The Effects of Different Concen Trations of Las on the Toxicity of Dieldrin to the Bluegill (Lefomis Macrochirus)

This dissertation has been microfilmed exactly as received 70-6795 HILLE, Kenneth Randolph, 1927- THE EFFECTS OF DIFFERENT CONCEN TRATIONS OF LAS ON THE TOXICITY OF DIELDRIN TO THE BLUEGILL (LEFOMIS MACROCHIRUS).

The Ohio State University, Ph.D., 1969 Zoology

University Microfilms, Inc., An:i Arbor, Michigan THE' EFFECTS OF DIFFERENT CONCENTRATIONS OF LAS ON THE

TOXICITY OF DIELDRIN TO THE BLUEGILL

(LEPOMIS MACROCHIRUS)

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Kenneth Randolph Hille, B.S, M.A,

* * * * * *

The Ohio State University 1969

Approved By

Adviser epartment of Zoology and Entomology ACKNOWLEDGMENTS

This study was supported, in part, by the Federal Water

Pollution Control Administration Training Grant Number 5TI-WP-39-04,

Recognition is due to Dr. Charles Dambach and the Natural Resources

Institute for the administration of the grant.

I wish to thank the staff of the Water Resources Center for the

use of a constant temperature room, equipment and supplies, without

which this project wouid have been impossible.

The assistance of Mrs. Elizabeth Thompson, with the chromatogra

phic techniques^ and Mr. Peter Nickolai, with the statistical analyses,

is greatly appreciated.

I should like to express my appreciation to Dr. L. S. Putnam,

Director of the Stone Institute of Hydrobiology, for his long

friendship and encouragement in my Ph. D. program.

I should like to express my appreciation also to Dr. Clarence

Taft for his constructive suggestions and guidance in the writing of

this dissertation.

Finally, I should like to express my sincere gratitude to

Dr. N. W, Britt for his long friendship, cooperation and encouragement,

and extreme patience while serving as my adviser. VITA

♦.Born, Brooklyn, New York

United States Navy

B. S. Wagner College, Staten Island, New York.

M. A,, Bowling Green State University, Bowling Green, Ohio.

Instructor, Fremont City Schools.

Water Pollution Control Administration Trainee, The Ohio State University, Columbus* Ohio.

Research Associate, Scioto River Project, The Ohio State University.

Assistant Professor, Bowling Green State University, Firelands Campus, Huron, Ohio.‘ TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS ...... - ii

VITA ...... iii

LIST OF TABLES ...... ;...... v

LIST OF FIGURES ...... vii

LIST OF PLATES ...... viii

INTRODUCTION...... 1

Pesticides ...... 1 Detergents ...... 11 Statement of problem ...... 17

METHODS AND MATERIALS ...... 20

Fish collection and maintainance .... 20 Continuous flow system ...... 21 Monitoring techniques ...... 27 Water quality...... 31 Gas chromatography...... 31 Preliminary investigations ...... 32 Pesticide extraction from fish tissues ...... 34 Static exposure studies ...... 36 Continuous flow studies ...... 37 Tissue extraction studies ...... 39 Testing procedure ...... 40

RESULTS ...... 42

Preliminary studies ...... 42 Static exposure studies ...... 47 Exposure studies using continuous flow ...... 52 Tissue extractions ...... 65

DISCUSSION ...... 80

LITERATURE C I T E D ...... 86

iv V \

LIST OP TABLES

No. Page

1 Chemical analysis of Columbus City water at the Water Resources Center, The Ohio StateUniversity ...... 45

2 The Mortality of Lepomis macrochirus of various weights and age exposed to 1 0 , 0 ppb. of dieldrin under continuous circulation ...... 46

3' Data from static exposure studies showing changes in dieldrin concentration in 96 hrs...... 48

4 Data from static exposure studies showing changes in LAS concentration in 96 hrs...... 51

5 Survival of Lepomis macrochirus exposed to various concen trations of LAS under static conditions ...... 54

6 Range and standard deviations of actual concentrations of dieldrin in TLm determinations (96 hr.) ...... 56

7 Survival of Lepomis macrochirus exposed to various concen trations of dieldrin under continuous flow conditions .... 58

8 Range and standard deviations of actual concentrations of . LAS in TLjjj determinations (96 hr.) ...... 61

9 Survival of Lepomis macrochirus exposed to various concen trations of LAS under continuous flow conditions ..... 62

10 Results of simultaneous exposure of Lepomis macrochirus to 1.0 ppb, dieldrin and various concentrations of LAS under continuous flow 64

11 Results of simultaneous exposure of Lepomis macrochirus to 5 t0 ppb. of dieldrin and various concentrations of LAS under continuous flow ..... 66

12 Results of simultaneous exposure of Lepomis macrochirus to 10.0 ppb, dieldrin and various concentrations of LAS under continuous flow 67

v LIST OF TABLES

No. ’ Page

13 Analysis of variance tests of differences in actual concentrations of dieldrin ...... 69

14 Accumulation of dieldrin in tissues of Lepomis macrochirus when exposed to different concentrations of LAS and 5.0 ppb, dieldrin simultaneously ...... 70

15 Accumulation of dieldrin in tissues of Lepomis macrochirus when exposed to different concentrations of LAS and 10.0 ppb. dieldrin simultaneously ...... 73

16 ■ Multiple analysis of variance ...... 79

Vi LIST OF FIGURES No. Page

1 Continuous flow s y s t e m ...... 21

2 : ■■ Mariotte bottle ,,,...». , ...... 24

3 Nixing chamber ...... 25

4 Siphon system ...... 28

5 Effects of various concentrations of LAS on monitoring efficiency of dieldrin in test flasks ...... 43

6 . Percent of dieldrin remaining in solution during 96 hr. acute static toxicity tests ...... 49

7 Percent of LAS remaining in solution during 96 hr. acute static toxicity tests ...... 53

8 . LAS-TLjjj values for Lepomis macrochirus under static conditions ..... 55

9 Dieldrin TL^ values for Lepomis macrochirus under .-.continuous flow application ...... 59

#' 10. LAS-TLjn value for Lepomis macrochirus under continuous flow application ...... 53

11 Dieldrin accumulation in tissues of Lepomis macrochirus after 24 hr. intervals of exposure to 5.0 ppb. dieldrin ... 72

12 Comparison of concentration of dieldrin in tissues of Lepomis macrochirus exposed to 5.0 and 10.0 ppb, of the pesticide 75

13 Dieldrin concentration in bluegills exposed to 5,0.ppm. of dieldrin and different concentrations, of LAS ...... ,;. 76

14 Dieldrin concentration in bluegills exposed to 1Q.0 ppm. of dieldrin and different concentrations of L A S ...... 78 •\

* LIST OF PLATES

No , Page 1 Needle assembly used in Continuous Flow System 30

viii \ \ I

' INTRODUCTION.

. ' Pesticides

Pesticides have been used in ever increasing quantities since the turn of the century. Concurrent with this expanded usage has been the discovery and development of a succession of new compounds designed for greater and more effective‘.control of pests. Prear (1963) lists

9428 pesticides that are marketed for use on land or in water.

In the early nineteen hundreds the main types of pesticides were nicotine, arsenous compounds, lime-sulfur, and petroleum oils. Fluor ine compounds, pyrethrum and rotenone were developed and used prior to W.W. H r Dinitro compounds and the thiocyanates were the first synthetic organic insecticides to appear (Jones, 1964) , Aside from the tests of mammalian toxicities, little attention was paid to the

effects of these compounds on non-target organisms in the environ ment.

During W.W. II the highly toxic, long residual, organic chlor

inated hydrocarbons were discovered to have insecticidal qualities,

DDT the first of these compounds' and still the most widely used, was

soon followed by aldrin, endrin, dieldrin, lindane, chlordane, toxa-

phene, heptachlor, and methoxychlor.

The discovery was soon made that these compounds could be

1 accumulated and concentrated by organisms in the environment. A great volume of literature attests to the toxicity of the chlorinated hydrocarbons, especially DDT, to man, domestic animals, and wildlife.

Dieldrin, a cyclodiene chlorinated hydrocarbon pesticide, was developed in the laboratories of the Julius Hyman Company and first

described by Kearns 11949) . This pesticide is also known as HEOD,

Compound 497 , and Octalox. Dieldrin, named after the German.chemist-

Diels, is the 6.7 epoxy-derivative of aldrin and the stereo isomer of

endrin. Dieldrin is reported to be non-phytotoxic.

The insecticidal activity of dieldrin, against the housefly, is

approximately 40 times greater than DDT and approximately 200 times

greater against the German roach. The exceptionally long residual

activity of dieldrin prevents its use on food crops. It is used

principally in the control of (1 ) flies and mosquitoes, (2 ) moths and ' * carpet beetles, (.3) cotton insects, (4) forest pests, (5) termites,

(6 ) pests in soil, (7) pests of lumber products, and (8 ) industrial

pests not actually affecting food products (Julius Hyman and Company,

1949.} , However, dieldrin has been found to be toxic to a wide var

iety of animals including man, domestic animals, and wildlife.

Man; The estimated fatal dose of dieldrin for a man of 70 kg, has

been estimated as 5 grams (DuBois, 1959.) . Dieldrin is readily ab

sorbed' through the skin.- Hayes (1957) reports that 9% of the spraymen in Ecuador , 18% in Venezuela , and 10% in Nigeria showed symptoms

,of dieldrin poisoning. The symptoms were; (1) headache, (2) blurred • ► » Vision, C3I dizziness and nausea, (4) slight involuntary muscular movements and sweating, and (5) difficulty in sleeping and general malaise, Fletcher, et al,, (1959) workiog with spraymen in a mos quito eradication program in Tanganyika, found that by placing ab sorbent filter pads on the skin, a sprayman using an average dose of

* 2,12 kg. (4.7 lbs.) received a dermal exposure of 1 . 8 mg./kg. of body weight per day. If, however, daily body cleanliness was maintained no clinical illness was produced in 16 spraymen exposed for 180 days.

The Clinical Handbook on Economic Poisons (Hayes, 1964) states that persons exposed to oral doses which exceed 1 0 mg./kg. body weight

frequently become acutely ill.

Domestic animals; The acute LD^q for 3 and 6 week old chicks was between 20 - 30 mg./kg. when dieldrin was mixed in commercial

starting mash. In chronic tests on chickens receiving 25, 50, and

. 100 ppm. in mash, all chickens in the 50 and 100 ppm. range died

before 90 days. The length of time of survival being inversely pro

portional to the amount of dieldrin in the diet (Eden, 1951), Steers

hogs, lambs, and chickens fed dieldrin in their diets for 1 2 weeks

were sacrificed and their tissues analyzed for the presence of the

pesticide. ' The concentration used in the diets of these animals were 0.1, 0,25, 0.75, and 2,25 ppm, Dieldrin was strongly detectable in the fat tissues of all animals studied, and when detectable in other tissues, the residues' were proportional to the content in the fat tissues, The amount stored was proportional to the rate of uptake for all animals, The greatest amount of dieldrin was stored by chick ens, followed by steers, hogs and lambs. Chicken eggs, however, con tained very little dieldrin (Gannon, et al., 1959). The toxicity of dieldrin to domestic rabbits by skin absorption was determined by immersing rabbits in water suspensions of the wettable powder. The

LD5 0 for dieldrin was 400-450 mg./kg. with the characteristic symptoms of loss of appetite, hyper-sensitivity, convulsions, and muscular spasms (Johnston, et al., 1953).

The acute oral LD5 0 for white rate ranges from 37-87 mg./kg. of body wdight (Rudd and Genelly, 1956). Barnes (1953) states the average single lethal dose of dieldrin when given orally is 50 mg./kg. for white rats. White rats were maintained for more than a year on water that contained 0 . 2 mg./I. of dieldrin without any apparent injury (McKee and Wolf, 1963). This same concentration is lethal to fish in a few hours.

Wildlife; In the spring of 1954, in Illinois, dieldrin was applied by the United States Department of Agriculture against a local population of the Japanese beetle (Popilla japonica) at 3 lbs./acre. Scottf at al,f (19591 made a routine study of sample plots in the

sprayed area and adjacent unsprayed areas. They reported severe

losses of wildlife during the week following application. Meadow-

larks, robins, brown thrashers, starlings, common grackles, and ring-

necked pheasants were virtually eliminated. Insect feeders and

ground dwellers were most severely hurt; seed feeders and arboreal

forms were least hurt. House sparrows and mourning doves were least

affected. Among the mammals, ground squirrels, muskrats, and rabbits

were eliminated and short-tailed shrews, fox squirrels, woodchucks,

and meadow mice were reduced in numbers. Domestic dogs and cats were

found dead in the treated areas. Wildlife populations appeared to

.have recovered by the following year.

Dieldrin applied from an airplane to control the fire-ant in Ala

bama caused the death of resident quail (Clawson, et al., 1959), Fans

set in the test areas indicated slightly less than 2 lbs,/acre appli

cation. This amount caused the death of nearly all resident quail in

sprayed plots while only 2 out of 76 disappeared from control plots.

Genelly and Rudd (1956) carried on experiment to determine the

effects of insecticide contaminated food on reproduction of ring-

necked .pheasants. They noted that egg production and egg fertility

were lower in the 25-50 ppm. range of dieldrin as compared to the

' controls. They also: noted that mortality of the young chicks was greatest in the first two weeks of their development. They concluded that the reproductive success of those birds receiving 50 ppm. dieldrin in their diet was 38% as compared to 78% for the control birds.

Evans, et al,, (1957) reported no apparent injury to field mice populations or fish in an area treated with 1.5 lbs./acre of dieldrin by aerial application. However, they.did note dead earthworms along . drainage ditches in the treated fields immediately after treatment.

Dieldrin pellets were air disseminated at a rate of 1 lb./acre over 2000 acres of Florida's east coast tidal marshes to destroy the

» t larvae of the sandfly (Culicoides). According to a study of the treated area by Harrington and Bidlingmayer (1958) , the fish kill was immediate and substantially complete. Along a 1200 foot segment of ditch, 20-30 tons of fish, or 1,175,000 fish of at least 30 species were recorded dead from the pesticide. Crustaceans, particularly the crab population was completely destroyed. The mollusks iii the area seemed unharmed. Large game fish succumbed most rapidly followed by herbivorous fishes and gobioid fishes. Repopulation of fishes began four weeks'after the kill and was initiated by the broad killifish

(Cyprinodon variegatus). * *

Oysters exposed to 1.0 ppb. dieldrin, lindane, and heptachlor, in continuous flowing water, for 60 days, showed no detectable lindane or heptachlor in their tissues; However, dieldrin. was present in their 7

I • • tissues in a concentration of 3,5 ppm, as determined by paper chroma tography (Loosanofff 1965). ■ %

Steiner and Gruch, C1959) reported on a study made by Adlung, et al., (1956), on the effects of dieldrin on the goldfish (Carassius auratus). The latter reported the fish showed the typical chlorinated hypersensitivity, and periodst.of resting on backs and sides, at a concentration of 0.2 ppm. At this concentration, all fish died in 12 hours. In the case of fish subjected to 1.0 ppm., the syndromes appeared in 30 minutes and death occured in 6 hours. In the 3 and 6 ppm. tests the fish were dead in 4.5 hours. Similar results were ob tained with the common guppy (Lebistes reticulatus).

Steiner and Gruch, (1959)' reported on another study made by Adlung and Kauth, (1956) in which the gastropods, Lymnaea auricularia and

Physa fontinalls were killed in 8 ppm. dieldrin in 16-18 hours exposure.

The mud snail Lymnaea stagnalis was damaged by 6 ppm. dieldrin but not killed.

. Dieldrin used in rice fields in Arkansas was toxic to goldfish and bluegills (Hogan and Gray, 1950). Water taken from rice fields

treated with 0. 1 lb./acre, 1 0 to.20 days previously, killed all fish

in'16 hours. The same water diluted with an equal amount of distilled water had no effect on the fish.

Dieldrin and its stereo isomer, endrin, are the most toxic chlorinated hydrocarbons to fish, however, Anderson (I960) reports that they are least toxic to Daphnia magnum. The concentrations required to immobilize Daphnia in 50 hours are 330 ppb. for dieldrin and 352 ppb, for endrin as compared to DDT which is only 1.4 ppb. Parathion,

an organic phosphate insecticide has little toxicity to fish yet is

toxic to Daphnia at 0.8 ppb.

In a recent study by Sanders and Cope, (1968) static bioassay

experiments were conducted to determine the acute toxicities of several pesticides to naiads of three.species of stoneflies; Pteronarcys

californica, Pteronarcella badla, and Claassenia sabulosa. Endrin and

dieldrin were most toxic of. the chlorinate hydrocarbons tested, and

DDT the least toxic to all three species. For dieldrin, species

differences were evident in regard to 24, 48, 96 hour LC50 values.

These being: .■

24 48 96 hour

.Pteronarcys californica 6.0 1.3 0.5

. Pteronarcella badia • 3.0 1.5. 0.5

, Claassenia sabulosa 4.5 2.3. 0,58 ppm.

Stock, (1950) tested the toxicity of toxaphene, chlordane, aldrin,

and dieldrin on 150 hatchery-reared brown trout (Salmo trutta fario)

averaging about 5 1/2 inches in length. Glass aquaria having four to

six fish in each 24 liters of water were used for these static tests.

Dieldrin was found to be the most toxic of those pesticides tested. At 1.0 ppm, the mortality was 100% In 9 hours, 100% at 0.025 ppm. In

24 hours, and 40% at 0.016 ppm, in 48 hours,

Weiss (1964) used C1^ tagged material to determine the locali zation of.dieldrin residues in goldfish itissues, The total uptake of tagged dieldrin was porportional to fish size and length of exposure.

Tissues most heavily endowed with dieldrin were blood, liver, brain, and digestive tract. The large amount in the digestive tract suggests that dieldrin, at least in part, is absorbed through the tract when the fish swallows water. Pish with the longest exposure time has a disproportionately high residue in the kidneys, suggesting that this accumulation might be indicative of the inability of this organ to secrete this particular molecular configuration. Dieldrin was present in decreasing concentrations, in the following tissue; heart, kidney, gills, muscle, spleen, gonads, skin, and spinal cord.

Work on the specific effects of dieldrin and other chlorinated hydrocarbons was performed by Tarzwell and Henderson (1957) and

Henderson, Pickering, and Tarzwell, (1960). They studied the effects

of ten different chlorinated hydrocarbon compounds on four different

species of fish,, Their study also included effects of different dil

ution waters, pH, alkalinity and hardness effects, and the.effects of

different formulations. The studies were carried on under similar

conditions and their results are useful comparatively. Their data >

indicate all the chlorinated pesticides, with the exception of BHC, , 10

are more toxic to.fathead minnows (Pimephales promelas), bluegills

(Lepomis macrochirus), goldfish (Carassius auratus), and guppies

(Lebistes reticulatus) , than the .organic phosphorus compounds. Endrin was by far the most toxic of all insecticides to all species of fish.

Toxaphene, the second most toxic, was followed by dieldrin, aldrin, and DDT, • Bluegills, the.most sensitive, were followed in order by fathead minnows, guppies, and' goldfish, Prom their static bioassay

.tests, .the TL^ for dieldrin was ascertained for bluegills at 7,9 ppb,, fathead minnows.at 16.0 ppb.,. guppies at 2 2 .0 . ppb.,'and goldfish at

37.0 ppb. They concluded that pH alkalinity and hardness, within the range tested, had no major effect on the toxicity of the chlorinated hydrocarbon insecticides to fish. Dieldrin, in particular, showed no change in the TL^ values for fathead minnows in soft of hard waters. Emulsifiablp concentrates were found to be equally as toxic as the acetone solutions in either soft or hard waters.

In a. field "run-off" study (Tarzwell and Henderson, 1956) in which a six acre grassy slope was treated with dieldrin at a concentration of

4.66 lbs./acre, bioassay studies were conducted using runoff water after each rain. In this study it was round that the runoff from the first rain contained. 0.13 ppm. of dieldrin and killed all fish even in a three 'fold dilution. Assuming an average farm pond of an acre size and'-bhree feet deep, the authors calculated from their run-off study the rate of application of dieldrin that would achieve the TL^ values . . \ ■ ■ ■ . - \ - ' ' \ . . ’.

established in their laboratory‘studies. For dieldrin, 0,07 lbs./acre ■ would approximate the TL^ value of 7.9 ppb. for bluegills,'

i, . The cruising speed and oxygen consumption of the pumpkinseed sunfish, existing in sublethal concentrations of dieldrin was measured in a recent study by, Cairns and Scheier (1964). They first performed acute bioassays to determine 24, 48, 72, and 96 hour TI^ concentrations which were recorded respectively as-15.5, 12, 7.5, and 6.7 ppb. Using four concentrations below the 96 hour TL^, 5.6, 3.2, 1.68, and 0.75 ppb., fish were, observed for 10 0 days, although most did not survive that length of timei Cruising speeds and oxygen consumptions, as compared to controls, were determined for those fish that did survive.

Within the first two weeks most of the fish in the two higher concen trations were dead or so uncoordinated that testing was impossible.

Fish exposed to 1.68, ppb. concentration had an oxygen consumption and cruising speed that was significantly different from that of the con trols. Fish that were exposed to 0.75'ppb, showed no significant difference from that of the controls.

Detergents

The history of detergents is most aptly summarized by Price (1952).

Shortly before World War II new surface active compounds which would not combine with calcium and magnesium salts'usually found in hard water were developed. This autonomy made them more efficient as ■ >:■ ■ ■ ' ‘ ' \ ■ . .

cleansing agents than the ordinary soaps. Detergents made a welcome

‘ substitute for soaps and their production soon exceeded that of the

soaps, These new products of synthetic detergents now compromise more

than 96% of the*total yolume of household and industrial cleansers.

The complete packaged household detergent of syndet consists

basically of two major ingredients; a surface active material called a

surfactant and alkaline phosphate builders such as tripolyphosphates

of tetrasodium pyrophosphates. Minor ingredients might consist of

such materials as; ammonium salts# sodium sulfate, both of which act as

an additional electrolyte to aid in wetting and dispersion, sodium

silicate, which controls corrosion of metals, and small amounts of

sodium carboxymethyl cellulose to prevent redeposition of soil. In

addition, bleaching agents, such as sodium perborate, organic dyes and

perfumes might be found, (Soap and Detergent association, 1962).

The most, widely used surfactants belong to a family of petro-chem-

ical compounds known.as the alkyl aryl sulfonates. These chemicals

are so named because they combine in one molecule both an "alkyl" (non-

cyclic) side chain and an "aryl" (cyclic or ring) grouping of carbon

and hydrogen atoms. Prior to July 1965 the most commonly used of these

compounds was alkyl benzene sulfonate, better known by its abbreviation—

ABS. . This "hard" surfactant possessed a low degradability and thus the

major portion of this chemical in raw sewage was unaffected by the

treatment process and passed into surface and ground waters. The occur-

ance of M S and visible foaming in streams, wells,' and drinking water \ ‘ ' 13

' * - . across the United States is well documented in the literature from

1950 tp 1960, In 1965 the major manufacturers of detergent products switched to a "soft” surfactant, linear alkyl sulfonate, abbreviated as LAS, and whose degradability has been documented principally by

Hanna, et al, (19651, Renn (1965), and Knapp (1965),.

Man and domestic animals; The United■States Public Health Service

established 0.5 ppm, as the maximum permissible concentration of ABS in drinking water. This value was established on the effect ABS had on the

aesthetic (foaming) quality of water and not on its mammalian toxicity.

Six human volunteers consumed 100 mg. of ABS, the equivalent of two

liters of water containing 50 ppm., daily for four months, with no ap parent ill effects (Walton, 1960),

Schmidt (1961) reports dogs kept on a diet of 1 gram of ABS per day

for six months showed.no effects deviating from the normal. In the same i report, rats whose, diet consisted of 0.5% ABS showed no ill effects

after 65 days and guinea pigs given 2000 mg./liter in their water for

six'months likewise showed no ill effects. ' Young pigs whose diet con

tained 0.1%, 0.2%, and 0.4% of ABS showed no effect in the 0.1% .and 0.4% • * * .concentrations and a.growth stimulation in the 0 .2 % concentration over a

period of 79 days.

Wildlife; Chase (1953) reported that detergents discharged with

sewage into salt.water bays and inlets have dissolved the oily coating

on duck feathers causing them to become water logged and drown, Mann

(1955) stated that fish exposed to surface active agents showed a marked 1 4

increase in sliming of gills and skin.

.Mayfly nymphs or the genera, Stenonema and Isonychia were killed . by 10 days exposure to ABS at 16 ppm. and were reduced in number by 8 ppm, Trichoptera larvae, Hydopsychidae were able to survive 10 day ex-, posures at 32 ppm., while the crayfish Orconectes rusticus, the fresh water shrimp Synurella, and sow bugs Lirceus were seriously reduced in numbers in 10 ppm. ABS after two weeks. The snail, Goniobasis, was completely tolerant of these concentrations, according to Surber and

Thatcher (1963).

Cairns, et al. (1964) tested the response of two diatom populations in laboratory culture to varying concentrations of ABS. Nitzschia linearis was reduced by 50% in five days in a concentration of 10 ppm., while Navicula seminulum, being more resistant, was reduced to 50% by

39.4 ppm. ABS in five days, • * Lemke and Mount (1963) studied the effects of long term exposure of bluegills to ABS under continuous flow conditions and found'the 30 day medium tolerance ranged between 15.5 and 18.3 ppm. During this 30 day period, the swimming ability of the fish was assayed and it was found that ABS does not affect any of the organs that determine swimming endur ance. Their findings also confirmed a previously reported observation that fish-became acclimated to higher concentrations of ABS. Pish that survived the 30 day tests at concentrations of 1.3, 1.9, 3.2, 6.5, and

13.0 ppm. were reexposed to concentrations varying, from 13 to 59 ppm.

ABS. Bluegills exposed at 13.0 ppm. for 30 days had a TL^ value 63% \.N

.higher than controls, and those exposed to 6.5 ppm. for 30 days exper ienced 50% mortality much sooner than those acclimated at 13.0 ppm.

Histological examinations of various organs of the test fish showed pronounced gill damage most evident at the higher concentrations. Epi thelial tissues of the gill lamellae, accompanied by a thickening of the cell walls, were as much as three times thicker than normal..

In a similar, study, Cairns and Scheier (1963) found that Lepomis macrochirus and Lepomis gibbosus»on acute exposure to ABS were very sim ilar in their responses. They found the 96 hour TLm to be 17,4 and 17.2 ppm. for macrochirus and gibbosus respectively, and the same for hard or soft water. In their acute tests there were no significant differ ences between the static or batch method and the continuous flow tech nique. Gill damage, viewed histologically, was evident in chronic . .

r * eiqposure at both 5.6 and 18,0 ppm. for both species studied.

Henderson, et al. .(1959) reported on the toxicity of syndets and soaps to fathead minnows and to bluegills,. in both soft and hard waters.

Through 96 hour tests they found•household soaps and syndets roughly comparable in their toxicities in soft water, but syndets were much higher in hard water. In testing the syndet.against the surfactant portion they found little difference, thus eliminating the builder por tion of syndets from the demonstrated toxicity of the syndets. They re ported the 96 hour TI^ for ABS to bluegills at 5.6 ppm. and the same time at 4,5 ppm. for fathead minnows. The 96 hour TL^ values to fathead min nows averaged 6 , 6 ppm. (range 3.6-9.2) in soft water and 4.3 ppm. (range 3.5-5,1) in hard water for ABS.

The work.of Wurtz*-Arlet (1960) as reported by McKee and Wolf (1963) with sodium alkyl aryl sulfonate on young rainbow trout, Salmo gairdnerii showed the 24 hour TLjh to be between 3 and 7 mg./liter. Trout aged 10 and 65 days were used in this study. In one and six hour tests using

10 day old trout, they found 10 and 5 mg./liter to be the dilution lim its and in the 65 day old animals the values were 10 and 7 mg,/liter.

At concentrations of 0.5 ppm., Bardach, et al. (1965) have shown that both ABS and LAS damage the chemoreceptors of yellow bullheads,

Ictalurus natalis. In histological preparations, taste, buds on the oral surfaces and barbels were found to be eroded after 4 weeks of exposure to surfactants ranging from 10 to 0.5 ppm. That these chemoreceptors were damaged physiologically was proved by recording action potentials from afferent nerve bundles at the base of the barbels of fish in surfac tant concentrations. Observations of the food finding ability qf exposed fish confirmed the damaged chemoreceptors.

' The surfactant ABS affects the aquatic ecosystem in a way other them direct toxicity (Kent and Hooper, 1966) by reducing the number of binding sites on organic amines which would normally, 'complexwith iron.

The.iron that is not.so bound is not available for photosynthetic pro duction thus reducing the primary productivity of such water.

Experiments performed at the Water Resources Center, The Ohio State

University, by Dugan (1967) showed that goldfish that were chronically exposed to surfactants were more susceptible to the toxic effects of ' . . ■ ' ' ■ ' ■ • - • A,. X . ‘

- 17

1 - /' dieldrin and DDT. The fish were exposed to ABS and LAS at concentra tions of 4 ppm, for periods varying from 51 to 96 days and then removed to aquaria containing 50 mg. dieldrin/ml. Mortalities were recorded over 12 hour periods, The fish exposed between 51 and 62 days to the surfactant ABS showed a higher susceptibility to 5 ppb. dieldrin than did the controls,over a 24 hour period. Fish exposed to ABS for 96 days, however, showed a greater susceptibility to the dieldrin up to about 16. hour when compared to controls, but from 16 hours to 24 hours the controls exceeded those exposed to ABS. Some fish in this 96 day-

ABS group failed to succumb even at 96 hours to the 5 ppb, dieldrin es- posure. In 96 hours all controls died. In another part of this work, fish exposed to LAS for 37 days were extremely susceptible to DDT as compared to., control fish, although the LAS had no direct observable effect on the fish.

STATEMENT OF THE PROBLEM

In the Transactions of the 1959 Seminar on Biological Problems in

Water Pollution, Cottam and Tarzwell point to some of the research needed for the establishment of water quality criteria for aquatic life; The following was written on the subject of toxicity:

"Studies,of the effects of repeated exposures and/or additive or accumulative effects would be of great value, Consideration should be given to the determination of synergism and antagonism and to the causes of such action when two or more materials are added to the aquatic environment." The following question, is from the previously cited paper by Dugan;

wWhat is the significance of the presence of trace quantities of these contaminants (pesticides)— particularly in relationship to the myriad of other types of water pollutants'1

The foregoing statements are evidence that there is a developing awareness of the needs for studying the interactions of.materials in the aquatic environment, The great bulk of information on the minimum lethal concentrations of .various effluents has set the stage for expan sion into the areas of interactions, modes of actions, and genetic adapt ations in relation to these pollutants. The frequency of occurrance of pesticides and detergents ranks them high on the list of pollutants in

American surface waters.

The use of fish as bioassay organisms has been documented by

Doudoroff, et al. (1951), Henderson and Pickering (1963), and Weiss

(1964). The use of the bluegill, Lepomis macrochirus, is most justi fiable because; (1) it is one of the most widely distributed species in the United States, (2) it is highly sensitive to most contaminants in water, (3) it has been widely used by other workers, an advantage for comparative purposes.

Dugan's work demonstrates some interaction exists in the toxicity of a chlorinated hydrocarbon in the presence of a surfactant.

Dieldrin was chosen because of the abundance of this chlorinated hydrocarbon in the major surface wafers of the United States. According to a report by Weaver, et al. (1965) on the results of a nation-wide pesticide surveillance project being carried out at the Taft Sanitary

Engineering Center, the following is stated: *-'ln order of- frequency of occurrence, dieldrin, ■ endrin, DDT, and DDE were found in all major United States river basins. Heptachlor and aldrin were less abundant and DDD was detected at only one sampling station.”

* x i v " * The purpose of this study was to investigate:

(1) the'possibility of interaction between the chlorinated hydro carbon pesticide— dieldrin, and the common surfactant, LAS, when admin

istered simultaneously to the bluegill (Lepomis macrochirus).

C21 this phenomenon under, continuous flow application of pesticide and surfactant.

C3) this phenomenon in relation to the assimilation of pesticide

in the tissues of. the fish as determined by electron capture gas chrom atography, and

(a) to determine, through preliminary investigations, the acute

TLr Values for LAS and dieldrin separately.

(b) to determine, through preliminary investigations, the possible

difference between TI^ values under static and continuous flow application

Cc) to determine, through preliminary investigations, the relation

ship of fish size, weight, and age", as well as water quality to the TL^ values. METHODS AND MATERIALS

Fish Collection and Maintainance: Lepomis macrochirus used in the static tests of this' study were collected by seine and hook and line from Aldrich Pond, Sandusky County, Ohio. Aldrich Pond is a

"borrow pit" created by the construction of the Ohio Turnpike and was used as a source of fish because of the overabundance of bluegills and the least likelihood of pesticide and detergent contamination.

Fish for the continuous flow studies were obtained from the fed eral fish hatchery at Hebron, Ohio. All fish were transported to the laboratory in plastic bags in 15 gallon styrofoam containers. Mortal ities were few during transport. ■

The fish were maintained, in the laboratory, in several galvan ized livestock watering tanks. These holding tanks, consisting of two

150 gallon tanks and one 300 gallon tank, were lined with 0.7 mil. plastic sheeting to prevent leaching of the toxic zinc from the gal vanized metal into - the water. Twice each week the bottoms of the tanks were siphoned clean along with the removal of about two thirds of the water volume. This was immediately replaced with an equal . amount of stored aereated water.

The fish were fed daily on a diet consisting of Purina Trout

Chow, shrimp pellets and flake fish food.

The dissolved oxygen was determined periodically in the holding

20 21 • tankst especially when the tanks were full of fish. An external air source was connected to the tanks and adjusted to maintain an oxygen concentration at an average of 7 ppm., never going below 6.4 ppm.

Mortality was less than one percent in fish maintained in the laboratory. Fish obviously deformed or in distress were removed and not used in any tests.

Continuous F^ow System; A continuous flow system, Figure 1, 'was constructed and maintained in a constant temperature room at the Water

Resources Center, The Ohio State University. Room temperature was adjusted to maintain a water temperature of 20° C.

Water supply was Columbus city water which entered a head tank (C) through a 1/4 inch line (A) to a float actuated control valve (B).

The valve was a replacement valve for a ‘furnace humidifier. Accurate control of wattr level was maintained in the head tank. The 20 gallon capacity head tank contained two submerged air operated filters con taining charcoal and glass wool. Retention time of the water in the head tank was sufficient to stabilize the temperature of the incoming water at 20° C.. Tyfo siphons removed water to an. aquarium filter out side the head tank which was also filled with charcoal and glass wool.

Water flowed from the base of this filter through 7 mm. glass tubing

(El to a horizontal manifold (F) from which it was distributed to six mixing chambers, (Gl. At each end of this manifold were sectiohs of

Tygon tubing with pinch clamps (X) to facilitate removal of air bubbles ^ P A A A A. i> A A 1 I 4 t 4 f 1 1 H I I 1 1 1 t

» 1 » f . 1 • ■ - C D < s I

‘ Figura 1. Continuous Flow System' - ■ : - . \ '' . v

' 23

at the start of each test.

Six glass carboys (H), each containing a different concentration of test material, were located on >a level below the head tank. Two holes were drilled in each carboy, one at the base and the other near the neck of the bottle. In the top hole was a "00" rubber stopper I . with a piece of 4 mm. glass tube extending down into the bottle ap proximately to 60 centimeters from the bottom. In the bottom hole was a similar size rubber stopper with a piece of 4 mm. glass tube extending into the bottle. To the outside end of this glass tube was. attached a piece of Tygon tubing which was connected to a latex bulb,

Luer adapter, and disposable syringe needle. These carboys utilize the principle of a Mariotte bottle. Figure 2 shows the Mariotte bot tle. The advantage of this bottle is a constant discharge rate re gardless of the height of the fluid in the bottle. The head pressure is determined by the volume of fluid between the bottom of the long inserted glass tube and the tip of the discharge needle. Head pres sure in the Mariotte bottle can be changed by raising or lowering the top inserted glass tube. Flow rates were equalized by this means in all six bottles,

The mixing chambers were glass cylinders, 65 mm. outside diameter and 205 mm. long, stoppered at both ends with #12 rubber stoppers. .

Each stopper contained four holes. In the top stopper one hole had a small funnel in it to facilitate rapid filling, of the mixing chamber. \ \

Stopper

4mm Glass Tubing .

— Carboy

Liters

Tubing

Syringe Needle

Figure 2. Mariotte bottle. Toxicant Dilution Water

Air s. i i Funnel i i / Stopper

— Glass Cylinder

Overflow

++--H — Stopper I I

Test Tank Drain # U Figure 3. Mixing chamber. ‘ . \ - ■

Another hole contained a. glass tube which projected almost to the bottom of the chamber and which carried an air stream for agitation

in the chamber, The two remaining holes had hollow plastic needles

obtained from disposable blood transfer units. Into each of the plastic needles was placed the disposable syringe needles; one from i * the water manifold and the other from the Mariotte bottle. In the bottom stopper, three holes contained short lengths of glass tubing.

Plastic tubing was attached to the outside end of each glass tube.

The distal end of each plastic tube has a disposable syringe attached

each syringe needle was affixed to the side of the test aquaria

(Figure 1, K). The fourth hole contained a longer piece of glass tube which extended into the. mixing chamber (Figure 1-L and Figure 3).

The test chambers consisted of six fifteen gallon aquaria, Figure

1-M, each with an air supply to oxygenate the water and create a cir

culation within each tank.

Water in each aquarium was maintained at 40 liters by an adjust

able siphon system that removed any fluid in excess of 40 liters after

all aquaria were leveled. The siphon consisted of a section of 7 mm.

glass tubing extending from near the bottom of each tank to a common

plastic cup (1-N). The upper lip of the cup was notched to a depth of

13 mm.' and for about an eighth the circumference. This plastic cup

was situated in a plastic, square freezer container which has a drain

hole in one corner of the bottom. A section of plastic tubing conn

nected this hole to a common drain (1-0). The cup and container were placed on a stand# the height of which could be adjusted, in a space between the six aquaria, With a siphon established from each aquaria

to the cup, the height of the cup was adjusted to the 40 liter levei

in all aquaria. If any tank exceeded its 40 liter level, water would

siphon into the cup and overflow through the notched area into the

container and out the drain. Figure 4 shows diagrammatically this

siphon system with only two tanks.

"Quick connectors" (1-P) were utilized throughout the tubing

structure to facilitate rapid disassembly and cleaning after each

test was completed.

The rate of flow from the common manifold to each mixing chamber

was controlled by affixing a disposable syringe needle at each outlet. i The needle was attached to a I'Luer" adapter which in turn was attached

to a latex rubber bulb connected to the manifold tubing. The adapter

and latex bulb were also components of a disposable blood transfer

unit. Different size needles altered the flow rate for the concen

tration desired. The number 21 needle allowed 1200 ml. per hour of

dilution water to enter the mixing chamber. A smaller needle, number

25, was used from the outflow of the Mariotte bottle and allowed a

flow of 50 ml. per hour. Plate 1 shows the needle assembly as a unit

and associated parts.

Monitoring techniques: LAS was determined by the'methylene blue

method. Individual calibration curves were made for each surfactant. Siphon Tubes

Notched Overflow 40 liter level Cup

Test 11 Test Tank 1/ Tank Dish VTT

Drain

Figure 4. Siphon System. K. The method used was that of Task Group 2662 P of the American Water

Works Association (.1958).. This process depends on the formation of a blue salt when methylene blue reacts with a surfactant. This salt is, soluble in chloroform but not in water, whereas the dye and sur factant are individually soluble in water and not in chloroform. The color intensity of the dye in the chloroform is proportional to the concentration of surfactant. This intensity is compared with known standards in a spectrophotometer. Spectrophometric analyses were made on a-Beckman DO model instrument equipped with a constant voltage chassis. New calibration curves were made approximately every month.

LAS used in all test and calibration curves was obtained from ; t I . 4 the American Soap and Detergent Association, 295 Madison Avenue, N. Y.

The LAS was a composite sample compounded from the products of the major detergent manufacturers. The LAS was from Lot #1-1 and contained the following by weight, LAS-60.8%, sodium sulate-36.1%, free oil-0.4%, water 2.7%, and equivalent weight-348. All surfactant concentrations .a: were calculated on the basis of percent active ingredients.

• Dieldrin determination: Dieldrin was determined by partitioning

a water sample against non-miscible solvents with higher affinity for

the pesticide. A 150 ml. water sample was removed from the test .

chamber and partitioned with three fractions of 25 ml. of redistilled

Skelly Solve B r Each fraction was poured through granular anhydrous

sodium sulfite and combined in a single beaker. About 10 ml. of solvent

was used to rinse the sodium sulfite at the termination of the third Plate I . Needle assembly used in continuous flow system. fraction, This total volume of about 85 ml. of solvent and pesticide * ' . was evaporated to dryness on a warm hot plate with a filtered air stream directed over the top of the beaker. The dieldrin remaining in the beakers was then redissolved by the addition of exactly 10 ml. of redistilled Skelly Solve B, and poured into 20 ml. screw cap vials.

A small piece of aluminum foil lined the inside of the screw cap of the vials. This condensed volume was sufficient to inject into the gas chromatograph for qualitative and quantitative analyses,

.Water quality: ' Water quality was determined by the use of the

Hach Chemical Kit. Analyses were made monthly. Parameters of water quality such as; phenol,and total alkalinity, chloride, chromate, - 1 , * copper, fluoride, calcium and magnesium hardness, iron, manganese, nitrate and nitrite nitrogen,, oxygen, ortho, meta, and total phosphates, silica, sulfates, and pH were determined.

Gas chromatography; Analyses of samples were made on a Varian

Aerograph HiFi model 600 C .gas chromatograph equipped with an electron capture detector, The .detector utilized a 250 me, radioactive tritium source. The 5 r x 1/8" coiled glass column was packed with 5% SE-30 on

Chromosorb W in 60/80 mesh size. Usual electrometer settings were; injector port temperature— 205° C., overi temperature— 200°. C., atten uation-;- 2-8, electron capture range— 1, and a nitrogen flow through the column at-.50 ml, per minute.

. The recorder was.a Leeds and Northrup Speedomax H.equipped with an Aerograph model 207 disc integrator..

.The injection instrument was a Hamilton #701-NW/G 10 microliter syringe equipped with. Teflon guides. After each injection the

syringe was rinsed in redistilled hexane followed by acetone and ag^in hexane, Petroleum ether was substituted for the hexane in the rinsing if petroleum ether was the solvent for the pesticide extract,

t in quantitative usage, the total amount of injection material was drawn completely into the glass calibrated syringe and the extract

amount determined.

Standards were routinely injected into the chromatograph to

ascertain the sensitivity of the instrument during the time of use.

The reference standard dieldrin was obtained from the Chemical Div

ision of the Shell Oil Company and was recrystalized 99+ % pure.* * . * preliminary Investigations; Although these studies were not

directiy related to the solution of a specific hypothesis, they were

important in dictating the conditions.of the experimentation.. All

too often such studies are not mentioned in the literature, which

leads one to speculate accuracy of the final results.

Efficiency of monitoring techniques: The efficiency of dieldrin'

water^hexane extraction was determined by inoculating six Erlenmeyer '

flasks 12 literl, each with' a different concentration of 99+ % purity

dieldrin in distilled water. The concentrations were 0.1, 0,5, 1.0,

5,0, 10,0 ppb. of dieldrin.. Dieldrin was immediately extracted and

determined quantitatively and qualitatively on the gas chromatograph.

Since dieldrin and surfactants would be mixed in the.same test

aquarium, it was imperative to know whether these compounds would complex so that neither could, be extracted efficiently for determin

ation . Six Erlenmeyer flasks 12 liter), each containing a liter of

waterf were inoculated to 6 ppb. concentration of dieldrin. Into

each o f five flasks was then placed a different amount of LAS to

acquire concentrations varying from 0.1 to 10,0 ppm,.' and a ‘control

without LAS. Extractions were made at 4, hours and 24 hours from

initial inoculation.

Duration of Surfactant and Dieldrin Residues: The ability of

dieldrin to remain in solution was observed over a period of five

months. An aquarium containing 40-liters of water was inoculated

with dieldrin to a concentration of 7.5 ppm. The 'aquarium was cov-

ered with aluminum foil to retard evaporation and only distilled water

was added to maintain the forty liter level. The water was sampled

for dieldrin after one month and after five months, 1

. A similar situation was observed with LAS at a concentration of

five parts per million over a period of a month.

. Cleaning Procedures: The continuous flow system and/or tanks

were disassembled and cleaned of any residual surfactant and pesticide

. at the completion of each toxicity test, and then reassembled for a .

new.test. Through washing with a standard laboratory detergent each

as Alconox, and three rinsings in tap water removed all recordable .

evidence of LAS and dieldrin.

. Age and Size of Fish: Several preliminary .tests were performed

to determine the relationship between time of mortality, and age and weight of fish. Fifty fish of various ages and weights were tested * in continuous flow at a dieldrin concentration of 10 ppb, The fish

Varied in age from one year to three years and in weight from 3.2 to

42,5 gins. Mortalities were recorded at 24,- 48r 72, and 96 hours.

When the fish died, scales were removed from the anterior lateral line for age determination, The results of this determination gov erned the weight and age of fish to be used in subsequent tests.

Pesticide Extraction From Fish Tissues; A fish was first weighed and cut into about 1/2 inch pieces. These pieces were then placed into a small stainless steel blender cup on.an explosion proof Waring blender. The tissue was blended with 20 ml. of acetone for approximately three minutes. This mixture was blended for about three minutes with 20 ml, of redistilled acetonitrile. This conglomerate of flesh and solvents was filtered through fine glass wool and then through

Whatman #1 filter paper.

■ The filtrate and 20 ml. of added redistilled petroleum ether and

10 ini. of 2% sulfate solution were poured into a 250 ml. Teflon stop pered separatpry funnel and shaken’vigorously for one minute. Upon standing, the petroleum ether separated out as the upper layer over the acetone-acetonitrlle-sodiura sulfate bottom aqueous layer. This aqueous iayer was introduced into another 250 ml. separatory funnel and partitioned with 20 ml. of petfoleum ether. Both petroleum ether fractions were combined and poured through a column of anhydrous sodium sulfate and the final volume of filtrate recorded. This was evaporated to dryness.and redissolved in 10 ml. of petroleum ether

» for Florisil column separation of dieldrin and DDE.

Glass columns of 20 mm. inside diameter and 400 mm. in length were used for Florisil separation. The columns contained frittered glass in the neck to facilitate holding of the packing. Two grams of anhydrous sodium sulfate were placed in the bottom of the column

followed by 20 grams of Florisil. The Florisil was actiyated by being stored in an oven at 120° C. until used. The Florisil used was obtained from the Kensington Scientific Company, Oakland, California.

It was 60-100 mesh that had been pre-activated at 650° C. The Flor

isil was topped by 5 grams, of sodium sulfate. The column was pre-1 washed with 50 ml, of diethyl'ether followed by two fractions of 25 ml. of petroleum ether. The column was allowed to drain dry before

approximately 10 ml. of pesticide extract was introduced to the col

umn, The column was then eluded with 150 ml. of petroleum ether

which extracted all to the DDE from the pesticide mixture. This was

followed by a second fraction consisting of 150 ml. of 2:1 petroleum

ether-diethyl ether mixture which extracted the remaining dieldrin.

Both fractions were brought to dryness under a filtered air stream

.on a warm hotplate. The pesticide was redissolved- in 10 ml. of

hexane and was ready for the gas chromatographic analysis.

Efficiency.of Tissue Extraction: The efficiency of the method

of extraction was determined by the following precedure. A bluegill

was killed, weighed, and the whole animal macerated in the blender. . 36 .

* * •

The macerated tissue was divided into halves. Ope half was analyzed for "background1' dieldrin, that is, the amount of pesticide present in the fish prior to experimentation. The other half was "spiked" with a known amount of dieldrin and analyzed in the same manner.

Efficiency of recovery was calculated from the following

Recovered am*t of dieldrin- (part 2) x 100 = % eff. of Ain't spiked (part 2 + Background (part 1) recovery

Florisil Standardization: The Florisil column was used to sep arate the pesticide DDE, a metabolite of DDT, and dieldrin. These two pesticides have the same retention on the: gas chromatograph thus

. * * ■ making qualitative and quantitative analysis of one ineffectual with out removal of the other.

Two Florisil columns were prepared and inoculated with known amounts of pesticide. To one column was added 0.9 NG of DDE and to the Other was added 0.9 NG of dieldrin. The column containing DDE was eluded with 200 ml. of petroleum ether and the filtrate collected in

25 ml. fractions. Each 25 ml. fraction was analyzed in the gas chrom atograph for DDE. The column containing dieldrin was eluded with 200 ml, of petroleum ether followed by 200 ml. of a 2:1 mixture of petro leum ether and diethyl ether. The filtrate was also: collected in 25 ml, fractions and analyzed in the; gas chromatograph for dieldrin.

Static exposure studies: Dieldrin: Two studies in which Lepomis macrochirus was exposed to dieldrin were conducted from Hay 15 to

*i:\; V ■' ■' : • ‘ May 26, 1967. Each, study was intended as a replicate of the other and eyery attempt was made to maintain identical experimental con ditions, Ten fish, ranging in weight from 10 to 20 grams, were placed in each of six aquaria. The 15 gallon aquaria were filled to

40 liter capacity. A different quantity of dieldrin was added to each tank to establish 5 different concentrations; with one tank being a control with no dieldrin. The five concentrations were 1.0,

3.2, 5.6, 8.7, and 10,0 mug./liter (ppb.). Mortalities were recorded at 24 hour intervals. The amount of dieldrin present in solution was determined at 24 - 48 - 72 - 96 hours.

Surfactant; Similar to that above, studies were carried out . * . * using LAS instead of dieldrin. Such tests were conducted from June 12 to June 30, 1967. .The five concentrations of LAS were 1.0, 5.6, 7.5,

10.0 and 20,0 mg./liter (ppm.).

Continuous Flow Studies: Dieldrin; Two tests were made exposing bluegills to dieldrin under continuous flow application. These tests were conducted from July 10 to July 21, 1967. The conditions of each test were, the same and thus considered as one test. Ten fish ranging from 10.2 to 20.0 grams weight were exposed at each concentration tested. The concentrations used were 1, 3,5, and 10 ppb. of dieldrin

Surfactant: Three studies were carried on using LAS as the tox icant, These studies were conducted form July 31 to Aug, 18, 1967. under similar conditions as those using dieldrin. The first study involved concentrations of 20, 13.2, 10,0 and 8.7 mg./I of LAS. Ten fish, ranging in weight from 10.5 to 18.8 grams were exposed at each concentration and control.

The second and third studies were replicates using,lower concen trations of 7,5, 6,5, 5,6, 3,2, and 1,0 ppm. LAS. Fish used in these tests ranged in weight from 11'.2 to 22,0 grams.

Dieldrin and surfactant mixture: Having established the expected mortalities (TLm) for pesticide and surfactant independently, tests were conducted exposing the fish to a mixture of the two toxicants.

These studies were carried on from Sept. 4, 1967 to Feb. 23, 1968.

In the first group of tests consisting of two replicates, the dieldrin concentration remained constant at 1,0 ppb, in all test

f I * . aquaria, except some controls, while the LAS varied in concentration between test aquaria. The LAS concentrations used were 0.1, 0.39,

1,5, and 5,6 ppm. There were three test aquaria as.controls, one re

ceiving no toxicant, only dilution (tap) water, one with 1.0 ppb . dieldrin and no LAS, and the third with no dieldrin and 5.6 ppm. LAS, the highest concentration of surfactant used. Twelve fish were placed

in each test aquarium, thus 24 fish were exposed to the same conditions during the three replicate studies. Fish used in these tests varied

in weight from 9.8 to 21,7 grams.

In the second group of tests, consisting of two replicates, the

dieldrin concentration remained the same through all test conditions

at 5 ppb. LAS concentrations remained the same as those in the first

group of tests. One control was changed to 5 ppb. dieldrin. The twelve fish, exposed at each, concentration ranged in weight from 10.6 to 18*1.grams,

In the third group of tests, consisting of two replicates, the dieldrin concentration was 10 ppb. while LAS concentrations remained unchanged. One control was changed to 10 ppb. dieldrin. The twelve fish exposed at each concentration ranged in weight from 9.7 to 20.5 grams.

In the first group of tests, each test consisted of an exposure time of 14 days under continuous flow. The second and third group of tests were terminated after 96 hours of exposure under continuous flow.

Tissue Extraction Studies; Two studies, conducted between May 6

and Sept. 6, 1968, were carried out to determine whether the uptake

and assimilation of dieldrin was.affected by the presence of different concentrations of surfactant. These studies were done under continuous

flow application.

in the first study, the dieldrin concentration was maintained at

5 ppb. in all test aquaria, while the LAS concentration was different

in each aquarium. The concentrations of LAS used were 0,1, 0,39, 1.5,

5.6 ppm. and a control with no LAS, The fish used in this study ranged

in weight from 6.2 to 31.1 grams.

After every twelve hours of exposure time, two fish were removed

from each concentration, killed, weighed, and quickly frozen. These

fish were analyzed for dieldrin in their tissues.

The second study was very similar to the first except the dieldrin concentration was maintained at 10 ppb. Fish in this study varied in weight from 7,7 to.32,7 grams. Two fish were removed from each con centration every 24 hours and stored for future tissue analyses.

Testing Procedure: Acute 96 hour tests were initiated on Monday and terminated on Friday. After each test the continuous flow system

•and tanks were cleaned and reestablished thus allowing two days for

the system to reach an equilibrium. Flow rates were checked and re

adjusted if necessary during this period.

Pesticide■and/or surfactant, of a volume calculated for dilution, was aidded to the carboys which were then filled to 40 liters with

distilled water. Distilled water was used to inhibit the growth of'

I , algae and fungi in the bottles. The dieldrin used in these tests

was a technical grade obtained from the City Chemical Company, N. Y.

and contained a dieldrin purity of 85%. concentrations were calculated

on the basis of the percent of dieldrin.

Pesticide and/or surfactant was also added to the aquaria in'

amounts that were necessary to. acquire the desired concentration.

After a period of equilibration, usually two days, fish were added to

the test aquaria. The fish were acclimated in holding tanks in the

constant temperature room for one week prior to each test.

The tanks were monitored each 24 hours for pesticide and/or sur- ) ' . factant concentration and each 48 hours the disposable needles were

replaced. At this time flow rates were checked and adjusted if neces

sary, The flow rates from the carboys and dilution water were deter-. 41;

mined by measuring ,• with, a stop watch, the time required to fill a 10 ml, volumetric flask.

Dead fish were removed from aquaria as soon as possible. A fish that died anytime before 24 hours was recordeel as a 24 hour mortality; between 24 and 48 was recorded as a 48 hour mortality, etc.

With tests in which tissue extracts were analyzed, the fish were killed, weighed, wrapped in aluminum foil and frozen for storage. ■ RESULTS

Preliminary investigations; The results of these studies, are presented here to substantiate and/or justify the methods used in the experimentation

Efficiency of monitoring techniques: The efficiency of the dieldrin-water-hexane extraction over the range of concentrations tested was 75+3%. .Seventy five percent represented the corrective factor used in all calculations determining the amound of dieldrin in aqueous- 1 solution.

In a mixture of dieldrin - LAS and water( foaming created a prob lem when the mixture was shaken with hexane in the liquid-liquid ex traction. The hexane portion became more cloudy as the concentration of LAS increased. However, the cloudy appearance of the hexane fraction did not.alter the extraction of dieldrin, ■ The cause of the cloudiness, in the hexane extrace is not known but it had no effect on the effic iency of dieldrin determination. Figure 5 shows dieldrin averaging

5.5 ppb. was present in the five different concentrations of LAS. In

24 hours, dieldrin showed a reduction in concentration probably due to adsorption on .the surface of the glass.

Water quality: The quality of the water posed no problems in maintaining stock fish and experimental controls. One large tank con- •. taining approximately fifty fish was maintained several months with constantly circulating tap water. V

X 6 24 hr

.o o x a 5 a. 48 hr 4 o c o 3 o

e 2

2 1 a> Q

.5 1.0 5.0 10.0 LAS Concentration--ppm.

Figure 5 . Effect of Various Concentrations of LAS on Monitoring Efficiency of Dieldrin in Test Flasks. The Columbus' city water received at the Water Resources building is of a medium hardness and alkalinity, but rather high in nitrogen# phosphates, and sulfates. At times, the pH was very high# reaching

a maximum of 1 0 .0 , ■

Analysis of Columbus city water showed no recordable amount of

dieldrin.

Table 1 summarizes the quality of water over the period of exper

imentation.' The wide ranges are probably caused by seasonal differ

ences in surface waters and inconsistencies in the Hach kit determina

tions, .

Age and Size of Fish: Fifty fish# verying in weight from 3.2 to

42.5 grams# were exposed to 10,0 ppb. of dieldrin under continuous

flow. All fish died in 96 hours. Fish were removed from tanks as

close as possible to time of death. The fish were weighed and age

determined-from scale samples.

The greatest number of fish died in 72 hours. The smaller fish

died within 24 hours while the largest ones survived beyond 72 hours.

Table 2 shows the weight and time of death of the individual fish.

The mean weight of those :fish dying within 24 hours was 3.85 grams,

within 48 hours — 8,46, within 72 hours — 10.36, and within 96 hours

— 20,94 grams. The results indicate that mortality to a toxic sub

stance, such as dieldrin, is weight dependent. -

' Age determinations revealed that fish in the weight range of 4,50

to 22,10 grams were two years old. Those fish that were heavier than Table 1. Chemical analysis* of Columbus City water at the Water Resources Center', The Ohio State University. (Results in milligrams per liter except pH and temperature)

5-26-67 8-8-67 2-5-68 8 —8 — 6 8

Alkalinity Phenol 19.0 0 . 0 2 2 . 0 2 0 . 0 Total 30.0 40.0 50.0 30.0 Carbon Dioxide 2 . 0 8 . 0 0 . 0 0 . 0 Chlorine 0 . 0 1 0 . 0 2 0.041 Chloride 35,0 7.2 . 43.0 50.0 Chromate 0.05 0.13 0.075 0 . 0 Copper 0.25 0.005 0.25 0.17 Fluoride 0 . 0 0.45 0.23 Hardness Calcium 80.0 70.0 55.0 81.0 Magnesium * 2 0 . 0 40.0 51.0 19,0. Total 1 0 0 . 0 1 1 0 . 0 106.0 1 0 0 . 0 Hydrogen sulfide 0 . 0 0 . 0 0 . 0 0 . 0 Iron 0 . 0 2 0.05 0 . 0 0 . 2 2 Manganese • 0.04 0,5 0.25 0 . 6 Nitrogen Nitrate 14.0 11.5 18.5 8.7 Nitrite 0 . 0 0.016 0 . 0 0 1 0 .0 0 - Oxygen 8 . 8 7.6 3.4 Phosphate • * • .Ortho 0 . 0 2 1 . 0 0 . 1 0 . 6 8 Meta 0.37 0.23 0.75 0.55 Total 0.39 1.23 0.85 1.23 Silica 3.0 2.5 3.7 1.4 Sulfate .. 97.0 180.0 180.0 75.0 PH 8 . 6 7.9 1 0 . 0 9.3

*Hach Chemical Kit Table 2. The mortality of Lepomjs macrochirus of various weights and age exposed to 1 0 ppb. of dieldrin under continuous circulation.

Weight of fish killed at

24 48 1 72 96 hours

. 9.25 11.95 37.85 7.00 14.35 2 2 . 1 0 8 . 2 0 ■ lOUOv 25.00 9.40 16.10 9.40 9.55 7,20 1 0 . 2 0 6.85 16.30 11.30 7.35 8,70 16.00 8 . 0 0 12.90 19.70 11.65 ■ 6.60' 10.40 8.55 8.30 33.30 9.55 8.70 13.60 * 8.50 9.90 42.50 9.60 10.80 6.30 14.40 8 . 2 0 11.50 7.60 6,30 1 0 . 1 0 10.70 - 8 . 1 0 7.10

Mean weight 3.85 8.46 10.36 20.94 grains Age 1-2 2 22 2-3 yrs. 47

2 2 , 1 0 grains were three years old.

.Pish in the 10-20 grains weight range were chosen because: (a) they were young enough to be within the period of active growth,

(bl they could survive 96 hour exposures in the intended concentra tions, (cl they were of a suitable size (3-4 inches), and (d) they were available.

Static Exposure Studies: . Dieldrin: No mortalities were recorded in either study among tihe ten fish present in each concentration from

1,0 to 10,0 mg./liter end control during 96 hours. The most prob able reason for the lack of mortality was the disappearance of di eldrin from the test medium. Since these tests were conducted under

static conditions, the adsorption and absorbtion of dieldrin resulted * in sublethal concentrations for the greater portion of 96 hours. When

the test aquaria were monitored at 24-48-72 arid 96 hours, the dield

rin concentration was found to decrease through the 96 hours. Data

from such monitorings are shown in Table 3. Since monitoring sam-. pies were taken in duplicates, and two studies were involved, mean values are represented for the actual concentration.

At the lower concentration ranges of 1 mg./liter and 3.2 mg./liter

no dieldrin was recordable at 96 hours. Less than 10 percent of the

dieldrin remaining in solution was less than 50 percent after 24

hours. Figure 6 shows! diagramatically the percent of dieldrin re

maining in solution during the 96 hour acute static tests.

When the tests were allowed to continue to 192 hours, mortalities 48

Table 3.. Data from static exposure studies showing changes in •dieldrin concentration in 96 hours.

Time Initial Actual Percent Concentration Percent (hours) Concentration Concentration Remaining Loss (mean) •

1 . 0 .86 8 6 4.4 • 3;2'.’ 1.40 , 43 57 24 5.6 1 . 2 0 ‘ 2 1 79 8,7 4.6 53 47 1 0 . 0 5.3 . 53 47

1 . 0 .29 29 71 3i2 . 8 6 C 26 74 48 • 5.6 .86 15 85 8.7 2 . 0 23 77 1 0 . 0 2 . 2 2 2 78

1 . 0 0.08 8 92 3.2 0.13 4 96 72 5.6 D.45 8 92 8.7 00,87 1 0 90 8 8 1 0 . 0 1 . 2 0 * 1 2

1 . 0 • 0 0 1 0 0 3.2 0 0 1 0 0 96 5,6 0.29 5 95 8.7 .0.58 6 . 6 93.4 1 0 . 0 0 . 8 6 8 . 6 91.4 \ \

100 100

1 Dg/liter '3.2 pg/liter

50 50

0 JH. 0 24 48 72 0 24 48 72

100 100

5.6 pg/liter 8.7 ng/liter

50 50

1 a B n 0 24 48 72 96 0 .2 4 48 72 96

100

10 Mg/liter

e to

0 24 48 72 96 Hours

Figure 6. Percent of Dieldrin Remaining in Solution During 96 Hour Acute Static Toxicity Tests did appear at 144 hours of exposure in the higher concentrations of

8.7 and 10,0 mg./liter. There was a 15 percent mortality at 144 hours in the 8,7 mg./liter concentration and a 35 percent mortality between 144 and 192 hours in the 10.0 mg./liter concentration.

Surfactant; Two identical studies were carried on using the surfactant — LAS, These studies were conducted under the same conditions as the studies using dieldrin. The concentrations of LAS consisted of 1,0, 5,6, 7.5, 10.0, and 20.0 ppm. (mg./I.) The concentration of LAS decreased through the 96 hour test period in a manner similar to dieldrin. Table 4 shows the data from these tests.

The observed concentrations represent mean values from the two sim ilar studies,

In the lower concentration of*1,0' and 5.6 ppm., the LAS concen trations were most reduced in 24 hours. Only 14 percent remained in solution in 24 hours in the 1.0, ppm. concentration of surfactant. In the same time period, 30 percent was present in the 5.6 ppm. concen tration. In the three higher concentrations of 7,5, 10.0, and 20.0 ppm., the concentrations of surfactant remaining after 24 hours was respectively, 50 percent,. 56, and 82 percent. Although the surfact ant concentration decreased from all initial concentrations, it was recordable - in lowered amounts only in the initially higher concen trations at 96 hours. In the lowest concentration, the surfactant was not recordable at 72 hours, and in the 5.6 ppm, initial concen tration at 96 hours. The 20.0 ppm. initial concentration showed the \ \

Table 4, Data from static exposure studies showing changes in LAS concentration in 96 hours

Time Initial Actual Percent Concentration Percent (hours) Concentration Concentration Remaining Loss (mean) . »

1.0 .14 14 8 6 5.6 1.68 30 70 24 7.5 3,75 50 50 10.0 5.60 56 44 20.0 16,40 82 18

1.0 .020 2 98: 5.6 .336 6 94 48" 7.5 .600 8 92 10.0 3.00 30 70 20.0 11.60 58 42

1.0 0 0 100 5,6 ‘ .112 2 98 72 • 7.5 .450 6 94 10.0 2240 24 76 20.0 7.20 36 64

1.0 0 0 100 5.6 . .0 - 0 100 96 7.5 .300 . - 4 96 10.0 .600 6 . 9 4 20.0 5.60 28 72 least loss in 96 hours. Figure 7 shows diagrammatically the percent of

LAS regaining in solution during these 96 hour aohte static tests.

Mortalities were' recorded at 24 hour intervals. The result of fish mortalities are summarized in Table 5, No mortalities appeared in 96 hours in the two lower concentrations of 1.0 and 5.6 ppm. There was 40 percent mortality iii 96 hours at the 10,0 ppm. concentration and 100 per cent mortality in 72 hours at the highest concentration of 20.0 ppm.

The TLm Cmedian tolerance limit) values for static exposure to LAS are shown in Figure 8 , The values are:

24 h o u r ------20 ppm, 48 hour — ------— — 20 ppm. 72 h o u r ------13.5 ppm. 96 hour — ---- — ------11.1 ppm.

Exposure Studies Using Continuous Flow. Dieldrin:- Two studies were conducted exposing bluegills to dieldrin under continuous flow applica tion, Due to the difficulty in maintaining exact concentrations, the actual concentrations deviated, frojn.the initial of intended concentration.

The range of actual concentrations is;! shown .on. Table .6 , The initial conrs centrations were 1,0, 3.0, 5,0, and 10.0 ppb. of dieldrin. Monitoring samples were taken in duplicates at 24 hour intervals. Between tests 1 and 2, the mean of actual concentrations varied only slightly over 96 hours,- In determining the TI^ values, the mean of the actual concentra tions of test 1 and 2 were used as representing the actual concentration's in the test aquaria. The actual concentrations in the test aquaria were

1.04, 3,4, 6,5, and 11,2 ppb. of dieldrin.

Ten fish, varying in weight between 10.2 and 20,0 gram3 , were exposed V

53 100 100 1.0 5.6

48 48 72

100 10.0

• P s

50 m iii '■"'■I &. IV- r,; err I Hi . b c t. 24

e <** .a 1 o in

24 48 72 96

Figure 7. Percent of LAS remaining In solution during 96 hr. acute static toxicity tests. Table 5. Survival of Lepomis macrochlrus exposed to various concentrations of l a s under static conditions.

Concentration Number of Number of fish surviving after Percent surviving after of LAS (ppm,) test animals 24 48 72 96 hours 24 48 72 96

.2 0 , 0 . 2 0 16 - 1 0 0 80 50 0 0 1 0 , 0 2 0 ‘ 2 0 2 0 18 1 2 1 0 0 1 0 0 90 60 7.5 2 0 2 0 2 0 18 18 1 0 0 . 1 0 0 .90 90 5.6 2 0 2 0 2 0 2 0 2 0 1 0 0 1 0 0 i o o 1 0 0 1 . 0 2 0 2 0 2 0 2 0 2 0 1 0 0 1 0 0 1 0 0 1 0 0

0 . 0 , 2 0 2 0 2 0 18 18 1 0 0 1 0 0 90 90 Percent Survival 100 50 i u e . A Tm aus o Lpms arciu Under macrochirus Lepomis for Values TLm LAS 8. Figure ttc Conditions. Static Table 6 , Range and standard deviations of actual concentrations of dieldrin in TLjjj determinations (96 hour), ,

Actual concentration of dieldrin (ppb) Initial concentration of Test I Test II • dieldrin Standard Standard (ppb) Range - Mean Deviation Range Mean Deviation

1 -.58-1,4 1 . 0 2 .13 .90-1.3 1.06 .15 ■

3 2,3-4,4 3,5 .57 2.7-3,9 3.3 .16 ID 00 • 5 ' • 5,8-8,0 6.5 1.07 5,8-7.6 6.4

. 1 0 11,3-12 11.5 .11 9.8-12.1 10.9. 1.15 567 at each concentration in tests 1 and 2, Table 7 summarizes the survival of bluegills during these tests. At the lowest concentrations of 1.04 . ppb,f 80 percent of the fish survived at 96 hours. At a concentration of 3.4 ppb, mortalities commenced at 48 hours and resulted in 50 percent surviving at 96 hours, At the two higher concentrations of 6.5 and

11,2 ppb.,.mortalities commenced at 48 and 24 hours respectively and resulted in nO survival at 96 hours.1 No deaths were recorded in the controls (no dieldrin in either test).

TLm values for Lepomis macrochirus under continuous flow application

appear in Figure 9. These values are:

.. 48 hour — — — ------10.1 ppb, 72 hour ------— — * 4,75 ppb. 96 h o u r ------3.5 ppb.

Surfactant: Three studies were conducted using LAS as the toxicant

As far as possible, these studies were similar to those using dieldrin

under continuous flow. •p The first study involved concentration of 20.0, 13.2, 10.0, and ,8.7 ppm. of LAS. At a concentration of 20.0 ppm., no fish surviyed longer

than 4 hours, At 13,2 and 10.0 ppm. concentration, 100 percent mortal

ity was attained in 8 and 12 hours, respectively. The lowest concentra

tion of 8,7 ppm, showed only 40 percent surviving at 48 hours, and no

survival at 72 hours. Since no fish survived to 96 hours, lower con

centrations were used in subsequent tests,

The second and third studies were replicates using the lower con

centrations of 7,5, 6,5, 5,6, 3.2 and 1.0 ppm. of LAS, Due to the Table 7, Survival of Lepomis roacrochirus exposed to various concentrations of dieldrin under continuous flow conditions.

Concentration Number of Number of' animals surviving after Percent surviving after dieldrin (ppb) test animals 24 48 72 96 hours 24 48 72 96

1.04 2 0 2 0 2 0 2 0 16 1 0 0 1 0 0 1 0 0 80 * 3.4 2 0 2 0 18 . 16 1 0 1 0 0 . 90 80 50 6.5 2 0 2 0 14 4 0 1 0 0 70 j 2 0 ■ 0

1 1 . 2 2 0 1 2 0 • 0 0 60 . 30 0 0

0 . 0 (c) 2 0 ; ‘ . 2 0 2 0 2 0 ' 2 0 1 0 0 ‘ 1 0 0 1 0 0 1 0 0 Percent Surviving 0 0 1 50 iue . ilrn L Vle fr eoi mcohrs Under macrochirus Lepomis for Values TLm Dieldrin 9. Figure 3 5 7 9 Cn, (ppb.) Cone, 1 9 8 7 6 5 4 3 2 otnos lw Application. Flow Continuous ( 3 . 5 ) 4 . 75 : ( 0 1 . 1 ) \ tn 60 difficulty of maintaining exact desired concentrations were slightly different from the initial concentrations. In Table 8 the actual con centrations represent the mean for fluctuations occurring in a given concentration-: 'over a 96 hour period. The range of fluctuation and standard deviations of observed concentrations is also shown. The actual concentrations were 7,5, 6.4, 5,2, 2.0 and 0.95 ppm. These values were used in determining the TLm for LAS,

Table 9 contains a summary of the experimental data of these three studies. At a concentration of'7.5 ppm. only 40 percent ..were surviving at 72 hours and only 30 percent at 96 hours. Fifty percent of the fish survived 96 hours at 6.5 ppm. No mortality was observed at the three lower concentrations of 5,6, 3,2, and 1.0 ppm, at 96 hours.

TLjh values are interpolated graphically in Figure 10. These values are: 48 hour ------8.3 ppm. (mg./I.) 72 h o u r ------— 7,2 ppm. 96 h o u r — ------6.5 ppm.

The slope'of the lines is much greater for LAS than for dieldrin, resulting in a much narrower TL^ range between 48 and 96 hours for the surfactant as compared to the pesticide.

Dieldrin and surfactant mixture: The results of the first group of tests in which dieldrin concentration was 1 . 0 ppb, are shown in

Table 10. There was 100 percent survival through the first 192 hours

(.8 days} of exposure. The first dead fish appeared on the 9th day in the 5.6 ppm, concentration of LAS. Mortalities continued to increase in this concentration until all fish were dead in 336 hours (14 days.) Table 8 , Range and standard deviations of actual concentrations of LAS in TL^ determinations, (96 hr.).■

Initial Actual Concentration of LAS (ppm) Concentrations of Test I Test II ■ . LAS. Standard Standard (ppm) Range Mean Deviation Range Mean Deviation

1 , 0 0,87-1,30 1.09 .05 ,69-.96 .82 .01

3,2 2;l-3.5 2.9 .07 2.7-3.4 2 . 8 ,16

5.6 . 3.9-6,0 5,3 ,1 0 5.2-6.2 5,1 .44

6,5 5.4-7.3 6,4 ,54 5,6-7.5 . 6,3 ,49

7.5 6 ,9-7,9 7,5 .26 6 ,8-7.9 7.4 .15 Table 9. Survival of Lepomis macrochirus exposed to various concentrations of LAS under continuous flow conditions.

Concentration Number of Number of animals surviving after Percent surviving after LAS-ppm, test animals 4 8 12 24 48 72 96 24 48 72 96 hr.

- 2 0 1 0 0 ' ' \ ' \ 13.2 1 0 ■ 1 0 0 1 0 . 0 . 1 0 1 0 6 0 • 8,7 . 1 0 • 1 0 1 0 1 0 . 7 4 0 70 40 0 : • 7.5 2 0 , 2 0 . 2 0 2 0 2 0 14 8 6 1 0 0 70 40 30 'k- ■ ' 6,5 2 0 2 0 2 0 2 0 2 0 2 0 16 1 0 1 0 0 1 0 0 80 50 - ■/: • ' ■ ■ ■ - 5.6 2 0 2 0 2 0 2 0 2 0 2 0 2 0 2 0 1 0 0 1 0 0 1 0 0 1 0 0 3.2 2 0 2 0 2 0 2 0 2 0 2 0 2 0 2 0 1 0 0 1 0 0 1 0 0 1 0 0 1 . 0 2 0 2 0 2 0 2 0 2 0 2 0 2 0 . 2 0 1 0 0 1 0 0 1 0 0 1 0 0 62 Cone, (ppm.)

Figure 10. LAS TLm for Lepomis macrochirus Under Continuous Flow Application. I

Table 10, Results of simultaneous exposure of Lepomis macrochirus to 1.0 ppb. dieldrin and various concentrations of LAS under continuous flow.

Concentration Number of Percent of animals surviving after (hour's) LAS - ppm. Dieldrin - ppb. . test animals 192 216 240 264 288 312 336

0 . 1 1 24 1 0 0 1 0 0 1 0 0 91 91 91 91 0.39 . 1 24 1 0 0 1 0 0 83 83 83 6 6 58 1,5 1 24 1 0 0 1 0 0 91 83 83 75 41

5.6 1 24 1 0 0 75 58 41 41 . 16 . 0 0 0 24 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 1 0 0 5.6 0 24 . . 100 100 100 100 100 100 100 0 .1 ■ 24 . 1 0 0 1 0 0 . . 1 0 0 1 0 0 91 75 6 6

✓ Mortalities appeared at 240 hours (1 0 th day) in the 0139 and 1.5 ppm.

LAS concentrations. There was 100 percent survival in the control with no toxicants (dilution;:; water only) and the control containing 5,6 ppm. of LAS and no dieldrin in 336 hours (14 days)'. In the control receiv ing only dieldrin, 6 6 percent survived to 336 hours. The highest sur vival was evident in the lowest concentration of LAS while the lowest survival appeared at the highest concentration of LAS.

The results of the second group of tests, in which the dieldrin concentration was 5 ppb,, are shown in Table 11. All" animals survived the first 24 hours. The lowest survival appeared in the highest concen tration of LAS with, only 13 percent surviving in 96 hours. The highest survival was evident in thellowest concentration of surfactant. All fish in both the control with no toxicants, and the control of LAS and no dieldrin, survived 96 hours. The control with dieldrin and no LAS had 46 percent survive in 96 hours..

The results of the third group of tests, in which the dieldrin con centration was 10 ppb,, are shown in Table 12. Survival was lowest in this group of tests due to the lethal level of dieldrin. Mortalities appeared in all concentration^fexcept water and LAS controls, within

24 hours. Only 22 percent survived in the control of dieldrin and no

LAS in 24 hours. Survival in the 5.6 ppm. concentration of LAS approx

imated the control with no LAS present. As the LAS concentration was

decreased from 5.6 to 0,1 ppm, the survival of fish increased. This Table lXt Results of simultaneous exposure of Lepomis macrochirus to 5.0 ppb, dieldrin and various concentrations of LAS under continuous flow.

Concentration Number of Number of animals surviving after Percent surviving after . . LAS-ppm. Dieldrin-ppb. test animals 24 48 72 96 hrs. 24 . 48 72 96 hrs.

0 . 1 5 24 24 2 2 2 0 15 1 0 0 91.7 8 6 -. 0 63.0 0.39 5 24 24 2 2 19 1 2 1 0 0 91.7 79.2 50.0 1.5 5 24 24 2 1 13 7 1 0 0 87.5 55.0 30.0 5,6 5 24 24 17 9 3 1 0 0 71.0 38.0 13.0 0 0 24 24 24 24 24 - 1 0 0 1 0 0 100 100 5.6 0 24 24 24 24 24 1 0 0 100 100 1 00 0 5 24 24 22 19 11 100 91.7 79.2 46.0

o\ cri Table 12. Results of simultaneous exposure of Lepomis macrochirus to 10,0 ppb. of-dieldrin and various concentrations of LAS under continuous flow.

Concentration Number of Number of animals surviving after Percent survival after LAS f ppm. Dieldriii'ppb. test animals 24 48 72 96 hrs. 24 48 72 .96

o ; r 10 36 35 25 17 11 97 . 69 ■ 47 31 0 .3 9 10 36 32. 23 19 11 89J- 64 ' 53 31 1 .5 10 . 36 23 10 4 1 64 28 11 3 5 .6 ■ 10 36 10 .5 1 0 28 13 3 • 0 0 . 0 36 36 36 36- ■ .36 100 100 100 100 5 .6 . 0 . 36 36 36 36 .36 100 100 100 100 0 .10 • 36 8 5 1 ’ 0. 22 3 3 0

cn . . • \ ■ ' s . '

.. 68

• indicates that small amounts of surfactant had an- inhibitory effect on

.the mortality rater while at higher concentrations it had little effect.

Surviyal decreased with, exposure time in.all concentrations of toxicants.

N o .fish survived to 96 hours in the highest concentration of LAS and the \ control with no LAS, Survival was complete in the controls with no tox

icants and with no dieldrin but only^LAS.

. The major difference in the three groups of tests was the concen

tration of dieldrin. In the first group with a dieldrin concentration

of 1 ppb,/.the presence of surfactant did decrease the survival of the

fish, as evidenced by. the decrease in survival with increased concen

tration of LAS, In the second group of tests containing 5 ppb, of diel

drin, a concentration close to the dieldrin TL^, the presence of the

higher amounts of LAS decreased survival while the lower concentrations

increased survival of fish.

The actual concentrations of dieldrin. varied from the intended

concentrations during the 96 hour period. Variation was also evident

i . , between replicate tests. There were, however, no significant differences

between the dieldrin concentrations within replicates and between re

plicates. Table 13 shows the results of analysis of variance of actual

concentrations of dieldrin.

Tissue extraction studies; Efficiency of extraction method: The

. method of extraction used recovered 81 percent of the "spiked” amount

of dieldrin from the fish tissue. This degree of recovery was repro-

, ducible in the analyses of three fish. The value of 81 percent was

the corrective factor used to determine absolute amounts of dieldrin in Table 13T Analysis of variance tests of differences in actual concentrations of dieldrin.

Dieldrin concentration . Source of df Mean square F variation

0 , 1 ppb. Between replicates 4. 0,08743 .4070 NS* Within replicates 2 0 0.10852 .5060 NS

5. ppb. Between replicates 4 0.00365 ‘ .2893 NS* Within replicates 2 0 . 0.01018 .7939 NS

. 1 0 ppb. Between replicates 4 0 . 0 0 1 0 1 .1047 NS Within replicates 2 0 0,02231 . 2.314 NS

* = Not significant 70 the tissue,

Florisil ’standardization; To elude with 150 ml, of petroleum ether removed all recordable DDE on:.the column, while the dieldrin remained.

If this were followed by 150 ml, of a mixture of 2:1 petroleum ether- di-ethyl ether, the dieldrin was eluded from the column. Thus dieldrin and DDE separation was accomplished .with a minimum loss of dieldrin.

• - Analysis- of dieldrin in the tissues of fish: The amount of diel drin found in the tissues of the fish increased in relation to the test concentration and the duration of exposure. The concentration of pesti cide in the tissues of fish exposed to 5.0 and 10.0 ppb. dieldrin, in all concentrations of LAS, at least doubled in 24 hours. An average of

0,42 ppm, of dieldrin was found in the tissues after 12 hours of expo sure in a test concentration of 5.0 ppb, of pesticide. Table 14 shows the accumulation of dieldrin in the tissues of the fish. Corrected concentrations in the tissue reflect the loss of dieldrin through the ' extraction procedure. The concentration increased in the next 12 hours of exposure to an average of 0,97 ppm. followed by 1.3 ppm. in 36 hours and 2,0 ppm. in 48 hours. Figure 11 shows the results of plotting the concentration in the tissues against exposure time, using only those fish in the 1 0 - 2 0 g, weight range.

In the similar test in which the dieldrin test concentration was

10,0 ppb,, an average of 1.4 ppm. of the pesticide was found in the tis

sues after 12 hours of exposure. Table 15 shows the accumulation of dieldrin in the tissues when exposed to different concentrations of I A S . V \

. 71 Table 14. Accumulation of dieldrin in tissues of Lepomis macrochirus vhen exposed to different concentrations of LAS and 5.0 ppb. dieldrin simultaneously.

Concentration Concentration * of dieldrin of dieldrin 0 M o to f? Concentration Pish . in tissue 3 Fish Wt . in tissue Av. time of LAS (ppm.)( No. (gram) (uncorrected) (corrected) (ppm.) (ppm.) (ppm.)

1 2 0 . 1 3-A 29.9 0.19 0.23 1 2 0 . 1 4-A 15.7 0.30 0.37 1 2 0.39 5-B 18.4 0.48 0.59 1 2 0.39 1-C 15.7 ' 0.46 0.57 1 2 1.5 1-B 24.4 0.163 0.20 0.42 1 2 1.5 2-B 15.0 0.34 0.42 1 2 5.6 1-A 14.6 0.31 0.38 1 2 5.6 2-A 2 0 . 6 0 . 2 0 0.25 1 2 0 . 0 3-B 17.6 0.48 0.59 1 2 0 . 0 '4-B 19.8 ■ 0.48 0.59 24 0 . 1 . 4-C 15.3 0.78 0.96 24 0 . 1 5-C 77.-9 1 . 1 1.40 24 0.39 3-D 20.5 • 0.60 0.74 24 0.39 4-D 17.1 0.76 0.94 24 1.5 2-C 19.8 0.70 0.86 0.97 24 1.5 3-C 1 2 . 0 0.83 .1 . 0 0 24 5.6 5-D 14.3 0.90 , 1 . 1 0 24 . 5 . 6 1-E 17.2 0.63 0.78 ,24 0 . 0 1-D 28.9 0.38 0.48 24 0 . 0 2-D 11.5 1 . 1 1.40 36 0 . 1 4-G 11.5 1.3 1.60 .36 0 . 1 4-G. 16.7 1 . 0 1 . 2 0 36 0.39 4-E 26.4 0.56 0.69 36 0.39 5-E 13.0 1 . 1 1.40 36 1.5 3-F 1 1 . 0 1.9 2.30 1.30 36 1.5 4-F 31.1 0.51 0.63 36 5.6 1-P 13.8 1.4 1.70 36 5.6 2-F 16.8 1.3 1.60 36 0 . 0 2-E 24.3 0.61 0.75 36 0 . 0 3-E 13.3 1 . 2 1.50 48 0 . 1 3-H 19.5 1.4 1.70 48 0 . 1 4-H 15.5 1.7 2 . 1 0 48 . 0.39 5-G <56^2 2 . 0 2.50 48 0.39 2-H 7.7 2 . 2 2.70 48 1.5 1-H 18.4 1.9 2.30 2.00 48 1.5 5-F 22.3 1 . 2 1.50 48 5.6 2 -G 15.7 1 . 6 2 . 0 0 48 5.6 5-H 17.7 1.5 1.90 48 0 . 0 1-G 2 2 . 6 1 . 0 1 . 2 0

• - Jr\ ■■ -*1! •; 3 Iv'*' ' ' '• TV . 0 Q. iue I Dedi Acmlto i Tsus of Tissues in Accumulation Dieldrin II. Figure . xoue o . pb o Pesticide. ppb. of 5.0 to Exposure Concentration of Dieldrin in Tissues eoi mcohrs fe 1 Hu Itras of Intervals Hour 12 After macrochirus Lepomis 1.0 0.1 12 24 36 48

:hours \ \

73 Table 15. Accumulation of dieldrin in tissues of Lepomls macrochirus when exposed to different concentrations of LAS and 10.0 ppb. dieldrin simultaneously.

Concentration Concentration of dieldrin of dieldrin Exposure Concentration Fish Fish Wt. in tissue inttissue Av. time of LAS (ppm.1) No. (gram) (uncorrected) (corrected) (ppm.) -- (ppm.) (ppm.)

1 2 0 . 1 5-1 13.2 1 . 2 1.5 1 2 0 . 1 1-J 25.2 0.87 1 . 1 12 0.39 2 - 1 32!i5 0.80 0.99 12 1 2 0.39 l-r 16.0 ' 1 . 6 2 . 0 1 2 1.5 4-1 15.6 1 . 6 2 . 0 1.4 1 2 1.5 3-1 27.8 0.39 0.48 1 2 5.6 4-j 18.6 1 . 1 1.4 1 2 5.6 5-J 17.4 1 . 2 1.5 1 2 0 . 0 4-H 2 0 . 6 0.84 1 . 0 1 2 0 . 0 • 5-H 9.6 1 . 8 2 . 2 24 0 , 1 2 -J 17.8 1.4 1.7 24 0 . 1 3-J 14.6 2.9 3.6 . 24 0.39 1 -K 23.9 . . 2 . 0 2.5 24 0.39 2 -K 18.0 2.7 3.3 24 1.5 . 5-K 20.3 2.7 3,3 24 . 1.5 1-L 32.7 1.4 1.7 2.4 24 5.6 3-K 27.9 1.9 2.3 24 .5.6 4-K 33.0 1.7 2 . 1 24 0 . 0 3-L 13.5 2.5 3.1 . • 24 0 . 0 4-L 40.5 0.56 0.69 36 0 . 1 4-N 25.4 3.1 3,8 .36 0 . 1 5-N 29.1 2.5 3.1 36 0.39 5-L 25.6 2 . 1 2 . 6 36 0.39 2-H 7.7 5.2 6.4 36 i.5 1-N 20.7 3.0 3.7 4.5 36 1.5 1-H 18.4 3.1 3.8 36 5.6 2-N 15.9 3.7 ' 4.6 36 5.6 3-N 15.0 5.0 6 . 2 36 0 . 0 2-L 14.5 3.1 3.8 36 0 . 0 4-0 23.3 5.5 6 . 8 48 0 . 1 4-H 19.2 5.2 6.4 48 0 . 1 3-M 2 2 . 6 6 . 6 8 . 1 48 0.39 1 -M 18.1 8 . 8 io.;9-i 48 0.39 5-0 14.3 6 . 2 7.7 48 1.5 1 - 0 24.1 8 . 2 1 0 . 1 7.8 48 1.5 2 - 0 16.9 9.4 1 1 . 6 48 5.6 2 -M 19.7 6.5 8 , 0 48 5.6 3-H 19.5 5.8 7.2 . 48 0 . 0 5rM 58.5 0.34 0.42 48 0 . 0 3-0' 21.5 6 . 0 7.4 jfewieir*' and 10,0 ppb, dieldrin simultaneously. The concentration in the tissue increased to 2,4 ppm. in 24 hours, 4.5 ppm. in 36 hours, and 7.8 ppm* in 48 hours.

The pesticide accumulated in the tissues.of fish maintained in the higher test concentration of 1 0 , 0 ppb. of dieldrin at a relatively faster rate than in those at the lower test concentration o'fi 5.0 ppb.

This, was more evident after 36 hours of exposure. Figure 12 is a comparison of the uptake of dieldrin by the fish when exposed to 5.0 and 10.0 ppb. The magnitude of difference was greater than two at 24 hours and greater than three at 48 hours.

In almost all fish examined, there was an inverse relationship between weight and concentration in the tissue. The larger fish had less pesticide accumulation than the smaller over the same period of exposure and test, concentration. A multiple analysis of variance in which the weight was a variable indicated that the weight factor was significant in the accumulation of pesticide in the tissues.

. In those tests in which the fish were exposed to LAS and dieldrin simultaneously, there was no correlation between the dieldrin in the tissues and the different- concentrations bf LAS. Although certain con centrations of LAS seemed to affect the toxicity of dieldrin to bluegills on the basis or mortality survival data, the concentration of dieldrin

in the tissues showed no such relationship.

Figure 13 shows the concentration of dieldrin in the tissues, plotted agdinst the concentrations of LAS for.12 hour intervals when \\

9 ppm 8

3 5ppm 2

12 24 36 48 hours

Duration of Exposure

Figure 12. Comparison of concentrations of dieldrjn in tissues of Lepomis macrochius exposed to 5.0 and 10.0 ppb. of the pesticide Concentration 'of dieldrin in tissues (ppm.) 2.3 2.0 1.7 1.3 2.1 1.7 1.5 l.l .3 .5 .7 .7

f ilrn n dfeet ocnrtos f A (2 hrs.) (12 LAS of concentrations different and dieldrin of iue 3 Dedi cnetain n bugle xoe t 50 ppm. 5.0 to exposed 'bluegllle In concentration Dieldrin 13. Figure ■ k 0.0 o o o i i • A cnetain (ppm.) concentration LAS o • o. 9 o 1 ' .1 , r \ '•> * ,* * ■ i - i i- i t - ' V ■- ’ ? ■■ '?■ ’ ' • ■ ♦30.

o o o o P . „ 1.5 o o o ‘ 0 0 1 I ' i 1 . _ . . 8 hr. 48 6 hr. 36 4 hr.24 2 hr. 12 76 5.6 o o o o o o • o o j } i I the fish were exposed to 5 ppb. dieldrin. Figure 14 shows the same parameters when the dieldrin concentration was maintained at 1 0 . 0 ppb.

Both figures include only fish in the 10.0 to 20,0 gram weight range.

It is obvious from the.position of plots that no pattern indicating a relationship between the dieldrin assimilation and the LAS concen tration in test aquaria exists, A multiple analysis of variance also indicates that the influence of LAS was not significant at the 10 per cent-level of accuracy, while the dieldrin was significant at one per cent. Tabie 16 summarizes the data of the statistical analysis. Concentration of dieldrin in tissues 8 hr. 48 Table 16, Multiple analysis of variance.

' Source of Degrees of Sum of Mean F variation freedom squares squares

Dieldrin 2 ’ 1468,06477 734,03239 55.273*

LAS 4 133.39662 33.34915 2,5111**

*' significant at 1 percent ** not significant at 1 0 percent ' DISCUSSION i ' Many factors must be considered in determining the effects of

toxicants on fish. Duodoroff, et al., (1951) described methods to be

followed in the evaluation of acute toxicities of industrial wastes on

fish. A review of the literature, however, reveals great differences

in methods and techniques used in toxicity studies. Notable among the

differences are (a) the manner in which the toxicant is administered,

(b) the size and weight of fish used, (c) the differences in monitoring procedures, and (d) the differences'in water quality.

Three basic methods are used in administering toxicants to fish.

One is the static method in which the toxicant is administered only

initially in the testing period. Second is the periodic renewal method

in thich the toxicant is systematically renewed during the course of

theptest, Third is the continuous* flow method in which the toxicant is

continually administered, in a quantity only sufficient to maintain a

constant concentration during the time of the test period.

The validity of static biosssay tests on aquatic organisms has been

seriously challenged by many workers, especially Holden (1962) and

Mount (1962). Due to adsorption, absorptioh, decomposition, and possi

bly coevaporation with the water medium, the toxic materials in a static

test decrease in concentration through a given period of time. However,

Cairns.and Scheier (1963) found little difference in results between

acute static and acute continuous flow in diheir, study of the effects of

ABS on bluegills. They recorded a 96 hr, TL^ of 17.44 ppqt. for static

tests and 17.37 ppm, for continuous flow using soft water in both tests.

■; ‘ ■ • 80 . * ■ Henderson, et al,, (19591 using a static, non-renewed test medium,

found the 96 hr, TI^ for ABS was 5,6 ppm. on bluegills. Lemke and

Mount C1963)., using continuous flow found.the 96 hr. TLm for ABS on

bluegilis to be 18,1 ppm. From the data presented in the foregoing

chapter of this study, it is^evident t£hat there is a loss of toxicant

in static tests. Both LAS and dieldrin concentrations were greatly

reduced in 96 hrs. under static conditions. No recordable dieldrin

or LAS remained at 96 hrs, at low concentrations and between 4 and 28

percent remained at the higher initial concentrations. This loss of

toxicant was evident also in the results of the TI^ determinations.

The difference between TL^ values in static and continuous flow con

ditions was.:very, noticeable. The TL^ values were higher for both LAS

and dieldrin under static conditions as compared to continuous flow.

For example, the 48, 72, and 96 hr. TLm values for LAS under static

conditions were 20.0, 13.5, and 11.1 ppijv, while under continuous flow

the same values were 8,2, 7.2, and 6.5 ppm. Henderson, et al., (I960),

using static bioassay tests, reported a 96 hr. TLm of 7.9 ppb. for

bluegills exposed to dieldrin.: The bluegills ranged in length from 1.5

to 2.5 inches and in weight from 1 to 2.grams. In the present study

the 96 hr, TLm ^or" bluegills-exposed to dieldrin was 3.5 ppb. using con

tinuous flow and fish of larger size and weight (more tolerant);. The

TI^ yalue is reduced more than 50 percent under continuous flow appli- :•

cation, Furthermore, Tarswell and Henderson (1956), in their field

"run-off" study, used the Tl^, value of 7.9 ppb. to compute the application rate that would contaminate an acre-size pond. An application rate of 0,07 lbs./acre was the computed value based on a TI^ of 7.9 . ' ppb. Using the value of 3.5 ppb, from continuous flow bioassay, the rate of application would be reduced to 0.03 ibs./acre. From these . facts it seems more realistic.to establish acute toxicity criteria • from studies in which the. toxicant is administered under continuous . flow rather than under static conditions,

* Descriptions of different continuous flow systems have appeared in .the literature, notably those descriptions of Herbert and Merkins

{.1952), Grenier (I960) , and Mount and Warner (1965) , The continuous flow system used in this study was different from those described in the literature and operated satisfactorily under the testing conditions used. The use of disposable syringe needles proved to be a reasonably accurate means of controlling flow rates throughout the gravity fed system. The latex bulbs and Luer adapters made cleaning and changing needles quick and efficient. •' v' ■ :v'’

The size of the fish is another factor of great importance’ in de termining toxicity values. As stated by Duodoroff, et.al., all fish :: should be of a uniform size and in an active growth stage.. In. subjecting bluegills of heterogenous .sized to dieldrin, the smaller fish showed a, greater susceptibility to the toxicant than did'the larger fish. For this reason fish, within the 110-20 gram weight range were used. To oh- tain a mor e steno-weight. range would be ^difficult without anesthesizing them for a closer: selection of weight; Fish within 50 and 70.iur. (2-3 inchesHin length ranged in weight between 6.7 and 31.0 grains. It is

not sufficient to select fish of a. given length in view of the broad

differences that can exist in weight at that given length.

The monitoring of toxicant concentrations is important in the

determination of toxic effects. Previous studies either overlooked

the need for monitoring or failed to report such in the literature.

Several tests in the present study were discarded because toxicant lev*

elsf as detected by constant monitoring of test aquaria, became aber- -

rant. The reason for this aberrance is unknown, but it does occur and

can seriously alter results. The actual concentrations differed from

the initial concentrations in all tests.. At concentrations of ppb.

. • variations are not considered unreasonable.- (Lamar, et al., 1966).

A knowledge of water quality, and in particular the hardness, is .

important in toxicity studies dealing with surface active agents, Henr

derson, et al. (1959) found the average 96• hr. TL^ for fathead minnows,

exposed to synthetic detergents, was 15*1 ppm. in soft water while the

comparable value in hard water was 8.5 ppm. The hard water had a total

hardness of 400 ppm, while the soft water was 20.0 ppm. In the present

study/' Columbus City water averaged '104 ppm, hardness.

. LeoOce. and Mount (19631 reported 18.1 ppm. as .the 96 hr. TL® for

bluegills exposed t o , ABS using continuous flow tests. . The ’fish weighed v between 1.75 and 2,25 grams. From the data in the foregoing chapter,

the 96 hr. TI^ for bluegills exposed to LAS is 6.5 ppm,. Pish of much • "<' '' ' " -greater sire and weight were used in the present study.; The biodegrad- . . . • • 84

' ’• ■ ' . ' . • : ' V • able surfactant - LAS seems to be more toxic to bluegills than the

. obsolete'non-biodegradable - ABS. *

When bluegrills were exposed to LAS and dieldrin simultaneously,

there was a difference in survival depending on the concentration of

surfactant. At,_the low concentration of 0.1 ppm. of LAS there was

- greater survival, in ali tests, than in the controls. The cause for

thisr phenomenon is unknown. Dugan (1967) found in one test that gold

fish exposed to ABS for 96 days, showed greater survival'than controls

exposed to dieldrin. He attributed the greater survival to a gain in

weight by those fish exposed to ABS, ' Less fish survived a t the high

.. concentration of 5.6 ppm. than in the controls. From the work reported

* ‘ . * • • '■ by Bardach (1965) and Cairns (1964), among others, the increased mor

tality could have been caused by eroded tissue as well as the toxic

effects of dieldrin on the central nervous system. Bardach (1965) re

ported that the taste buds of yellow bullheads were eroded when exposed

to ABS and LAS at concentrations between'0.5 and 10VO ppm. for periods

up to 4 .weeks. Cairns, et al. (1964) , reported gill -damage, in acute:;

" and chronic studies, starting-at 24 hr. In this study no attempt.was

made to examine any fish histologically,

Since LAS reducesi the surface tepsion of water, the possibility

,’existed that such a surface active agent might increase the absorptive .

• capability of the membranes of fish and thus increase the uptake of di

eldrin. This ' decrease in survival of fish' exposed to high concentrations of LAS and sublethal concentrations of dieldrin appears, at first, to . further substantiate this possibility. However, if this possibility were true, then there would exist a correlation between the dieldrin in the tissues of the fish and the different concentrations of LAS when fish are exposed to both simultaneously. To check this hypothesis, the concentration of dieldrin was ascertained from the body of each fish

, * ' • ■ • exposed to dieldrin and LAS simultaneously. Ho correlation was found between the concentration of dieldrin in the fish and the different concentrations of LAS. There was no more or less dieldrin in fish exposed to both dieldrin and LAS together than in fish exposed to di eldrin alone. Thus the possibility that a surface active agent, such as LAS, might increase the absorptive capability of fish membrane is unfounded. ' . ,

The concentration of dieldrin in the tissues of fish increased with length of exposure from 24 hr. to 96 hrs. Dieldrin concentration in the tissues also increased between fish exposed to 5.0 ppb. and 10.0 ppb.

BluegillPf as well as many other aquatic organisms are capable of con- centratingsatoxicant such as dieldrin'in their tissues to a level much higher than that of the surrounding medium. ;

Further study is needed to find a practical use for the phenomenon a . ' of increased survival in low concentrations of thie surfactant LAS. ^. . • ‘ 86

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The Effects of Different Concen­ Trations of Las on the Toxicity of Dieldrin to the Bluegill (Lefomis Macrochirus)

The Effects of Different Concen Trations of Las on the Toxicity of Dieldrin to the Bluegill (Lefomis Macrochirus)