 

of the trout become darker, and the suckers 

appear mottled. Death follows after a variable 

period.

Some of the trout which survived the 

120-hour bioassays at 1.5° C. were placed in 

fresh aerated tap water. With tactile or 

vibrational stimuli, many individuals showed 

symptoms of Thiodan poisoning. Few lived 

longer than 5 to 7 days after the transfer. 

Ludemann and Neumann (1960a) found that the 

effects of Thiodan on carp, with one exception, 

were irreversible. One carp exposed to 14 ppb
Richard A. Schoettger: Toxicology of Thiodan in Several Fish and Aquatic Invertebrates 

WESTERN WHITE SUCKER TROUT

11

<r

< 5.0-

0-

10 C. 

19* C.

24 48 72 96 120 144 

HOURS

1.5 C.

IQ'C.

24 48 72 96 120 144 

HOURS

DAPHNIA MAGNA

cti

10 C. 

19* C.

24 48 72 96 120 

HOURS

CO

30Oco Io:

< 2SOCL

DAMSELFLY NAIADS

ao" c. 

i sfc.

24 48 72 96 120 144 

HOURS

Figure 1: Effects of time and temperature on the TLm's of rainbow trout, western white 

sucker, Daphnia magna t and damselfly naiads to Thiodan.
12 Investigations in Fish Control 35: Bureau of Sport Fisheries and Wildlife

lay on its side after 40 hours, but recovered 

completely in fresh water.

Thiodan is one of the more toxic chlorinated insecticides to fish, but it is one of the 

least toxic to mammals. Linquist and Dahm 

(1957) reported an acute oral LD50 of Thiodan 

in corn oil to rats of 40 to 50 mg per kg body 

weight. The manufacturer obtained a value of 

110 mg per kg when the insecticide was administered orally in cottonseed oil, and an LD50 

of 359 mg per kg when it was applied to the skin 

of rabbits (P.M.C. Corporation, 1964). 

Negherbon (1959) cited oral toxicity values in 

mg per kg for similar chlorinated insecticides 

dissolved in peanut oil: endrin 16.8; dieldrin 

38.3; toxaphene40.0; and aldrin45.9.

Loeb(1963) determined the oral toxic ity 

of Thiodan to carp. Technical Thiodan was not 

toxic in 120 hours at doses of 119 to 234 mg per 

kg, but doses of 49 and 195 mg per kg of 24 - 

percent miscible Thiodan were lethal within 118 

and 22 hours respectively. A dust form of the 

insecticide was twice as toxic as the miscible 

formulation. The lethal oral levels for carp 

are similar to the toxic doses for rats. Although the doses for carp cannot be compared 

directly with lethal concentrations in water, they 

suggest th^t Thiodan is more easily absorbed 

across gill and oral membranes than by the 

intestine.

According to my results, and those of 

others, Thiodan appears to have little value as 

a selective piscicide against suckers or carp. 

Concentrations which are lethal to so-called 

rough or problem species are also toxic to game 

fish such as trout and northern pike. Fishery 

biologists recognize, however, that in many 

instances where fish control is indicated, the 

proportion of game fish may be insignificant. 

Here, a nonselective poison such as Thiodan 

could be used provided it was not overly detrimental to other aquatic organisms and decomposed rapidly into a harmless product.

Fertilized fish eggs--The exposure of 

rainbow trout eggs to various concentrations of 

Thiodan for 30 or 120 minutes appears to have

no adverse effect on their hatching success 

(table 3). Development in the eggs had progressed to approximately the 32- or 64-cell 

stage at the time of treatment. The eggs 

hatched 29 days after fertilization, and mortalities in the control groups were similar to 

those observed in groups exposed to concentrations of up to 50,000 ppb. I was unable to 

test higher concentrations because Thiodan could 

not be maintained in the test solutions . It 

formed a noticeable precipitate in the water and 

adhered to the sides of the containers .

Neither gross nor microscopical observations of treated and control eggs revealed 

any abnormal development that was attributed 

to exposure to Thiodan. Large numbers of 

young fish in the test and control groups died 

46 days after hatching. Fish from the same 

lot of eggs, but used in other experiments at 

the Fish Control Laboratory, also died. Some 

factor other than Thiodan was believed responsible for the mortality, although a causative agent 

could not be established.

Rainbow trout eggs are extremely resistant to Thiodan in comparison with 2-inch 

fish of the same species . This resistance may 

be related to selective permeability of the egg 

chorion or to underdevelopment of Thiodansensitive structures at the time of treatment, 

or both. Antimycin A, a registered fish toxicant wnich inhibits oxidative-phosphorylation 

(Walker, et al., 1964), was tested against eggs 

from the same lot as those in the Thiodan trials. 

It killed the eggs at a concentration of 10 ppb, 

which suggests selective permeability of the 

egg chorion, or at least a mode of action 

different from that of Thiodan. Exploratory 

tests with Thiodan and antimycin against the 

eggs of northern pike support this finding.

Berger_/ also tested Thiodan against 

eggs of rainbow trout, but exposed them continuously to the toxicant until they hatched.

3_/ Personal communication from Bernard L. 

Berger, Chemist, Bureau of Sport Fisheries 

and Wildlife, Fish Control Laboratory, 

La Crosse, Wis., 1965.
Richard A . Schoettger: Toxicology of Thiodan in Several Fish and Aquatic Invertebrates 13

Table 3: Hatching success of rainbow trout eggs after 30- or 120-minute exposures to 

various concentrations of Thiodan.

Number 

of 

Concentration and exposure egg s

Control:

10

100

1,000

10,000

50,000

30 minutes

120 minutes

ppb: 

. 30 minutes

120 minutes

ppb: 

30 minutes

120 minutes

ppb: 

30 minutes

120 minutes

ppb: 

30 minutes

120 minutes 

ppb:i/ 

30 minutes

120 minutes

100

100

100

100

100

100

100

100

100

100 

100

100

Number 

unfertilized

1

7

5

5

7

5

7

2

6

0 

3

3

Number 

Number remaining 

examined before 

microscopically hatching

35

28

31

29

31

29

31

23

31

28 

32

23

64

65

64

66

62

65

62

70

63

72 

65

69

Percent 

hatching

76.6

70.8

84.4

69.7

51.6

86.2

79.0

68.2

76.2

94.4 

73.8

85.5

_!/ Thiodan formed a white precipitate in these solutions.

The hatched fish were held in the same solutions . He found that levels up to 50,000 ppb 

were not lethal over the 25-day incubation 

period, but that the fish became more susceptible after hatching and as they grew and absorbed 

the yolk sac. Concentrations of 750, 500, and 

250 ppb caused 100-percent mortality of trout 

fry after 7, 12, and 20 days respectively. None 

of the treated fish survived after 30 days in 

100 ppb of Thiodan, although about 30 percent of 

the controls also died at this time. lyatomi et 

al. (1958) observed similar toxicity of endrin to 

newly hatched carp and snakehead fish (Channa 

argus).

Daphnia magna--Liidemann and Neumann 

(1960b, 1960c, 1961b, and 1962) and Cuerrier 

(1960) indicate that most aquatic invertebrates, 

including Daphnia, are less sensitive than fish 

to Thiodan. My bioassays support this finding 

(table 2). The 24- to 120-hour TLm's at 19° C. 

range from 68 .0 to 53 .5 ppb, and at 10° the 

values are 178 to 47.5 ppb. The lowest value 

at 10° indicates that D. magna are at least six

times as resistant to Thiodan as suckers or 

trout. Thiodan used in fish control at concentrations of approximately 50 ppb, as it was in 

one experimental field trial, could have an 

adverse effect on Daphnia populations.

Liidemann and Neumann (1960b) suggest 

that D. magna are even more resistant to 

Thiodan than was indicated by my bioassays . 

They found that 100 ppb caused only partial 

mortality, and 1,000 ppb killed all of the animals 

in 24 hours at 18° to 21° C. Extrapolation of 

the regression for the 24-hour TLm at 19° 

yields a 100-percent lethal concentration of 

about 90 to 100 ppb. Since they did not measure 

the toxicity of concentrations between 100 and 

1,000 ppb, levels considerably below the latter 

value also may be 100-percent lethal.

Thiodan does not appear to have a significant chronic effect on D. magna at 19° C . The 

TLm values change little with longer exposures 

(fig. 1). The chemical degradation of Thiodan 

is a possible explanation. The addition of
14 Investigations in Fish Control 35: Bureau of Sport Fisheries and Wildlife

sodium bicarbonate to the media containing 

Daphnia increased pH to about 7.9. In later 

tests pH 8 .4 promoted decomposition of Thiodan, and conceivably pH 7.9 had a similar 

effect.

At 10° C. Thiodan is initially 2.5 times 

less toxic to D. magna than at 19° (fig. 1). 

With longer exposures the toxicity of the compound increases and after 96 hours gives TLm 

values similar to those at the higher temperatures . The chronic effect of Thiodan at 10° 

suggests that degradation at alkaline pH is 

temperature dependent.

Damselfly naiads - -Damselfly naiads 

appear to be the most resistant to Thiodan of the 

four species of animals tested, except for trout 

eggs. However, in these trials the chemical 

was more toxic to naiads in cold water than in 

warm water. The TLm's at 8° C. ranged from 

235 to 62 ppb, whereas at 19° the values were 

275 to 75 ppb (table 1). In tests with other 

species, the compound is usually less toxic at 

colder temperatures . The greater toxicity of 

Thiodan to naiads at 8° was unexpected, but a 

second series of tests supported the earlier 

results . Since the bioassays extended over a 

period of 4 to 5 weeks, older naiads were used 

in the 8° trials . These specimens may have 

been physiologically more susceptible to Thiodan 

than younger individuals. McDonald and Jacobson 

(1958) demonstrated differences in the susceptibilities of army cutworms of various ages to 

endrin. They found that older larval instars 

were more resistant. The older damselfly 

naiads in my trials may have been less resistant. 

Thiodan appears to have a chronic effect on the 

insects with time, but levels off at about 60 to 

70 ppb after 120 hours of exposure at both temperatures (fig. 1).

According to the results of Stringer and 

McMynn (1956), and Schoettger and Olive (1961) 

toxaphene appears relatively more toxic than 

Thiodan to damselfly naiads . They found that 

levels of 10 to 30 ppb of the former compound 

were lethal. However, insects such as 

Chironomus and Corethra (Chaoborus) are more 

sensitive to Thiodan than to toxaphene (Ludemann 

and Neumann, 1962).

Extrapolations of the 24- and 48-hour 

regressions indicate that approximately 425 ppb 

of Thiodan are required to kill 100 percent of 

the damselfly naiads . This concentration is 

relatively independent of temperature and 

exposure.

Effects of water quality on toxicity

Calcium and magnesium salts--Salts of 

calcium and magnesium in concentrations of 

500 ppm, as calcium carbonate hardness, do 

not alter the toxicity of 20 ppb of Thiodan to 

suckers (table 4). The mortalities of fish in 

solutions containing salts and Thiodan, and in 

those without salts are similar and appear unrelated to aging of the solutions . Statistically, 

the probability of observing this set of data 

under the null hypothesis is 0.81. Solutions 

containing only the salts are not toxic.

pH--Trials with D_. magna suggested 

that alkaline pH may influence the toxicity of 

Thiodan. Solutions containing 20 ppb of Thiodan 

were adjusted to pH 6.4 or 8 .4 and bioassayed 

with suckers at 19° C. The results in table 5 

indicate that pH has no effect on toxicity. Since 

the solutions were open to the air and aerated, 

pH was measured periodically. It declined 

about 2 to 4 tenths of a pH unit in both acidic and 

basic solutions during the first few hours of the 

test. After 24 hours the pH of acidic solutions 

was approximately 6.2 and that of the basic 

solutions was 7.4 . Although pH control was 

unsatisfactory, the insecticide did not degrade 

rapidly in the early segment of the test.

The later determinations of effects of 

pH on the toxicity of Thiodan were performed 

by adjusting pH of the solutions and then sealing 

and storing them for various periods before 

bioassay. The change in pH was less than 0.75 

of a unit over periods of 240 hours using this 

technique. The chemical degradation of Thiodan 

with time rather than the immediate effect of 

pH on toxicity was measured.

The storage of solutions containing 

20 ppb of Thiodan at pH 6.4 for periods up to 

240 hours has no effect on their toxicity to 

suckers (table 6). A statistical evaluation of
Richard A . Schoettger: Toxicology of Thiodan in Several Fish and Aquatic Invertebrates 15

Table 4: Effects of calcium and magnesium salt solutions, and solution age, on the 

24-hour toxicity of 20 ppb of Thiodan to western white suckers at 19° C.

Age of

solutions

prior to

bioassay

48 hours

192 hours

43.6 hours

Number

of fish

per jar

10

10

10

Tap

water

control

10

10

10

Number of fish surviving in 

Tap water containing

500 ppm of

CaCl 2

10

10

10

MgCl 2

10

10

10

CaSO/4 IS

10

10

10

igs<\

10

10

10

20 ppb of Thiodan

20 ppb of and 500 ppm of

Thiodan

0

3

1

CaCl 2

2

2

0

MgCl 2 (

2

1

0

_*d ow 1.

0

2

1

MgS04

2

0

0

Table 5: Effects of pH on the 24-hour toxicity of 20 ppb of Thiodan to western white 

suckers at 19° C.

Number of fish surviving in 

Test

No. 1 

No. 2

Number 

of fish 

per jar

10 

10

Tap water 

control: 

pH 6.4 

replicates

123

10 10 10 

10 5 10

Tap water 

control : 

pH 8.4 

replicates

123

10 10 10 

10 10 10

20 ppb of 

Thiodan: 

pH 6.4 

replicates

123

100 

000

20 ppb of 

Thiodan: 

pH 8.4 

replicates

123

111 

101

Table 6 : .Effects of pH 6.4 and 7.4, and solution age on the 24-hour toxicity of 20 ppb 

of Thiodan to western white suckers at 19° C.

Number of

Age of 

solutions 

prior 

to 

bioassay

120 hours 

168 hours 

240 hours

Number 

of fish 

per jar

10 

10 

10

Tap water 

control: 

pH 6.4 

replicates

1 2

10 10 

10 10 

10 9

3

10 

10 

10

fish surviving

1

10 

10 

10

Tap water 

control : 

pH 7.4 

replicates

2

10 

10 

10

3

10 

10 

9

in 

1

0 

6 

0

20 ppb of 

Thiodan: 

pH 6.4 

replicates

2

0 

2 

0

3

1

7 

0

1

0 

0

1

20 ppb of 

Thiodan: 

pH 7.4 

replicates

2 3

0 1 

1 0 

4 1

mortalities in toxic solutions at pH 6.4 and 7.4 

gave a probability under the null hypothesis of 

0.04. This is a low probability in comparison 

with that calculated above for the effect of 

calcium and magnesium salts. It suggests 

some degradation of Thiodan in solutions of 

pH 6.4; however, survival in these vessels did 

not appear random, but predominated in those 

aged for 168 hours. The relatively high survival in two jars may be related to an error in the 

preparation of solutions, or to an inadvertent 

selection of resistant fish. In general, I considered the experiment adequate and the original 

conclusion valid.

The aging of alkaline solutions containing 

20 ppb of Thiodan reduces or completely eliminates their toxicity to suckers (tables 7 and 8). 

Solutions of pH 8 .4 were only slightly toxic 

after 72 hours of aging. The mortalities of fish 

in solutions aged for 24 and 48 hours averaged 

60 and 15 percent respectively. Interpolation 

of these percentages on the 24-hour concentrationmortality regression gives Thiodan concentrations 

of about 7 .3 and 3 .8 ppb and indicates a threeto five-fold degradation of the insecticide.

Nearly all of the toxicant degrades within 

24 hours at pH 9.4 (table 8). Mortalities were
16 Investigations in Fish Control 35: Bureau of Sport Fisheries and Wildlife

Table 7: Effects of pH 7.4 and 8.4, and solution age on the 24-hour toxicity of 20 ppb 

of Thiodan to western whLte suckers at 19° C.

Number of fish surviving

Age of 

solutions 

prior to

bioassay

24

48

72

96

hours

Test

Test

hours

Test

Test

hpurs

Test

Test

hours

Test

Test

.

No,

No.

j

No.

No.

:

No.

No.

:

No.

No,

1

2

3

4

5

6

7

8

Number 

of fish

per jar

10

10

10

10

10

10

10

10

Tap 

water 

control :

pH 7.4

10

10

10

10

10

10

10

10

Tap 

water 

control :

pH 8.4

10

10

10

10

10

10

10

10

20 ppb 

of 

Thiodan:

pH 7.4

1

1

2

0

1

1

2

1

in 

20 ppb of 

Thiodan: 

pH 8.4 

replicates

1

0

8

10

8

10

7

10

10

2

10

3

10

10

10

10

8

10

3

0

7

10

9

10

10

10

10

4

3

2

6

6

10

10

10

10

Table 8:--Effects of pH 7.4 and 9.4, and solution age on the 24-hour toxicity of 20 ppb 

of Thiodan to western white suckers at 19° C.

Number of fish surviving in 

Age of 

solutions 

prior to

bioassay

24

48

72

96

hours

Test

Test

hours

Test

Test

hours

Test

Test

hours

Test

Test

.

No.

No.

:

No,

No.

:

No.

No.

:

No.

No.

1

2

3

4

5

6

7

8

Number 

of fish

per jar

10

10

10

10

10

10

10

10

Tap 

water 

control :

pH 7.4

10

10

10

10

10

10

10

10

Tap 

water 

control :

pH 9.4

 10

10

10

10

10

10

10

10

20 ppb 

of 

Thiodan:

pH 7.4

5

2

3

0

3

0

1

0

20 ppb of 

Thiodan: 

pH 9.4 

replicates

1

10

8

10

10

10

10

10

10

2

10

10

10

10

10

10

10

10

3

10

10

8

10

10

10

10

10

4

10

10

9

10

10

8

10

10

observed in only 4 of 32 potentially toxic solutions and none exceeded 20 percent. All of the 

control fish survived at pH 7.4 to 9 .4 and the 

mortalities in 20 ppb of Thiodan at pH 7.4 

ranged from 50 to 100 percent.

Statistical probabilities for observing 

certain blocks of data in tables 7 and 8 were

computed under the null hypothesis (table 9). 

The relative magnitude of the probabilities was 

considered in assigning significance to the 

various comparisons. The very low probability 

that the survival of suckers in Thiodan solutions 

aged for 24 hours at pH 8 .4 was no different from 

that of fish in the control (comparison 2) indicates residual toxicity. The higher probability
Richard A . Schoettger: Toxicology of Thiodan in Several Fish and Aquatic Invertebrates 17

Table 9: Probabilities, under the null hypothesis, of observing various sets of data 

taken from experiments on pH.

Comparison

Table 

number

Age of

solutions

(hours) Comparison Probability

No. 1

No, 2

No, 3

No. 4

No. 5

No. 6

24

8.4

48, 72, 96

48, 72, 96

24, 48, 72, 96

24, 48, 72, 96

Survival in 20 ppb of 

Thiodan, pH 7.4,

vs. 

Survival in 20 ppb of

Thiodan, pH 8.4 

Survival in control, pH

vs. 

Survival in 20 ppb of

Thiodan, pH 8.4

Survival in 20 ppb of

Thiodan, pH 7.4

vs. 

Survival in 20 ppb of

Thiodan, pH 8.4 

Survival in control, pH 8.4

vs. 

Survival in 20 ppb of

Thiodan, pH 8.4

Survival in 20 ppb of

Thiodan, pH 7.4

vs. 

Survival in 20 ppb of

Thiodan, pH 9.4 

Survival in control, pH 9.4

0.0064.

Approximately

Approximately 

1.03" 36 .

0.0254.

Approximately 

5.33- 53 .

0.2069.

3urvtv.il in 20 ppb of 

Thiodan, pH 9.4

for comparison 1, however, suggests that some 

detoxification of the chemical occurs with 24 

hours . The aging of similar solutions for 

longer periods (comparisons 3 and 4) results in 

further detoxification which is demonstrated by 

a reversal in magnitude of probabilities for the 

respective comparisons. The values for comparisons 5 and 6 indicate extensive detoxification 

of Thiodan at pH 9.4 .

Of the various water-quality factors 

which were considered as possibly important 

in the detoxification of Thiodan, alkaline pH is 

the most significant. Calcium and magnesium 

ions and mildly acid media appear to have no 

effect on toxicity. Thus, the residual toxicity 

of Thiodan can be minimized when treatments 

are made just prior to anticipated increases in

basicity. We must not discount the interaction 

of temperature and pH on the residual toxicity 

of Thiodan. The efficacy of alkaline pH on de - 

toxif ication of Thiodan at temperatures lower 

than 19° C . may be considerably less . Bergerl./ 

found that 9 days were required to detoxify 

10 ppb of Thiodan to rainbow trout at pH 9 and 

12° C.

According to Frensch et al. (1959) the 

fate of Thiodan in water is hydrolysis to Thiodan 

alcohol. Apparently little is known of the 

biological toxicity of the alcohol, although it

4/ Personal communication from Bernard L. 

Berger, Chemist, Bureau of Sport Fisheries 

and Wildlife, Fish Control Laboratory, 

La Crosse, Wis.
18 Investigations in Fish Control 35: Bureau of Sport Fisheries and Wildlife

must be relatively nontoxic to fish. Lindquist 

and Dahm (1957) tested it against one rat, and 

an oral dose of 1370 mg per kg was not lethal.

Deposition and metabolism of Thiodan

Radioactive Thiodan--The exposure of 

suckers to 20 ppb of 14C-labeled Thiodan results 

in the deposition of significant amounts of radioactive substances in their tissues (tables 10, 11, 

and 12). The mean concentrations of labeled 

substances in ug per g of dry tissue during 12 

hours of exposure are: liver 8 .4; gut and feces 

5.8; blood4.4; heart 4.1; gill 3.1; kidney 2.7; 

gut (empty) 2.7; brain 2.6; skin 1.8; and muscle 1.1.

A concentration of 80 ppb of 14C-labeled 

Thiodan killed suckers within 2.25 and 9 .25 

hours of exposure. The mean levels of radioactive compound in their tissues in jig per g of 

dry tissue are: liver 22.9; gut and feces 14 .5; 

blood 8.9; brain 6.5; kidney 6.0; heart 5.6; 

gut (empty) 5.3; gill 4 .0; skin 2.6; and muscle 

1.8 (tables 10, 11, and 12). The livers, guts, 

blood, brains, and kidneys of fish treated with 

80 ppb of C-labeled Thiodan contain approximately 2 to 3 times as much radioactive 

material as was present in the same tissues 

of fish treated with, but not killed by, a concentration of 20 ppb. The activity levels in 

several tissues of the former seem highly 

variable and poorly correlated with exposure. 

Those in brain, on the other hand, were 

relatively consistent and may represent the 

concentration necessary to cause death.

The concentrations of radioactive substances, presumably l^c-labeled Thiodan, in 

muscles of suckers are relatively low in comparison with those in other tissues. This tissue, 

however, is probably one of the larger reservoirs for Thiodan deposition because of its 

relatively greater mass. In order to estimate 

the total Thiodan deposited in muscle in relation 

to that added to the external medium, the percent of muscle in the fish and its water content 

must be considered. Holden(1962) assumed 

that muscle amounted to 75 percent of the total 

body weight of brown trout, and Spector (1956)

placed the water concentrations in muscles of 

several freshwater fish at about 70 percent. 

Using these percentages, an average weight of 

fish of 4 0 grams, and average levels of Thiodan 

in muscle, I calculated that 9 .9 jag of Thiodan 

were present in the muscle of each fish treated 

at 20 ppb and 16.2 ;jg in those killed by 80 ppb. 

The deposits in muscle comprise 33 and 14 percent respectively of the total ^C-labeled Thiodan 

added to the external media. A fourfold increase 

of the Thiodan concentration in the water did not 

elevate muscle residues by a similar amount.

The small difference between muscle 

residues of fish treated at 20 or 80 ppb of 

14C -labeled Thiodan suggests that the insecticide may reach a level of saturation in muscle 

lipids. Ludwig, Weis, and Korte (1964) found 

that aldrin reached a level of saturation in the 

lipids of rats after which no additional toxicant 

was stored.

Linear regressions based on the unaveraged tissue concentrations of radioactive 

substances and exposures were calculated for 

fish treated at 20 ppb of 14C-labeled Thiodan 

to estimate correlation and rate of uptake 

(fig. 2 and table 13). The correlation coefficients for liver, gut and feces, blood, heart, 

gill, kidney, and brain range from 0.67 to 0.97 

and are statistically significant at the 0.05 level o 

probability. The concentrations of labeled 

substances in gut (empty), skin, and muscle 

are not significantly correlated with exposure, 

particularly those in muscle, which had a 

coefficient of only 0.29 . The tissues containing 

the greatest concentrations of radioactivity are 

not necessarily the ones with the highest rates 

of uptake. For example, gut and feces contain 

a lower average concentration than liver, but 

the rate of uptake in the former was approximately 22 percent greater. The intercepts and 

slopes of the regressions suggest that the uptake 

of l^C -labeled Thiodan by the various tissues is 

rapid and probably nonlinear during the first 

hour of exposure. Thus, the linear regressions 

cannot be extrapolated linearly to determine 

uptake rates for the first hour.
Richard A. Schoettger: Toxicology of Thiodan in Several Fish and Aquatic Invertebrates 19

Table 10: Concentrations of radioactive substances in liver, kidney, and gut of western 

white suckers following various exposures to two concentrations of C-labeled 

Thiodan at 19° C.

Concentration 

of

- a e e Hours 

Thiodan Exposure

20 ppb

20 ppb

20 ppb 

20 ppb 

20 ppb

20 ppb

20 ppb

20 ppb

Overall mean

80 ppb 

80 ppb 

80 ppb

Overall mean

1.00

2,00

3.00 

4.00 

6.00

8.00

9.00

12.00

2.75V

5.50V 

9.25V

Number 

of 

fish

2

1

2 

I 

2

1

2

1

1 

2 

1

Radioactive

Liver

Mean

4.3

4.2

6.3 

12. 2l/

10.4

13.9

11.1

8.4

18.8 

35. oi/ 

14.8

22.9

Counting 

error!/ 

(i)

0.4

0,4

0.5 

0.5 

0.4

0.8

0.5

0.9

0.6

0,5 

0.9 

0.7

0.7

substances (ug/g dry weight of tissue) in

Kidney

Mean

1.5

2,2

2.6 

1.3 

3,9

4.0

2.3

4.4

2.7

4.0 

9.5 

4.5

6.0

Counting 

error.!/ 

U)

0.6

0.9

0.5 

0.5 

0,7

1.2

0.4

1.2

O.S

0.6 

0.6 

0.5

0.6

Gut

Mean

1.2 i.8.y

3.7

2. a!/

7.2

3.&1/

10.9

3,o2/

5,8

2.7l/

5.9 

22.1

5. al/

14.5

5. a!/

Counting 

error.!/ 

U)

0.4

0.9

0.5 

1.1 

0.5

1.2

0.7

1.1

0.3

1.1

0.1 

1.0 

0.8

0.6 

0.8

I/ 0.05 probability limit. 

_2/ One sample. 

3/ Gut contents removed. 

4/ Tima of death.

Table 11: Concentrations of radioactive substances in blood, heart, and brain of

western white suckers following various exposures to two concentrations of 

l^C-labeled Thiodan at 19° C.

Concentration

of

14C-labeled Hours

Thiodan Exposure

20 ppb

20 ppb

20 ppb

20 ppb

20 ppb

20 ppb

20 ppb

20 ppb

Overall mean

80 ppb

80 ppb

80 ppb

Overall mean

1.00

2.00

3.00

4.00

6.00

8.00

9.00

12.00

2.251/

5.501/

9.251/

Number 

of

fish

2

1

2

1

2

1

2

1

1

2

1

Radioactive substances (ug/g dry weight of tissue)

Heart

Mean

2<

N, 3.

3.

5.

7.

4.

11.

6.

8.

]£/

5

-

9

-

3

-

0

0

1

7

9

Counting 

error!/

(i)

0.2

0.9

_-_

0.7

 

1.7

0.9

0.9

0.1

0.4

0.3

Mean

2.2

4.5

2.6

2,5

4.2

5.3

4.0

9.7

4.1

5.9

7.3

3.7

5.6

Counting 

error.!/

(4

0.

1.

0.

1.

0.

1.

0.

4.

1.

0.

1.

1.

1.

:)

7

6

8

7

9

7

7

0

5

6

1

2

0

in

Brain

Mean

1.5

2.3

1.8

1.6

3.5

2.8

3.4

3.7

2.6

6.2

7.3

5.0

6.5

Counting 

errorl/

(i)

0.4

0.7

0.4

0.6

0.4

0.7

0.4

0.1

0.6

0.4

0.6

0.7

0.6

JL/ 0.05 probability limit. 2/ One sample. _3/ Time of death.
20 Investigations in Fish Control 35: Bureau of Sport Fisheries and Wildlife

Table 12:--Concentrations of radioactive substances in skin, gill and muscle of western

1 4 white suckers following various exposures to two concentrations of C-labeled

Thiodan at 19° C.

Concentration

of

14 C-labeled

Thiodan

20 ppb

20 ppb

20 ppb

20 ppb

20 ppb

20 ppb

20 ppb

20 ppb

Overall mean

80 ppb

80 ppb

80 ppb

Overall mean

Radioactive substances

Skin

Hours

Exposure

1,00

2.00

3.00

4.00

6.00

8.00

9.00

12.00

2 25-2

5.5Q!/

9.2S.2/

Number 

of

fish

2

1

2

1

2

1

2

1

1

2

1

Mean

1.1

0.6

2.5

1.1

2.4

1.2

1.3

4.9

1.8

3.1

3.3

1.3

2.6

Counting 

errori'

(* )

0.8

0.7

1.1

1.1

0.9

0.5

0.8

2.9

1.1

0.9

0.4

1.1

0.8

(ug/g dry weight of

Gill

Mean

1.9

2.7

3.7

3.1

2.8

2.4

3.2

6.4

3.1

4.1

4.9

3.1

4.0

Counting 

error '

(-*-)

0.5

1,6

1.7

1.5

1.1

0.5

0.9

1.7

1.2

0.4

0.7

0.5

0.5

tissue) in

Muscle

Mean

1.2

0.5

0.8

0.6

1.8

1.3

1.1

1.3

1.1

1.4

3.3

0.8

1.8

Counting 

error.!/

(±)

0.3

0.2

0.2

0.2

0.3

0.4

0.3

0.4

0.3

0.2

0.3

0.2

0.2

_!/ 0.05 probability limit 

2/ Time of death.

15 

14-1

12-

Q- 9.

08.

</> 6. 

I''

S 4 . Q

Q* O .

liver

Table 13: Slopes and correlation coefficients for regressions in figure 2.

Tissue

Liver

Gut and feces

Heart

Blood

Gill

Kidney

Brain

Gut (empty)

Skin

Muscle

Slope 

(b)

0.93

1.18

0.48

0.34

0.29

0.33

0.25

0.14

0.23

0.05

Correlation 

coefficient 

(r)

0.85

0.97

0.78

0.89

0.67

0.84

0.82

0.66

0.48

0.29

muse le

567 

HOURS

9 10 11 12

Figure 2: Uptake of radioactive substances 

in tissues of western white suckers during 

their exposure to 20 ppb of C-labeled 

Thiodan at 19° C.

In general, with the exception of gut 

and feces, the higher concentrations and rates 

of uptake of labeled chemical occur in blood and 

those tissues containing relatively large amounts 

of blood. The actual volumes of blood in sucker 

tissues were not measured; however, Hoffert 

(1966) found that livers, gills, kidneys, and 

muscles of lake trout contained 17.0, 16.5, 

8.9, and 0.4 percent blood respectively. If
Richard A. Schoettger: Toxicology of Thiodan in Several Fish and Aquatic Invertebrates 21

similar amounts of blood are present in sucker 

tissues, and if the concentrations of radioactive 

substances were wholly dependent on blood content in tissue, then one would expect to detect 

concentrations of radioactivity in tissues which 

vary according to blood content, and the radio - 

activity in blood. The regressions in figure 2 

indicate that radioactive substances in liver, 

gill, kidney, and muscle were not sufficiently 

lower than those in blood to be correlated entirely with the blood content of the tissues. 

Since the ranking for concentrations of 

radioactivity in gill, kidney, and muscle 

was similar to Hoffert's blood values, the 

blood content may have some influence on 

the level of radioacitivity in the tissue.

The observation that the levels of 

labeled substances in the liver were much 

greater than those in blood suggests that Thiodan is removed from the blood by this organ 

and stored. The lower deposition and uptake in 

other tissues , except gut and feces, may be 

due to this function of the liver.

The removal of the contents of the gut 

lowers the concentration of radioactivity in the 

gut (table lOandfig. 2). This demonstrates 

that the majority of the radioactivity present 

in the intestine is contained in the feces rather 

than in the tissue itself. Mount (1962) treated 

bluntnose minnows with endrin and detected it 

within the gut. In his opinion, endrin may have 

entered the tract along with ingested water. 

Because few freshwater teleosts drink significant amounts of water (Black, 1957), another 

avenue for the entrance of Thiodan into the intestinal tracts of suckers was sought. Since 

the samples of gut used in the analyses were 

collected posterior to the entrance of the gallbladder, I postulated that the radioactivity may 

have entered with the bile.

A second series of experiments was 

necessary to test the hypothesis that Thiodan is 

excreted in bile. This also provided an opportunity to evaluate the solubility characteristics 

of the labeled materials in tissues and bile of 

14C-labeled Thiodan treated fish. Northern

creek chubs and western white suckers were 

exposed to 40 ppb of ^C -labeled Thiodan for 

3 or 5 hours at 21° C. The concentrations and 

relative benzene-water solubilities of radioactive substances in the bile, and in chub 

tissues are shown in tables 14 and 15. Simple, 

ethanolic extracts of chub tissues removed 

approximately 62 to 100 percent of the radioactivity (table 14). The majority of the labeled 

substances in the lipid extracts are separated, 

after benzene-water partition, into the benzene 

fraction. The remainder, ranging from 9 to 

20 percent of the total, appear water soluble; 

however, a portion or all of these may have 

been benzene soluble since emulsions were 

included with this fraction.

The concentrations of benzene-soluble 

radioactive substances are highest in liver, 

brain, kidney, and blood, in that order (table 14) 

Extension of the exposure from 3 to 5 hours 

did not increase the amounts deposited in the 

tissues.

Conclusions about distribution of Thiodan 

based on measurements of radioactivity must 

be qualified because of the possible metabolic 

destruction of the chemical. The fact that the 

radioactive substances extracted from chub 

tissues were soluble in ethanol and benzene 

indicates that activity levels in chubs and those 

measured earlier in suckers , represent 

deposits of Thiodan, or at least the aromatic 

portion of the molecule. According to Hawk, 

Oser, and Summerson (1954), aromatic compounds are fairly resistant to oxidation, whereas 

their acyclic side chains maybe catabolized.

Radioactive materials are present in 

the bile of chubs and suckers which are treated 

with 14C-labeled Thiodan (table 15). Ninetyfour to 100 percent of the activity is separated, 

by benzene-water partition, into the water 

fraction. The concentrations in this fraction 

range from 88 to 158 ug per g of dried bile for 

chub and 36.5 to 48 .0 ug per g for sucker. 

Semiqualitative measurements were made of the 

water-soluble metabolite in sucker bile. Incubation of the bile with beta-glucuronidase 

converted the labeled metabolite to a benzene-
22 Investigations in Fish Control 35: Bureau of Sport Fisheries and Wildlife

Table 14: Benzene- and water-soluble radioactive substances in extracts of tissues of 

northern creek chubs exposed to 40 ppb of ^C-labeled Thiodan at 21° C.

Radioactive substances (pg/g

Benzene 

soluble 

fraction

Tissue 

and 

exposure

Liver:

3 hours

5 hours

Kidney:

3 hours

5 hours

Blood:

3 hours

5 hours

Brain:

3 hours

5 hours

Number 

of 

fish Mean

2

2

2

2

2

1

2

2

8.2

4.5

3.3

4.2

2.3

2.0

5.3

3.1

Water 

soluble 

fraction

Cou.nti.ng 

error.!/ 

( ±.) Mean

0.4

0.2

0.5

0.8

0.3

0.5

0.6

0.6

1.

0.

0.

0

0.

0.

0

0

0

7

9

6

4

dry wt . of tissue) in

Extracted 

tissue

Counting Counting 

error! error!/ 

(±) Mean (±)

0.2

0.2

0.4

-,

0.3

0.3

-

-

1.7

1.8

0.3

2.6

0.4

0

0

0

0.

0.

0.

0.

0.

-

-

-

4

3

3

6

2

Percent 

in 

benzene

75.

64.

73.

61.

69.

83.

100

100

0

0

3

8

7

3

Percent 

in 

water

9.

10.

20.

0

18.

16.

0

0

0

0

0

2

7

Percent 

in 

tissue

15.

25.

6.

38.

12.

0

0

0

6

7

7

2

1

_!/ 0.05 probability limit

Table 15:--Benzene- and water-soluble radioactive substances in gallbladder bile of two 

species of fish exposed to 40 ppb of C-labeled Thiodan, and the effect of 

beta-glucuronidase on substance solubility.

Before incubation of bile with 

enzyme; radioactive substances 

(ug/g of dry bile) inAfter incubation of bile with 

enzyme; radioactive substances 

(ug/g of dry bile) in 

Benzene soluble Water soluble Benzene soluble Water soluble 

Species fraction fraction fraction fraction

and Confish cen- Counting

num- Expo- tra- error!/

ber sure tion (+)

Northern creek chub:

No. 1 3 5.6 0.9

No. 2 3 0.0

No. 3 5 4.5 0.9

Western white sucker:

No. 4 5 1.5 0.5

No. 5 5 2.2 0.5

Conl cenPer- tracent tion

6.0 88.0

0.0 158.0

3.5 124.0

3.9 36.5

4.4 48.0

Con- ConCounting cen- Counting cen- Counting

error!/ Per- tra- error.!/ Per- tra- error!/

(±)

5.5

5.8

5.9

1.7

1.7

cent tion (±) cent tion (±)

94.0 - -

100.0 - -

96.5 - -

96.1 46.0 1.7 99.4 0.3 0.6

95.6 41.4 1.0 97.2 0.1 0.6

Percent

-

-

-

0.6

2.8

I/ 0.05 probability limit.

soluble form. Thus, the results suggest that 

the liver removes Thiodan from the blood 

stream, changes it to an aromatic metabolite, 

and conjugates it with glucuronic acid. Then 

the conjugate is discharged with bile into the

gallbladder. The subsequent release of bile 

containing the radioactive conjugate into the gut 

may explain the high levels of radioactivity 

present in feces of suckers (fig. 2).
Richard A. Schoettger: Toxicology of Thiodan in Several Fish and Aquatic Invertebrates 23

Metabolism and excretion of Thiodan in 

fish appears to be similar to that observed in 

rats and rabbits for the excretion of other 

chlorinated insecticides, such as C-labeled 

aldrin and ^C-labeled dieldrin. These compounds are metabolized by the liver into more 

hydrophilic compounds and excreted via the 

bile into the intestinal canal (Korte, Ludwig, 

and Vogel, 1962; and Morsdorf et al., 1963).

Ludwig et al. (1964) found that rats fed 

low doses of ^C-labeled aldrin over long 

periods excreted aldrin, dieldrin, and unidentified hydrophilic products in both feces and 

urine. The total activity excreted daily over 

12 weeks increased from approximately 50 to 

100 percent of the daily dose. The amount 

excreted in the feces was 10 to 20 times as 

great as that in urine. The hydrophilic compounds in feces and urine were not alike 

chromatographically, and alkaline hydrolysis 

of the urinary metabolites gave a compound with 

acidic properties . Aromatic acids such as 

benzoic acid are conjugated with glucuronic 

acid (Long, 1961).

Additional evidence supporting the 

proposed pathway for the metabolism and excretion of Thiodan by fish will be discussed in 

the next section.

Nonradioactive Thiodan--As stated 

earlier, further trials with radioactive Thiodan 

were impossible owing to its degradation to 

what appeared to be Thiodan alcohol. To 

strengthen the findings of the radiation experiments , additional fish were treated with technical Thiodan, and its residues or metabolites 

in tissues and bile were analyzed chemically.

In preliminary experiments, goldfish 

and carp were killed with 20 ppb of Thiodan and 

their tissues analyzed. Traces of the insecticide were found in muscle, but the levels were 

beyond the range of accurate measurement by 

the analytical method. The possibility that concentrations of Thiodan in the tissues could be 

elevated by exposing the fish for longer periods 

to lower concentrations was tested by treating 

one goldfish and one carp daily with 7.0 ppb.

The carp died after the second treatment and 

the goldfish after ten. Only traces of the toxicant were found in carp, but 1.1 pg per g wet 

weight were measured in muscle of the goldfish 

and 0.5 ug per g in its gills. Seven ug per ml 

were found in the bile after incubation with 

beta-glucuronidase. Since only traces of 

Thiodan were found in other goldfish tissues, 

samples from a number of fish were combined 

in later trials in order to measure Thiodan in 

these tissues satisfactorily. Repeated treat - 

ments of goldfish with ordinarily subacute 

concentrations apparently cause mortality and 

induce measurable residues of Thiodan.

Ten goldfish weighing 96 to 337 grams 

were exposed daily and individually to a concentration of 7.0 ppb of Thiodan. The order 

in which the fish died according to their 

weight, sex, length of exposure, and total 

Thiodan treatment is shown in table 16. The 

two smallest specimens were males, and they 

succumbed on the fourth and fifth days of treatment after exposure to combined concentrations 

of 28 and 35 ppb of Thiodan respectively. The 

remaining fish were females which died at 

intervals up to 13 days after the initial exposure. 

Two of the largest individuals survived 20 

applications, or exposure to a total of 2.10 mg 

of Thiodan.

The largest deposits of Thiodan residues 

in goldfish tissues ranged from 3.40 to 12.76 jig 

per g of wet tissue in the bile, liver, brain, 

and abdominal fat respectively (table 16). The 

mean residues in ug per g of wet tissue for 

other tissues are: scales and fins 0.45; gonad 

0.67; gill 0.86; heart, kidney, spleen, and 

blood 1.39; muscle 1.67; and skin 2.48 . 

Thiodan was not detected in the gut and feces .

Susceptibility of goldfish to repeated 

subacute concentrations of Thiodan is strongly 

related to their weight. The lightest individuals 

are the most susceptible. The correlation of 

weight of fish with number of treatments before 

their death gives a coefficient of 0.89.

The correlation of weight with number 

of treatments suggests an accumulation of
24 Investigations in Fish Control 35: Bureau of Sport Fisheries and Wildlife

Table 16:--Residues of Thiodan in goldfish treated daily in fresh solutions containing 

7 ppb of Thiodan at 12° C.

Thiodan Concentration of Thiodan in ;ug 

treatments weight in -

Fish Weight

Amount 

Number before 

before death MusSex death (mg)(ppb) cle Bilel/Gill

Gut 

and 

Scales conSkin fins Liver tent

per g wet

Heart Perikidney tospleen neal 

Gonad blood Brairw'fatl/

No. 1 109

No. 2 96 M

No. 3 129 

No. 4

4 0.42 28 1.41

5 0.53 35 2.54

7 0.74 49 2.10

Samples composed of tissues of fish 1 to 5 

0.69 3.28 0.77§76100.731.23

0

No. 5 188 F 11 

No. 6 191 F 13

8 0.84 56 1.15 

1.16 77 

3.67

1.37 91 1.99 Samples composed of tissues of fish 6 to 10 _____

No. 7 184 F 13

No. 8 277 F 13 

No, 9 241^ / F 20

No.10 338^/ F 20 2.10 140 

1.37 91 2.13

3.40

1.37 91 0.95 1.03 1.69 0.21 5.32 0 0.61 1.54 

2.10 140 1.09 12.76

1.67 3.53 0.86 2.48 0.45 5.M-7 0 0.67 1.39 8.55 12.76

_!/ Samples composed ot bile of two fish. Bile incubated with beta-glucuronidase. Mean

based on positive values only. 

_2/ Sample composed of brains of 10 fish, 

_3/ Sample composed of fat from fish 9 and 10. 

4-7 Alive after 20 treatments.

Thiodan with time; larger fish, because of 

greater mass, require a longer exposure to 

attain the same concentration of Thiodan in 

their tissues as smaller fish. Residues in fish 

treated for up to 20 days are essentially no 

greater than those in individuals receiving up to 

11 treatments. The coefficient of correlation 

for residues in muscle, based on wet weight, 

with days of exposure is 0.38.

The differences in sizes of the goldfish 

are due primarily to weight rather than length. 

The heaviest fish weighed approximately three 

times as much as the smallest but was only 

30 percent longer. This length-weight relation 

suggests that the heavier and more resistant 

individuals also contained the greatest amount 

of lipids . The lipids in goldfish muscle were 

calculated as a percent of the wet weight of the 

tissue and for fish 1 to 10 (table 16), are:

1.2, 3.5, 2.1, 3.4, 6.7, 3.0, 5.6, 3.7, 6.8, 

and 6.1 percent. In terms of dry weight, most 

of the values are between 14 and 26 percent. 

The correlation of lipid content with the number 

of treatments which they survived gives a 

coefficient of 0.77 indicating that individuals 

with more muscle lipids survive longest.

The male goldfish were the most sus - 

ceptible to Thiodan, but also they were the 

smallest specimens . These two variables were 

confounded, and the independent influence of 

sex could not be evaluated.

The micrograms of Thiodan per gram 

of muscle lipids extracted from each of the 10 

fish are, in order: 113.28, 71.93, 97.73, 

33.68, 64 .37, 37.92, 25.39, and 16.15 . The 

concentrations are negatively correlated with 

the number of treatments, weight of fish, and 

the lipid content of the muscle. The coefficients 

are 0.78, 0.75, and 0.85 respectively. Whereas 

the quantity of the Thiodan in muscle was relatively constant, an increase in lipid content
Richard A . Schoettger: Toxicology of Thiodan in Several Fish and Aquatic Invertebrates 25

allows a decline of Thiodan in muscle lipids of 

heavier fish. These data indicate that muscle 

lipids aid in the "detoxication" of Thiodan 

by providing a reservoir for its storage and 

dilution. However, since all but the two most 

resistant goldfish died during the experiment 

the actual amount of poison in muscle appears 

to be more closely allied with mortality than 

its concentration in lipids. Thus, a physically 

greater mass of lipids in heavier fish contribute more to their survival than the degree of 

saturation of lipids with Thiodan. The high 

concentration of Thiodan in abdominal fat of 

fish 9 and 10 (table 16) indicates that insecticide 

storage in this tissue may also contribute to 

the resistance of the heavier individuals .

Thiodan concentrations are considerably 

greater in brain than those in gill, liver, and 

a composite of heart, kidney, spleen, and blood 

(table 16). This is contrary to observations 

made on suckers where the high concentrations 

of what was presumed to be Thiodan were 

associated with blood containing tissues . Also, 

the skin of suckers contained less Thiodan than 

that of goldfish. This apparent inconsistency 

may be related to a greater lipid content of 

goldfish, and their longer exposure to Thiodan.

Thiodan is adsorbed on or absorbed into 

the scales and fins of goldfish (table 16). 

Although the levels are comparatively low, 

they demonstrate an ability of the body surfaces 

to remove and concentrate the insecticide from 

the external medium. Holden (1962) found that 

DDT was concentrated by the external mucus of 

brown trout, and he pointed out the possibility 

of its entry into fish through the skin.

The gonads of suckers treated with 

C-labeled Thiodan were not assayed for 

radioactivity, but composite samples of goldfish gonads contain 0.67 to 0.73 yag of Thiodan 

per g of wet tissue (table 16). In toxicity 

trials, the fertilized eggs of rainbow trout 

were extremely resistant to Thiodan; however, 

incorporation of the insecticide into fish eggs 

before spawning may have an adverse effect on 

their fertility or survival. Residues of chlorinated insecticides such as DDT in fish eggs are

suspected of reducing their reproductive 

success (Burdick et al., 1964; Cuerrier, 

Keith, and Stone, 1967; and Macek, 1968).

The water content of samples of goldfish 

muscle ranged from 71.3 to 78 .1 percent and 

averaged 75.2 percent. The two fish receiving 

20 treatments with Thiodan (table 16) had the 

lowest water concentrations in their muscles . 

In general, however, there appeared to be no 

relation between the mortality of fish and 

tissue water.

The measurement of Thiodan in various 

tissues of goldfish by relatively specific 

chemical methods supports an earlier belief 

that the majority of the radioactive substances 

detected in suckers were indeed ^C -labeled 

Thiodan. According to Barnes and Ware (1965) 

technical Thiodan contains four components, 

the high and low melting point isomers, 

Thiodan alcohol, and Thiodan ether. The 

chemical method used in my investigation 

measures both isomers and the Thiodan alcohol 

but not the ether. The low melting point isomer 

and the alcohol are not recoverable from the 

Florisil-carbon column. The possibility that 

a small portion of the radioactivity in suckers 

did not represent Thiodan is suggested by the 

incomplete extractions of radioactive substances 

from some chub tissues with ethanol (table 14). 

The remaining activity may possibly have been 

associated with water-soluble metabolites.

Thiodan is not detected colorimetrically 

in petroleum ether extracts of goldfish bile 

until after the samples are incubated with betaglucuronidase, nor in extracts of gut and content 

(table 16). This was expected considering the 

existence of water-soluble radioactive substances found in the bile of suckers and chubs 

(table 15). The wavelength of maximum 

absorption for the colored product is 534 mu, 

the exact wavelength used for measurements of 

Thiodan. Its average concentration in bile is 

3.5 ug per ml, after applying a correction for 

a background of unidentified constituents of the 

bile. The uncorrected concentrations agree 

closely with that measured in bile during the 

preliminary investigation. The average
26 Investigations in Fish Control 35: Bureau of Sport Fisheries and Wildlife

background was 3.6^ig per ml. It was derived 

by measuring the absorbance, at 534 mu, of 

two samples which apparently contained no 

Thiodan; one made up of bile from fish 1 and 2 

and another from fish 3 and 4 . In these sam - 

pies, peaks of maximum light absorption were 

not detected at 534 mu.

The apparent lack of Thiodan metabolites 

in the bile of some goldfish may be attributed 

to: amounts below the limits of detectability 

due to small volumes of bile or slow rates of 

conjugate formation; or inhibition of betaglucuronidase. The volumes of bile in fish 1 

to 4 were 0.4 to 0.5 ml whereas those in the 

remaining fish usually exceeded 0.7 ml. 

Ludwig et al. (1964) found that rats excreted 

greater amounts of aldrin metabolites the 

longer they were maintained on an aldrin containing diet. Beta-glucuronidase is inhibited by 

several substances including a product of the 

hydrolysis, glucuronic acid (Long, 1961).

The hydrolysis of water-soluble metabolites of Thiodan, in experiments with both 

suckers and goldfish, with beta -glucuronidase 

indicates that these species are able to metabolize the insecticide and conjugate it with glucuronic acid. According to Harper (1959), in 

mammals uridine diphosphate glucuronic acid 

(UDPGA) is the active form of the acid involved 

in the conjugation of chemicals containing carboxyl and hydroxyl groups . It is formed by the 

oxidation of UDP-glucose which is catalyzed by 

a diphosphopyridine nucleotide-dependent UDPG 

dehydrogenase. The conjugation reaction requires glucuronyl transferase. My results 

imply that not only are both enzymes present 

in the livers of these species, but also that 

this organ is able to convert the insecticide to 

a substance containing carboxyl or hydroxyl 

groups. The conjugable substance is probably 

Thiodan alcohol since it, like Thiodan, reacts 

colorimetr ically.

Barnes and Ware (1965) studied the 

metabolism of Thiodan in house flies. It was 

oxidized to Thiodan sulfate, a slightly less 

toxic compound which was considered to be an 

intermediate in the detoxication process .

Their analyses of feces revealed water- and 

acetone-soluble substances which they believed 

were glucoside, or glucosiduronic conjugates 

of Thiodan metabolites . Although they did 

not establish the identity of the conjugate, they 

did eliminate the possibility of conjugation at 

the terminal sulfur of Thiodan. Thus, experiments with fish and house flies suggest at least 

one pathway for the biochemical degradation of 

Thiodan. First, oxidation to Thiodan sulfate, 

then conversion to Thiodan alcohol, followed 

by conjugation with glucuronic acid and excretion in the feces . The results of Barnes and 

Ware (1965) also indicate that the resistance 

of flies to Thiodan may be controlled by the 

rate of its oxidation, or the rate of conjugation 

of the metabolite.

The lip ids of fish may serve as a 

reservoir for depositing the quantities of 

Thiodan which exceed those that can be 

immediately and effectively detoxified in the 

degradation pathway. Then, the deposits may 

be released and detoxified gradually during the 

turnover of lipids. Ludwig et al. (1964) 

reported that rats which were given daily sublethal doses of aldrin in their food were eventuall 

able to excrete the total daily dose as hydrophilic metabolites. After the last dose, the 

rate of metabolite excretion declined slowly 

as did the concentration of aldrin in the fat.

In the trials with goldfish, Thiodan may 

have been released from muscle lipids and its 

toxicity added to that of later treatments because the fish were fasted throughout the tests . 

The replenishment of muscle and liver glycogen 

in fish is dependent on food or conversion from 

protein and fat (Black et al., 1960; Change and 

Idler, 1960), and it is conceivable that Thiodan 

was liberated during fat catabolism. Graham 

(1960) believed that poor condition and 

physiological stress of fish may result in 

stores of DOT becoming lethal.

Another nutritional aspect to be considered in the toxic ity of Thiodan to fish is the 

systemic level of precursors available for the 

formation of glucuronic acid. As mentioned 

earlier, glucose is the forerunner of glucuronic
Richard A. Schoettger: Toxicology of Thiodan in Several Fish and Aquatic Invertebrates 27

acid in the uronic acid pathway. Therefore, 

deficiencies in glucose or glycogen might retard the conjugation of Thiodan metabolites because of insufficient glucuronic acid. Brodie 

and Maickel (1962) found that frogs which were 

pre-fed glucose converted phenols largely to 

glucosiduronic acids . Without glucose, the 

phenols were changed primarily to ethereal 

sulfates.

Although conjugation reactions involving 

glucuronic acid are well known in mammals 

(Harper, 1959), there is conflicting evidence 

in the literature regarding the abilities of fish 

to form glucosiduronic acids with other compounds . Brodie and Maickel (1962) reported 

that livers of several species, including goldfish, do not conjugate phenols with glucuronic 

acid in vitro, or, in vivo after intraperitoneal 

injections of phenol. They found that liver 

microsomes contained glucuronyl transerase, 

and phenyl glucuronids were formed when 

UDPGA was added to liver preparations . However , the microsomes apparently lack UDPG - 

dehydrogenase for conversion of UDPG to 

UDPGA. On the other hand, Grajcer and Idler 

(1963) measured conjugates of testosterone in 

the blood and testes of sockeye salmon. The 

hormone was released by the action of betaglucuronidase, and they concluded that it had 

been conjugated with glucuronic acid.

The results of my investigations and 

those of Grajcer and Idler (1963) do not support 

the conclusions which Brodie and Maickel (1962) 

have drawn concerning the inability of fish to 

form glucosiduronic acids. Certainly, further 

research is indicated to resolve these 

apparently contradictory findings . Perhaps 

tissues other than the liver, such as the kidney, 

are also involved in the conjugation and excretion of Thiodan metabolites in fish.

SUMMARY

The median tolerance limits (TLm) of 

rainbow trout and western white suckers to 

Thiodan are 0.3 to 8.1 ppb, depending on tern - 

perature and exposure. Daphnia and damselfly 

naiads are at least seven times as resistant

than fish. The naiads, in contrast to other 

species, appear more susceptible at colder 

temperatures . The effect of temperature on 

the toxicity of Thiodan to fish and invertebrates diminishes with exposure. Thiodan is 

nontoxic to trout eggs, but after hatching, the 

fish become increasingly susceptible with age.

The toxicity of Thiodan to suckers is 

not influenced by calcium or magnesium salts 

in the water or by mildly acidic conditions . 

A pH of 8 .4 or 9.4 reduces or eliminates the 

toxicity of the insecticide withint 24 to 96 hours 

at 19° C. Degradation at these pH's is slower 

at cooler temperatures.

Investigations with carbon C-labeled 

Thiodan indicate that the insecticide is taken 

up and deposited in various tissues of fish. 

The uptake is highest in liver and in the gut and 

feces . Rates of uptake are slower in heart, 

blood, gill, kidney, and brain. Western white 

suckers exposed to 20 ppb of labeled compound 

contained mean concentrations in the tissues 

of 1.1 to 8 .4 ug per g of dry tissue. The 

residues in muscle and skin correlate poorly 

with exposure.

Goldfish are killed by daily exposures 

to fresh solutions containing 7 ppb of Thiodan. 

They succumb according to their size and the 

lipid content of muscle. Residues of 0.95 to 

2.54 ;ig per g of wet tissue are detected colorimetrically in muscle. They correlate poorly 

with the number of treatments . Lesser 

amounts are found in the gonad, gill, heart, 

kidney, spleen, and blood, and greater levels 

are found in skin, liver, brain, and peritoneal 

fat.

Radio-tracer and chemical techniques 

reveal a water-soluble metabolite of Thiodan 

in the bile of western white suckers, northern 

creek chubs, and goldfish. Analyses suggest 

that Thiodan is degraded metabolically to its 

alcohol which is then conjugated with glucuronic 

acid and excreted via the bile into the feces .

In general, Thiodan appears useless as 

a selective piscicide against so-called trash
28 Investigations in Fish Control 35: Bureau of Sport Fisheries and Wildlife

species. The chemical may be useful, however, 

where selectivity among fish is unimportant 

because: it is highly toxic to fish and less toxic 

to many aquatic invertebrates; it is less toxic 

to mammals than several other chlorinated insecticides; and it does not persist long in water 

of relatively high pH and temperature. The 

exposure of fish to Thiodan probably renders 

them unfit for human consumption since residues occur in edible tissues such as the muscle.

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