CHRONIC EFFECTS OF ENDRIN ON BLUNTNOSE MINNOWS AND GUPPIES

Donald Irvin Mount

RESEARCH REPORT 58 UNITED STATES DEPARTMENT OF THE INTERIOR Stewart L. Udall, Secretary Frank P. Briggs, Assistant Secretary for Fish and Wildlife FISH AND WILDLIFE SERVICE Clarence F. Pautzke, Commissioner BUREAU OF SPORT FISHERIES AND WILDLIFE Daniel H. Janzen, Director

Published by the U.S. Fish and Wildlife Service • Washington • 1962 Printed at the U.S. Government Printing Office, Washington, D.C.

For sale by the Superintendent of Documents, U.S. Government Printing Office Washington 25, D.C. - Price 35 cents CONTENTS Page Abstract iv Introduction 1 Chronic-toxicity tests on bluntnose minnows 5 Chronic-toxicity tests on guppies_ 14 Acute-toxicity tests on bluntnose minnows_ 17 Effect of endrin on oxygen consumption 20 Effect of endrin on swimming of bluntnose minnows 24 Port of entry into and distribution of endrin in the fish's body 25 Mode of action of endrin on animals 29 Summary 31 Conclusions 32 Literature cited 33 Appendix—Raw data from tests 36 ABSTRACT In an investigation of chronic toxicity of endrin to bluntnose minnows and guppies, conducted for 291 days, neither species could withstand concentrations greater than 0.5 parts per billion of endrin in water for more than a few days. Less than 50 percent of the test animals could live in 0.5 p.p.b. for more than 30 days. Approximately 65 percent could live in 0.4 p.p.b. for periods exceeding 30 days, while there was little mortality due to endrin in 0.25 and 0.1 p.p.b. endrin in water. Fish suffering from endrin poisoning exhibited symptoms (increased ventilation rate, convulsions, and loss of equilibrium) which indicate that it affects the central nervous system. Sublethal concentration caused increased activity and hypersensitivity to stimuli. No cell damage was found in fish which were able to survive in endrin con- centrations, and essentially no physical effects other than increased activity were noted. Adult female guppies ceased to have young when placed in endrin concentra- tions, but the gonads of the bluntnose minnows were approaching sexual maturity when the test was terminated. In continuous-flow acute-toxicity tests, it was found that the 96-hour TLm values ranged from 0.27 p.p.b., for fish 31) mm. standard length, to 0.47 p.p.b., for fish 60 mm. standard length. Endrin had no effect on oxygen consumption in the seven bluntnose minnows tested. Neither did acute exposure to endrin significantly affect the ability of the fish to swim against a current of water. In carp exposed to concentrations of 2.5 to 10 p.p.b. endrin during periods varying from 2.5 to 28 days, the digestive tract, liver, heart-spleen-blood, and kidney contained the highest accumulations of endrin. The maximum concentrations of endrin found in the tissues were approximately 160 times greater than that in the water in which the fish were living. The concentration of endrin in the heart-spleen of a carp exposed 28 days to 2.5 p.p.b. was found to be 400 p.p.b. Muscle tissue was consistently low in endrin content. The gills were always low or negative in endrin content, whereas the digestive tract was consistently high, indicating that endrin probably enters the body through the intestine. Endrin does not appear to be cumulative in its effects on guppies and on bluntnose minnows. Little or no tissue damage was found in fish which could live in water con- taining endrin, and fish seemed to recover completely from one exposure to endrin. The increased activity caused by very low concentrations of endrin could be very damaging to fishes in natural waters, disrupting spawning and subjecting the fish to predation and other decimating factors. iv CHRONIC EFFECTS OF ENDRIN ON BLUNTNOSE MINNOWS AND GUPPIES

By Donald Irvin Mount

Ohio State University

In the past 15 years, the use of pesticides boats; floor waxes contain pesticides for has increased rapidly, and the number of ant and roach control. Some insecticides compounds used for pest control has grown have been found very effective in rodent at an astounding rate. Abstracting jour- control, especially in orchards. Many nals in recent years have had to add a sub- conservation departments use chemicals to title for articles about pesticides, and each eradicate fish populations in large bodies year this section becomes larger. Herbi- of water, and it appears at present that ef- cides have increased in use in a similar fective control of the sea lamprey in the manner, and at present are replating cul- Great Lakes may be achieved by use of tivation in the production of some agricul- chemicals. In short, one will find pesti- tural crops (corn, for instance). On the cides used in almost any place he may commercial market, one finds a whole ar- go. ray of compounds for combating roaches, Such widespread use has caused many ants, spiders, mites, and other pests. people to wonder what effect these chemi- Cities now have spraying programs in cals may have on the intricate balance of which every alley is sprayed for fly and animal populations. Unfortunately, mosquito control. Highway departments many of the pesticides used are not very spray roadsides with herbicides instead specific in their effects. These pesticides of mowing. The Federal and State gov- may alter food chains by removing a cer- ernments, often in cooperation with local tain group of animals. A long residual governments or agencies, spray large areas life as well as high toxicity are sought- for various insect pests. The use of after features of pesticides, and for ex- chemicals, especially DDT, for the con- tended periods of time after spraying they trol of forest pests is of ever-increasing are dangerous to aiiimals other than those importance. being controlled. Recently a huge aerial-spraying pro- Aquatic animals in particular are (Tram has been conducted in the southeast- highly susceptible to pesticides. As an ex- ern United States in an attempt to control ample, Fitzwater (1958) reports that in the imported fire ant. Various extermi- 1953 an airplane carrying a spray mixture nating agencies now use new chemicals to containing a little over 1 gallon of actual control termite damage to buildings, and endrin crashed into a Louisiana bayou. often give a guaranteed protection period This resulted in the death of all fishes and which may last several years. Marine reptiles, including 42 alligators, for a dis- paints on the market contain chemicals to tance of 35 mile, from the point of the control arthropod damage to wooden crash. Aquatic a(nimals are often exposed 1 2 to insecticides as a result of runoff from second. - They found concentrations as treated areas, as well as from direct ap- high as 2,275 parts per million in the fat plication to the water. of apparently healthy largemouth bass, There is a dearth of information on the and concentrations as high as 42 parts per residual life of insecticides in soils and million in the fat of yearling largemouth in organisms. Young and Nicholson bass hatched 7 to 9 months after the last (1951) reported what they thought to be application. Carnivorous fish had higher the first recorded large-scale kill of aquatic levels of DÐÐ in their tissue than did animals caused by agricultural use of in- plankton feeders. secticides. There weie 15 major fish kills All these investigations and many more in northern Alabama in one summer, and not cited point up the need for cautious in Flint Creek in Alabama, in a 13-mile use of insecticides, and for extensive section varying from 6 to 20 feet in depth, studies to determine what the total effect of there was an apparent total eradication of their use may be over a long period. the fish population. These kills were Certainly in coming years, as the world thought to be due to frequent applications population increases, the value of our in- of insecticides, followed by several heavy land waters for recreation as well as food rains in August which washed the insecti- supply will be increasingly important, and cides from the soil into the streams. the aquatic habitat must be protected. At Barker (1958) found robins in Illinois the present increased rate of use of pesti- dying from feeding on earthworms after cides, it is not impossible that even the heavy rains in the spring. Analysis of oceans along the continental coasts may the worms showed high concentrations of receive enough to reach a toxic level. The DDT, which had been sprayed on the area unfortunate fact is that in general there almost 1 year earlier. Barker found the is little concern about the danger until de- DDT content of the soil to increase in the sirable organisms begin to die, and often autumn, presumably owing to the residue it is then too late to remedy the situation. on the falling leaves. Barlow and Hada- Acute testing of new chemicals on vari- way (1955) found DDT recoverable from ous animals is rapidly becoming standard soil 12 months after sorption was com- procedure before the chemicals are re- plete. Harrington and Bidlingmayer leased to the public. Toxicity is usually (1958) found extensive kills of aquatic measured by the number of deaths, but sub- animals resulting from aerial applications lethal effects also can be very damaging. of 1 pound of dieldrin pellets per acre The need for chronic-toxicity studies is be- in a tidal marsh. They noted crabs eating coming more pressing and is gaining some moribund fishes and dying themselves the support. The importance of finding next day. symptoms of poisoning at a time when Hunt and Bischoff (1960) have pre- complete recovery is still possible is slowly sented a very complete picture of the ac- being recognized. In an effort to focus cumulation of DDD (dichloro-diphenyl- attention on this area, as well as to de- dichloroethane) in animal tissues. In this termine some of the effects of sublethal case, Clear Lake in California was treated doses, an investigation of the chronic tox- three times with DDD. The first treat- icity of endrin to fishes was initiated. En- ment was applied in 1949 at a concentra- drin was chosen because of its extreme tion of 1 part in 70 million, the second in toxicity to fishes and its increasing use as 1951 at 1 part in 50 million, and the third. a pesticide. Henderson, Pickering, and in 1957 at the same concentration as the Tarzwell (1959) state, "Endrin was by far 3 the most toxic of the insecticides to all p.p.m. in the runoffs from the first and species of fish [tested]. In fact, it is the third rains in the treated area mentioned most toxic chemical that has been tested above. These two experiments, together in this laboratory." They found endrin with many published accounts of fish mor- to be lethal in 96 hours to 50 percent of tality, indicate that these insecticides do the bluegills tested at a concentration of reach the streams in sufficient quantity to 0.6 p.p.b. (parts per billion) in the water. be toxic. Mr. Henderson has informed Endrin is 1,2,3,4,10,10-hexachloro-exo- me that "quite constant amounts" of insec- 6,7-epoxy-1,4,4a,5,6,7,8,8a-oct a hy dr o-1,4- ticides are found in some streams. Un- endo,endo-5-8-dimethanonaphthalene. It doubtedly the concentrations increase in is a yellowish-white crystal with a sweet periods of widespread spraying (April odor when it is in a dry form. Endrin is and May), but it is significant that there slightly soluble in water and moderately is a measurable level of insecticides in the soluble in alcohol. streams at all times. Presumably these The use of endrin has increased each levels are maintained by residues of year since it was first introduced in the insecticides in the soil which have been early 1950's. Officials of one agricultural shown to last for at least 1 year (Barlow chemical company informed me that they and Hadaway, 1955). Seasonal spraying sold several thousand pounds of active renews this supply in the soil. This fact endrin in a 3-State area during 1958. makes the need for studies of chronic Endrin has been used effectively to control effects even more pressing. many pests. A few are listed below to A literature search revealed no studies illustrate its broad spectrum of toxicity. on the chronic effects of endrin on fishes, Other pests such as army cutworms, although some experiments have been con- plant-lice, tent caterpillars, and white- ducted on birds and mammals. Treon, pine weevils have been effectively Cleveland, and Cappel (1955) found that controlled. all rats fatally poisoned by endrin had Although such application rates may "diffuse degenerative changes in the liver, kidney, and brain." In a rabbit given 50 seem low, Tarz well and Henderson (1956) oral doses (1 mg./k1.), endrin caused "dif- working with a closely related compound, fuse degeneration and fatty vacuolation of dieldrin, found runoff from the first and hepatic and renal cells and degeneration of third rainfalls from an area treated with the heart." Survival times of male rats 4.66 pounds of dieldrin per acre to be fed 100 and 50 p.p.m. endrin in their food, lethal to 50 percent of the bioassay fish and of females fed 100, 50, and 25 p.p.m., in 96 hours. Runoff from the fourth rain for periods up to 106 weeks were signifi- still contained some dieldrin, as indicated cantly shorter than for those animals fed by the reactions of fish placed in a sample 1 and 5 p.p.m. Concentrations of 50 and of it. Rosen and Middleton (1959), by 100 p.p.m. in food caused the ratio between use of a carbon filter, found. 0.1 and 0.013 liver weight and body weight to be larger

USE OF ENDRIN TO CONTROL CROP PESTS

Pest Crop Dosage

Spiny bollworn ------Cotton ------500 grams per hectare. Lesser peach borer ------Peaches ------1 qt. emulsified endrin per 100 gal. (0.5 gal, per tree.) Potato tuber moth ------Potatoes ------0.25-percent spray. Red-banded thrips ------1 pt. per 100 gal. Potato leafhopper_ ------Potatoes ------2 cc. per gallon of 19.5-percent endrin. Citrus psylla ------Citrus fruit__ ------0.01-percent endrin. Pine mice ------Orchards ------2.5 lbs. per 52 trees. 4 than normal. Male rats fed 5 and 25 Lepomis macrochirus, 1 p.p.b. for fathead p.p.m. for 2 years also showed an increased minnows, Pimeph,ales promelas, and 1.9 liver weight to body weight ratio. p.p.b. for goldfish, Carassius auratus. They also stated that, in general, the These fishes were 1.5 to 2.5 inches long. only effect in female dogs fed 4, 3, and 1 Guppies, Lebistes reticulatus, 0.75 to 1 inch p.p.m. for 6 to 19 months was some slight long, had a 96-hour TL,T, value of 1.5 p.p.b. renal tubular vacuolation. Some dogs fed under the above-mentioned conditions. 3 p.p.m. for 19 months had enlarged kid- These were static tests with five guppies neys and hearts. Those dogs which sur- in 2 liters of test solution and five fishes vived on 8 p.p.m. had normal viscera, ex- in 10 liters of solution for the other three cept for slight diffuse degeneration of the species. In constant-flow tests of 20 days' distal convoluted tubules in the kidneys. duration, they found endrin to be toxic at DeWitt (1956) reported that 10 p.p.m. levels of half the 96-hour TI,Li value. endrin fed to adult pheasants during the Their conclusion was that endrin had a reproductive period reduced hatchability cumulative or chronic effect on fishes. of eggs and survival of the young. One They concluded also that natural varia- p.p.m. fed to quail before the reproductive tions in pH, alkalinity, and hardness have season, but discontinued during reproduc- no major effect on the toxicity of the chlo- tion, reduced the fertility and number of rinated hydrocarbon insecticides to fishes, eggs, as well as the survival of the young. although endrin, among others, was Sherman and Rosenberg (1953) found slightly more toxic in soft water. 3.5 mg./kg. of endrin in 7-day chickens to Iyatomi et al. (1958) determined the be lethal between 6 hours and 5 days. It 48-hour TL; for 5- to 7-centimeter carp to caused degeneration of the liver and kid- range from 140 p.p.b. at 7°-8° C. to 5 ney, and following long exposures was p.p.b. at 27°-28° C. They found that the stored in peritoneal fat and the liver. 24-hour TL for day-old carp was 8,500 Kunze and Laug (1953) detected stor- p.p.b., which decreased to 46 p.p.b. at 6 age of endrin in the brain and liver of rats. days of age. It is quite probable that these Females contained more fat-stored endrin TL,T, values are high, since five fishes (5 to than males. These investigators recovered 7 centimeters long) were placed in 2 liters considerable amounts of endrin from the of water. Such large fish in 2 liters of kidneys, but this was of a "different chem- water would absorb endrin and thereby ical nature." reduce the concentration. Ely, Moore, Carter, and App (1957) Both of the last two papers mentioned considered the effect of the form in which reported that 20 mg. or more per day fed the endrin was administered (powdered, to cattle caused measurable amounts of granular, or emulsion) on the lethal dose. to occur in the milk. One and one- endrin Form was not considered in the current half milligrams per kilogram per day investigation. Form probably affects the caused the cattle to exhibit toxic symp- amount dissolved in the water and not the toms. actual toxicity. Data on the effect of endrin on fish are very scanty. Henderson, Pickering, and Objectives Tarzwell (1959) state that in soft water The objectives of this investigation (alkalinity 18 p.p.m., hardness 20 p.p.m.) were- at 25°C. the 96-hour TIAT, (median toler- 1. To determine the maximum concen- ance limit) was 0.6 p.p.b. for bluegills, tration in which guppies and bluntnose 5 minnows could survive for extended ately with each phase of the investigation. periods. Raw data are given in the appendix. As 2. To determine reproductive and used here, the terms "chronic test" and physiological effects of sublethal concen- "acute test" refer to tests of long and short trations of endrin on fishes. duration, respectively. 3. To determine the place of entry into, This report was prepared in partial ful- and the movement through, the fish's body. fillment of the requirements for the degree 4. To determine any differences in the of doctor of philosophy in the Graduate acute toxicity of endrin to various size School of Ohio State University. Dr. groups. Wilson Britt was my major advisor and 5. To determine whether there is a po- Dr. Eugene Dustman was my coadvisor tential danger to human beings or to other during my doctoral study. In addition, I animals which eat fishes that have been am indebted to Drs. William Spoor, David exposed to sublethal doses of endrin. F. Miller, George Ware, Frank Fisk, and The methods and equipment employed William McIntosh for advice and in this investigation are described separ- assistance.

CHRONIC-TOXICITY TESTS ON BLUNTNOSE MINNOWS

Objective to control the water flow into the constant- level chamber; the level did not vary by The objective of this experiment was to 1/4 determine the effects of sublethal levels of more than inch during operation. Au- tomotive valve-grinding compound was endrin on bluntnose minnows exposed for used to seat the ground-glass surfaces. extended periods of time. No sealing compound was used on the valve seats; only a slow seepage occurred Methods and equipment when the valve was closed. Large cork The chronic-toxicity testing was based stoppers coated with plastic resin served on a continuous flow system. The appa- as floats. Ten of these valves functioned ratus consisted of a reservoir capable of for periods of 6 to 11 months with approx- holding ,a 4-day supply of the test solu- imately 10 failures due to particles of dirt. tion, a constant-head chamber, and a test Water was siphoned from the constant- chamber, all shown in figures 1 and 2. level chamber into the test chamber, and The reservoirs were made of 3/4-inch the flow was controlled by small glass jets. marine plywood coated with four coats of These jets were made by heating and draw- plastic resin in which glass cloth was im- ing glass tubing to a very small diameter. bedded for strength (Mount, 1959). Ten The jet was fastened to the water line, and such reservoirs, 16 by 16 by 24 inches, small pieces were broken off until the de- were arranged in two batteries of five each. sired flow rate was obtained. Glass stop- They were supported 4 feet above the floor cocks and screw clamps were tried, but by six 4-by-4 legs and eight 2-by-6 planks. both failed to maintain a constant water Glass tubing was bent as shown in figure flow and became clogged quickly with dust 2 to serve as water-level gauges. particles. Flow rates were measured by a Water was siphoned from the reservoirs 10-milliliter graduated cylinder. All wa- into a constant-level chamber. Figure 3 ter lines were made of glass tubing with shows the details of the float valve used tygon tubing at the joints.

620654 0-62-2 6

FIGURE 1.—Continuous-flow system used for the chronic-toxicity testing. 7 Since the endrin was added directly to LEVEL GAUGE RESERVOIR the reservoir, the level gauge of each reser- voir was calibrated so that exactly 80 liters of water could be measured into it. In this way the desired concentration could SIPHON be made directly in the reservoir. There were 2 inches of water under the zero level mark in the level gauge, so that when the reservoir was lowered 80 liters the col- FLOAT VALVE CONSTANT HEAD umns of water in the level gauge and CHAMBER — siphon tube were not broken. A change in SIPHONS volume of 0.1 percent could be detected on the level gauge. Each reservoir held a 4.5-day supply of solution which flowed out at the rate of 720 ml. per hour. This TEST CONSTANT LEVEL rate amounted to the approximate capac- CHAMBER CHAMBER ity of the test chamber in 24 hours. FIGURE 2.—Diagram of continuous-flow system In 1.5 hours, all 10 reservoirs could be used for chronic-toxicity testing. drained and refilled with fresh solutions. During the filling, the siphon tubes were The bluntnose-minnow test chambers, clamped to prevent an improper concen- 10 by 24 by 6 inches, were constructed in tration from reaching the test chambers. the same way as the reservoirs, except that the front was glass, and white pigment was added to the plastic resin. A narrow retaining strip was placed around the front to hold the glass in place. Double- strength window glass was cut so that it — ALIGNMENT was as large as the entire front, and was COLLAR sealed in place with aquarium cement. A 1 strip of aquarium cement /8 inch wide or less, around the glass, was exposed to the GROUND GLASS test solution. Each test chamber, when VALVE SEAT filled to normal level, had a capacity of ap- proximately 18 liters. A U-shaped glass tube siphoned the water from the test chamber into a constant-level chamber, and from there it was emptied into the drain. The end of the tube in the test GLASS TUBE- CLOSED chamber was inserted into a cylinder made AT LOWER END of plastic screen in order to prevent fish from entering the siphon tube. The test FLOAT chambers were joined in batteries of three to seven, and arranged in three banks or levels, all lower than the reservoirs. Com- pressed air was bubbled into the water in FIGURE 3.—Float-valve assembly for regulating each chamber through a glass jet. water-flow. 8

This entire unit was set up in a base- the test fishes. During all months except ment room built especially for the ex- June, July, and August the temperature periment. The walls were made of two rarely varied by more than plus or minus sheets of polyethylene-plastic film with 0.5° C. Continuous temperature records 4 inches of dead-air space between them. were kept by a recording thermograph. Sunlight from a basement window entered Examination of these records showed that through the plastic wall; thermostatically no large temperature fluctuations occurred controlled electric heaters held the temper- at any time. ature constant. It was found that small Water chemistry in the test chambers variations in air temperature did not alter was determined periodically, and the pH water temperature measurably. There and alkalinity of the tap water were meas- was temperature stratification of the air; ured each time before the reservoirs were but this proved to be advantageous be- filled. Chemical analyses of the tap water 0 cause the water in the reservoirs was 1 to upon leaving the Morse Road water-treat- 2° C. warmer than that in the test cham- ment plant are shown in table 1. This in- bers. Influent was introduced at the water formation was made available through the surface in the test chamber, and since it courtesy of Mr. Richard Melick, Division was less dense it spread out over the sur- of Water, Columbus, Ohio. An analysis face and settled to the bottom as it cooled. by the State health department on No- The outlet siphon drew the water from vember 19, 1959, showed, in addition, the bottom. This movement, plus the tur- fluorides 0.2 p.p.m., silica 4 p.p.m., nitrate bulence from aeration, gave good wider nitrogen 0.5 p.p.m., and sodium 7 p.p.m. circulation through the test chamber as de- termined by methylene-blue dye. Four TABLE 1.—Chemical analyses of Columbus tap test chambers and four reservoirs were water [All values except pH are expressed in p.p.m. These values used for the bluntnose-minnow tests. were determined by the Columbus Division of Water on the Three test chambers contained endrin and water as it left the Morse Road treatment plant] were designated TB-6, TB-7, and TB-8; Oct. 21, Nov. Dec. 28, Feb. 2, one designated CB-10, without endrin, 1959 25, 1959 1959 1960 served as a control. Total alkalinity ------22 22 20 21 Phenolphthalein alkalin- ity ------4 3 12 10 Physical and chemical conditions Noncarbonate hardness - 52 54 62 71 Total hardness ------74 76 82 92 Calcium ------18 21 24 26 The chronic-toxicity tests were con- Magnesium ------7. 5 6 5 7 Iron ------0 o o 0 ducted at a constant temperature of 22.2° Chlorides ------7.5 8 7.8 9. 5 Sulphates ------52 58 62 66 C., plus or minus 1° C. The summer of Free C 02 ------0 0 0 0 pH value ------10. 1 8. 5 10. 2 10.0 1959 was unusually warm, and on several Turbidity ------.2 .25 0 0 Total solids ------153 139 148 166 days the capacity of the cooling unit was Manganese ------.3 0 0 0 exceeded. For 3 days in August, the tem- perature reached 25° C. in the late after- According to the analysis made at my noon, and for a total of 18 days in the laboratory before the reservoirs were months of June, July, and August the filled, the chemical characteristics of the temperature exceeded 23.2° C. but did not tap water used in the tests, based. on 73 reach 25° C. I feel that this variation did samples over a period from April 11, 1959, not affect the results significantly, since to January 27, 1960, were as follows : this was a chronic test, and because any Range Mean variation due to temperature fluctuation pH ------6.5— 8.5 p.p.m_ 7.8 p.p.m. would affect the control group as well as Total alkalinity_ 15.0-42.0 p.p.m_ 23.9 p.p.m. 9 Only two alkalinity determinations ex- as much as they would. The excess food ceeded 30 p.p.m., and only two pH read- and excrement were siphoned daily. ings were below 7.0. Phenolphthalein al- This species fights very little under lab- kalinity was always present in tap water, oratory conditions, and its behavior, plus but disappeared during aeration as the its tolerance to laboratory conditions, reservoirs were filled. Free chlorine ap- makes it a very desirable test animal. All peared in the water for the first time on fish used in the chronic test were collected November 29, 1959, and filtering through in a small pool in Rocky Fork Creek, on charcoal was necessary from then until the the Franklin-Delaware County line, on end of the test. The filter consisted of a March 16, 1959. The fish were held in the 4-foot length of glass tubing, 100 mm. in- laboratory at 22° C. for 26 days before the side diameter, packed with granular bone test for chronic effects was begun. Those charcoal. Chlorine determinations were not used immediately in the tests were held made according to the starch-iodine test at 22° C. for later use. described in Standard Methods for the Ex- amination of Water and Sewage (1949) . TABLE 2.—Chemical analyses of water in the test chambers based on determinations made Table 2 summarizes the chemical char- approximately every 9 days during the 291- acteristics of the solutions in the test cham- day test period bers. No phenolphthalein alkalinity was [All values except pH are expressed in p.p.m found in the test chambers. The unmodi- fied Winkler method was used for dis- Chemical character Range Mean solved-oxygen determinations, and Total alkalinity - - - 18-33 (One reading over 28)---- 24.0 Dissolved oxygen_ _ 5. 3-7. 2 ------6.2 dissolved carbon dioxide and alkalinity Dissolved CO2 - - - 1-3 ------2.3 pH ------7. 0-7. 8 ------7.4 were determined by standard methods described by Welch (1948). Indicator solutions with a color chart were used to Ninety-six percent pure reference determine pH. A comparison of this standard endrin was used in the tests. A method with a pH meter indicated that the stock solution of 1 mg. endrin per milli- method was sensitive to 0.25 pH units. liter of 95-percent ethyl alcohol was pre- The chronic testing was begun on pared, and the desired amounts were meas- April 11, 1959, and was conducted for 291 ured with a 1—milliliter-graduated pipette. days, ending on January 27, 1960. Five Initially, 200 ml. of the stock solution were bluntnose minnows, Pinzephales notatus, prepared. After this had been used, one were placed in each of the four test cham- liter of solution was made and this was bers. They were assigned to chambers at sufficient for the rest of the chronic test random by the use of a table of random and for all of the acute testing. digits. Rocks of similar composition When filling the reservoirs, I dropped were placed in each chamber to serve as the appropriate amount of the stock solu- cover. Hardware cloth of 1/4-inch mesh tion into a beaker containing approxi- was placed over the top to keep the fish mately 100 ml. of water. The beaker was from jumping out. Ground commercial then emptied into the reservoir and washed dog-chow checkers were free-fed twice for several minutes under the water daily. This diet was supplemented from stream entering the reservoir. It was time to time with chopped earthworms necessary to keep the alcohol content as taken from an area known to be free from low as possible, because it permitted a insecticides. The fish were overfed inten- considerable growth of bacteria, even in tionally, so that all fish were able to eat low concentrations. A volume of pure 10 95-percent ethyl alcohol, equal to the concentrations are very near the threshold largest volume of alcohol put into the of tolerance, the fact that no fish died in test chamber of highest endrin concentra- the TB-6 effluent may not be significant. tion, was put in the control chamber. After the chronic test was ended, five fish This originally amounted to 0.72 ml. per ranging from 29 to 38 nun, standard length 80 liters of water, but later when the maxi- were placed directly into TB-7, and all mum test concentration was dropped to ,died within 3 days. Five others were 0.5 p.p.b. endrin, it was decreased to 0.4 placed in TB-6. One disappeared, but ml. per 80 liters of water. Even this the other four died within 3 days, indicat- amount permitted an appreciably greater ing that the concentrations were as ex- bacterial growth than did the 0.08 ml. pected. (Despite precautions, fish were put into TB-8. The stock solution was occasionally lost by jumping out onto the measured near the floor to avoid splashing floor.) Although the actual concentra- endrin into the test chambers. The bottle tions may have been slightly lower than containing the stock solution, and the the calculated concentrations in some pipette, were frequently wiped with a chambers, this does not introduce serious cloth saturated with pure 95-percent errors because the concentrations in TB-6 alcohol to remove any traces of endrin and TB-7 we're the maximum that the fish residue. could withstand. During the testing period the need for The small loss of endrin may have been determining the concentration of endrin a result of two factors: absorption by the in the test chambers became apparent. fish and loss because of aeration. The The best chemical test for endrin is sensi- manufacturers both of the plastic resin tive only to 5 p.p.b., but by bioassay it used to coat the reservoirs and chambers, was possible to determine concentrations and of the endrin, gave assurance that as low as 0.4 p.p.b. In November 1959, there would be no reaction between the during acute-toxicity testing, it was found endrin and the plastic: The concentration that 100 percent of the fish 20 to 40 mm. was changed in several chambers and, so standard length would die in concentra- long as they were washed thoroughly with tions as low as .4 p.p.b. in less than 4 days. soap and water, there was no residual in- Fish of similar sensitivity were used to secticide found in the water. determine the concentrations in the test TABLE 3.—Bioct88ay of test concentrations of the chambers by bioassay techniques. In chronic-toxicity tests acute tests in which the concentration was [Young bluntnose minnows were used as bioassay animals. Survival rates in known concentrations are taken from acute- better known, fish died in 1 to 2 days at testing results. See appendix table 1] 0.6 p.p.b. and in 2 to 3 days at 0.4 p.p.b. Number Size Average A 5-gallon glass jug with the top cut off Source of water of fish range, survival was placed so that the effluent from TB-7 mm. S.L. in days flowed through it. Later, this was also Effluent from TB-7 ------5 25-30 2.2 Do ------4 54-59 4.0 done with TB-6 and with the influent to Influent to TB-7 ------5 22-33 2.4 Effluent from TB-6 ------5 25-30 (I) each of these chambers. The results are Influent to TB-6 ------3 29-35 5.3 Known concentration, .6 p.p.b. 5 29-34 1.0 summarized in table 3. By comparison Do ------5 26-31 2.0 Known concentration, .4 p.p.b_ 5 25-34 2.4 with known concentrations, these data Do ------5 26-32 3.0 show that the concentration in TB-7 was approximately 0.5 p.p.b., while the concen- 1 No deaths. tration in TB-6 seems to have been some- In order to determine the effect of aera- what lower than 0.4 p.p.b. Since these tion on the loss of endrin from water, 9 11 liters of a 5-p.p.b. solution were made and TABLE 4.—Summary of the chronic-toxicity tests divided equally into two battery jars. on, bluntnose minnows One was aerated vigorously with five air Concentra- Initial Number Growth tion of endrin Chamber Sex size (mm. of days (mm.) jets for 72 hours; the other solution was S.L.) lived not aerated. At the end of 72 hours, five 0.9 p.p.b - - - - TB-8 F 46 9 ------fish were put into each jar. All fish died F 36 11 ------M 42 6 ------in 12 hours in the unaerated solution, but 42 3 ------36 5 ------none died within 48 hours in the aerated 36 4 ------one. This evidence indicates that aera- 0.75 p.p.b ...... TB-6 F 41 9 ------M 36 9 ------tion does remove at least some endrin. M 37 10 ------M 38 29 ------Aeration was very slow in the test cham- 38 9 ------bers and previously cited evidence indi- 0.5 p.p.b - - - - TB-7 F 50 Mi-,

F 36

cates that there was a minimal loss. , F 49 8 F 35 ...._ 23 ,

F 56 ggWOCI

Results F 57 1- F 60

F 45 .. Initial test concentrations were selected F 48 ,-, 3 ,

M 43 M 51 on the basis of 48-hour screening tests. , 5 ...„. -,- 1 0,0,4104-4M...... 11- M 45 , 5 Concentrations of 0.9, 0.5 and 0.1 p.p.b. 33 .00...0

38 Cla. were used for chronic testing. The sur- I I I I 0.4 p.p.b - - - - TB-6 F 59 1 194 0 F 39 196 13 vival times are listed in table 4. As fish F 45 10 ------F 39 '107 3 died in the various chambers, other fish M 53 '231 19 M 47 '196 8 were fin-clipped for identification and 55 11 ------37 8 ------were placed in the chambers to maintain 36 10 ------five animals per chamber. 36 12 ------0.1 p.p.b - - - TB-8 M 50 247 21 A summary of the growth data on fish M 49 '291 23 M 37 '291 32 living for extended periods is given in M 35 ' 291 33 table 5. Means are expressed as individual M 31 '291 35 0 p.p.b - - - - - CB-10 F 45 274 14 growth per fish per day. The mean for F 35 269 23 F 36 ' 291 19 each group was calculated as follows : M 69 '51 5 M 47 '291 20 =mean daily growth. 39 2 62 ------F ' Killed at the end of the test. T Total number of days that n. fish 2 Jumped out of test chamber. lived. G=Total growth of /1 fish. n=Number of fish in the group. TABLE 5.—Mean daily growth of bluntnose Discussion minnows [In millimeters. Number in parentheses is number of fish on Fish poisoned by endrin exhibited which the mean is based] symptoms clearly indicative of central- Mean daily growth of— nervous-system disorders. The first indi- Concentration Chamber of endrin cation of poisoning was an increase in ven- Males Females Both sexes tilation rate accompanied by rapid, jerky 0.5 p.p.b ------TB-7 - - - 0.047 0.050 0.049 movements of the body and fins. Almost (2) (3) (5) 0.4 p.p.b ------TB-6 - - - .063 . 032 . 047 simultaneously, an increased sensitivity to (2) (3) (5) 0.1 p.p.b ------TB-8 - - - .102 ------. 102 external stimuli would be noticeable. (5) ------(5) 0 p.p.b ------CB-10_ ___ .073 .067 . 069 Next, the fish would move to the surface, (2) (3) (5) and shortly thereafter begin turning 12 around and around rather slowly, fre- Because of the limited facilities, only quently swimming backwards. Convul- a very small number of fish could be used sions would follow, and between them the in the test; therefore, any conclusions fish would vibrate. The fins and body drawn from the data must be made with were not still for a second, and the fish caution. Small numbers were employed jumped out of the water frequently. Con- to avoid crowding with the hope that un- vulsions would become more severe and of der these conditions the fish would spawn. longer duration; finally, loss of equilib- The high growth rates of those fish in rium would occur. The fish often lived 0.1 p.p.b. stand out as the most significant several hours after loss of equilibrium, difference in table 5. This growth was and would make periodic darts around the greater than that of the controls, and be- chamber, often "barrel rolling" in the gan very early in the test period. Fish process. Breathing would then stop, but were not measured during the test period usually the heart would continue to beat because of the danger of injury. Unfor- for 10 to 15 minutes. The body would be tunately, all the fish in 0.1 p.p.b. were limp at this time, but as rigor mortis set males, and the higher growth rate may in, a characteristic bend in the spine would be due to this fact. It is true, however, occur at about the region of the anus, so that the females grew more than the males that the body formed an obtuse angle. in one out of three chambers in which both During the period of poisoning, there was sexes were present. This is an indication a progressive darkening of body colora- that the larger length-increase in 0.1 p.p.b. tion until the fish died. endrin was not attributable to the fact In weaker concentrations, the above that all were males. One might postulate symptoms were prolonged over several many reasons for this growth rate, but the weeks. Some fish were in the stage of hy- significant point is that these fish were able peractivity for many days. The two fe- to grow in 0.1 p.p.b. endrin and, as will males that lived 290 and 291 days in 0.5 be pointed out later, showed some indica- p.p.b. (table 4) were in such a state for 2 tion of reproductive activity. months. They became emaciated; their The fish in 0.1 p.p.b. endrin appeared to abdomens caved in so that they appeared be healthy, and behaved in a normal man- humpbacked. (This may have been an ner. They fed readily as soon as food was illusion due to the hollow abdomens.) introduced. When they were examined When food was dropped into the chamber, after being killed, there were many fat they attempted to feed, but usually they deposits, especially around the abdominal were unable to grasp the food, often miss- cavity. These fish were much fatter than ing it completely, as if their coordination any of the other fish tested, including the were impaired. For a period of several controls. Whether there was any relation weeks they ate little if anything. On June between this growth and the endrin is not 10, 1959, two fish were put into the cham- known. This effect should be tested fur- ber with them. The latter two died within ther with a larger number of fish. 11 days, but the original two females be- If we assume similar growth rates for gan to feed, and by July 1, had begun to both sexes, the combined mean growth in- gain weight and appeared normal. The crements can be compared. In 0.5 and behavior of these females during the first 0.4 p.p.b., the growth rates were lower 2 months was typical of many other fish than the control group by factors of ap- which had a prolonged period of poisoning proximately 0171 and 0.68 respectively. before death. These two concentrations were the ones in 13 which a number of fish died before five 90 percent of the surface area of a section specimens were found that would live in was blank space, which I assume repre- them, whereas the first five put into 0.1 sents lipid material removed in the alco- p.p.b. lived without visible signs of poison- hol dehydration process. Sections of liver ing. Reference to table 4 will show that from fish recently taken from the stream in 0.5 p.p.b. 9 out of 14 or 64.3 percent died, did not show such fat deposits. In view and that 5 out of 10 or 50 percent died in of the low liver-fat content found in fish 0.4 p.p.b. These percentages approximate in 0.4 p.p.b. (which were in the test cham- the 96—hour TL F,-, value's determined in the bers for shorter periods of time) and in acute-toxicity testing. DeWitt et al. fish taken directly from the stream, it (1960) stated that in pheasants the con- seems probable that this fatty vacuolation centrations of endrin producing acute and was caused by a high lipid content in the chronic effects were almost identical. A diet. comparison of table 4 and table 9 indi- The effect of diet and reproductive con- cates that the concentrations producing dition on stored fat is discussed by W. S. chronic and acute effects were very close Hoar in Brown's Physiology of the Fishes together for the bluntnose minnow. In (1957). According to this author, fact, those producing chronic effects are a changes in liver size due to a change in fat little higher. This is thought to be attrib- content, and correlated with the seasons, utable to the time of year, and to varia- tions in individual fishes. has been observed in several species. Ap- There were five deaths which occurred parently, a high fat content is normal for near the end of the chronic testing. A some fishes, but whether or not the condi- male in 0.1 p.p.b. appeared to be in breed- tion found in these fish was normal is not ing condition before losing equilibrium known. after 247 days in the test chamber. An One male in 0.4 p.p.b., one in 0.1 p.p.b., autopsy showed excessive stored fat in all and one in the control group had sperma- body parts, and sections of the liver re- tids in the testes and moderately well de- vealed almost complete fatty vacuolation veloped tubercles on the head. The other of the liver. When sections of the spleen, males had both primary and secondary kidney, heart, and brain were compared spermatocytes in the testes. Fighting with sections from normal tissue, no dif- was common among the males in 0.1 p.p.b., ference was noted. indicating sexual activity. Four females from the other test cham- bers died in the last 20 days of the test. All the females had ovaries in a state of They exhibited bulging eyes and rapid maturity comparable to fishes taken in the ventilation rates before death. The sec stream. Several females in TB-6, TB-7, tioned viscera appeared normal, the and CB-10 showed distention of the ab- ovaries were well developed, and there domen resulting from ovarian develop- were considerable fat deposits. ment. It appears, therefore, that endrin Sections of the liver and gonads of all in concentrations of 0.5 p.p.b. or less will fish which were living at the end of the not affect gonadal development in indi- test were prepared using the paraffin viduals which can tolerate such concentra- method and staining with hemotoxylin and, tions. More data are needed on this aspect eosin. Most of the fish, except those in of the subject before conclusions can be 0.4 p.p.b., had considerable lipid deposits reached. It must be emphasized that only in the cells of the liver. In some cases, a fraction of the fish were able to live in

620654 0-62-8 14 0.5 and 0.4 p.p.b. One fish in each of these lethal concentrations of endrin were pres- concentrations was slightly emaciated, but ent in a natural body of water, they could no other physical effects were noted. quite possibly cause the fish to leave their Little difference in activity was noted normal habitat niche and be subjected to between fish in 0.1 p.p.b. and the control predation and the effects of current. The group, but fish in 0.5 and 0.4 p.p.b. rarely bluntnose spawns under objects, and such stayed under the rocks. On the other an effect on the activity could upset the hand, the fish in 0.1 p.p.b. and in the con- spawning behavior and result in infertile trol chamber normally stayed under the eggs in this species. It is also important rocks during daylight hours. After sun- to bear in mind that even though the down, all fish rested quietly on the bottom gonads were maturing, spawning did not with little movement. Even fish having occur, so that it cannot be concluded that convulsions became quiescent after the endrin had no effect on spawning. These room was darkened. are matters that must be investigated be- The increase in activity mentioned above fore any conclusions can be drawn about could have a very detrimental effect on the the effects of sublethal concentrations on fish in their natural environment. If sub- reproduction.

CHRONIC-TOXICITY TEST ON GUPPIES

Methods and equipment random to the test chambers. Initially, This experiment was conducted simul- endrin concentrations of 0.9, 0.75, 0.5, 0.25, taneously with the chronic-toxicity test on and 0.1 p.p.b. were used. It was soon ap- bluntnose minnows, from April 11, 1959, parent that 0.9 and 0.75 p.p.b. were too to October 24, 1959, under the same condi- high, so a 0.4 p.p.b. concentration was in- tions. The water chemistry was the same cluded. The guppies used were produced as described earlier. The only differences from breeders obtained from a local trop- were in the number and size of test cham- ical-fish store. bers, and the endrin concentrations used. Results Guppy test chambers were 10 by 12 by 6 inches and held approximately 9 liters Before proceeding with the discussion at test level; the flow rate was 6 per of the data, it must be stated that the minute. Paired chambers (designated by guppy test data should be interpreted cau- the letters "A" and "B" after the number) tiously because the animals became dis- were fed by one reservoir and emptied eased, which probably affected some of the through a common constant-level cham- results. I first became aware of this dis- ber. Test chambers were half as large as ease early in September when the females those used for bluntnose chambers; two in the control group began to appear dark test chambers could be supplied for the in color, behave abnormally, and lose same period of time by one reservoir of the weight. They swam slowly about and same size as those used for the bluntnose stayed near the bottom, frequently with minnow tests. Six pairs of chambers (five the head lower than the rest of the body. pairs of test chambers and one pair for Immediately before they died, their ab- controls) were used in these tests. domens became distended, their scales The chronic-toxicity test was begun by stood perpendicular to the body, and the assigning five 1- to 7-day-old guppies at fish came to the water surface. The same 15 symptoms appeared in fish in other cham- Perhaps the most significant point to be bers and were followed by death. Exami- inferred from the data is that if diseased nation of the dead fish revealed that the fish were able to grow and reach sexual kidneys were enlarged and that there was maturity in various 6oncentrations from an edema of the body cavity. Axelrod and 0.5 to 0.1 p.p.b., then normal fish ought to Schultz (1955, p. 142) describe a disease survive and grow in at least those concen- called dropsy, which has symptoms similar trations, if not in slightly higher concen- to those observed in these test fish. The trations. cause of this disease is thought to be a primary virus infection accompanied by a Discussion of data secondary bacterial infection. Upon Inspection of the data given in table 6 checking back in my records, I found that for the two highest concentrations indi- some of the recorded descriptions of dying cates that the adults were significantly fish indicated that deaths may have been more tolerant than the young. The rea- due to the disease as far back as the first sons for this increased tolerance in the of August. The disease seemed to affect larger fish are not known. sexually maturing females first and then A comparison of mean survival times in the males. Because of this disease, the all concentrations shows an inverse rela- chronic study on the guppies had to be tion between concentration and survival. discontinued on October 24. 1959. If the mean survival times (tables 6 and The question arises how the results of 7) are plotted against the logarithm of the the guppy tests should be treated. Table concentration, the points fall along a 6 lists the fish which died in 0.9 and 0.75 straight line, which is often the case in p.p.b. Since the fish in these tests did not toxicological studies. live for long, and since these tests were completed at least a full month before the TABLE 6.—Survival of guppies in 0.9 and 0.75 disease was first observed, the results are p.p.b. ends-in probably reasonably accurate. At the time Survival time Mean sur- Initial age of each fish vival in these tests were conducted, all fish in all in days days chambers appeared healthy and normal, 0.9 p.p.b. concentration of en- and those in the lower concentrations of drin: 1-7 days ------3-3-3-3-2 - - - 2.8 endrin and in the control chambers were 1-7 days ------3-4-4-5-6 - - - 4.4 1-7 days ------4-4-4-4 - - - - 4.0 growing normally. 1-7 days ------4-4-4-4-4 - - - 4.0 Adult males ------7-9 ------8.0 Table 7 presents the data for the lower Adult females ------8-11-13 - - - - 10.6 concentrations of endrin tested. Here Mean for 1-7 day fish - - - - 3. 8 the disease no doubt was an important 0.75 p.p.b. concentration of en- drin: factor. For instance, in 0.25 p.p.b. there 1-7 days ------3-3-4-4 - - - - 3. 5 1-7 days ------11-16-18-18-18_ 16.2 is a rather uniform progression of deaths 1-7 days ------3-3-3-4-4 - - - 3. 4 1-7 days ------3-3-4-4 - - - - 3.5 with time; this is probably due to the dis- Adult males ------8-10-18-24 - - 15.0 ease, since the more usual pattern of deaths Adult females ------11-14-17-20_ _-- 15. 5 due to endrin, in both species, was numer- Mean for 1-7 day fish - - - - 6.6 ous deaths within the first few weeks and then long periods before other deaths oc- The comparison of mean survival does curred. In view of the disease factor, no not give a true picture of the toxicity of data have been given on growth rates, endrin, however. It was mentioned ear- other than to indicate the fishes which at- lier that most deaths occurred at the be- tained sexual maturity. ginning of the test period. This is es- 16

TABLE 7.-Survival of guppies in 0.5, 0.4, 0.25 and p.p.b. endrin. Above this threshold the 0.1 p.p.b. of endrin fish die, and below it the fish live with few Mean physical effects. Initial age Number of days each fish survival survived In The data are too scanty to draw any days** conclusions concerning a sexual difference 0.5 p.p.b. concentra- in tolerance. On the basis of a cursory tion of endrin: 1-7 days ------4-44-5-172* ------4.3 examination of the tables, it seems that Do ------4-5-8-6-6 ------5.4 Do ------6-6-7-8-8-9-9-9 ------7.8 there is no appreciable difference between Do ------5-6-7-7-8-9-9-9-10 - - - - - 7.8 Do ------5-5-6-6-7-7-7-11-14-28 - - - 9.6 the sexes. Do ------13-17-20-122 -122 -122 - 16.6 122*-122*-122*-122* The only data on reproduction were ob- Do ------8-11-12-13-18-24-24-25-41 - 21. 7 41 tained from the adults used in the tests. Do ------25 - 45 - 122* - 122* - 122* - 122*-122*-122*-122*-122*. 35.0 Some of the adult females were pregnant Do ------5-8-11-82-101 ------41.4 Do ------5-105-106e-120cr-193d' - - 105.8 when placed in the test solutions, but of Adult males - - - - - 25-26-24-154* ------25.0 Adult females - - - - 31-39-40-44-154*-154* - - - 38. 5 six females which bore young, only one Mean for 1-7 day bore a second litter. This female was in fish ------25. 5 0.4 p.p.b. concentra- 0.5 p.p.b. of endrin. There were six in tion of endrin: 1-7 days 6 - 10 - 15 - 30 - 32 - 32 - 20.8 137*-137*-137*. the first litter, all normal in appearance. Do 7-10-26-42-92-129 9 -137* 9 -- 51.0 In the second litter, 6 of the 12 had large Do 62 - 83 -87 - 101* 9 -1019- 77. 3 1019 - love - love - 101•e-101*e. yolk sacs and were probably premature. However, the rate of survival of these Mean 49. 7 0.25 p.p.b. concentra- young was about the same as for normal tion of endrin: 1-7 days 5-80-146e-163*c?-175*e---. - 77.0 ones. The young born from the other five Do 55-61-163 9-196 o 196 d'- - - - 93.0 Do 12 - 44 - 45 - 60 - 64 - 70 - 81- 61.8 83-97-122*. females (two in 0.75 p.p.b. and three in Mean 77.3 0.5 p.p.b.) had survival rates similar to 0.1 p.p.b. concentra- tion of endrin: normal young also. (No adult females 1-7 days 108 - 102 - 122* - 122 - 108 122*-122*-122*-122*-122*. were put into the two lower concentra- Do 41-70-1329-188 9 -196*e _ _ _ _ 108 Do 110 9 -118 9 -169 9 -Nee__ 132.3 tions.) Whether the effect of the endrin Mean 116.1 is on the egg or on the sperm is not known. 0 p.p.b. concentra- tion of endrin: Males frequently attempted to copulate 1-7 days 12-70 9 -159 9 -191 0*-196* - 108 Do 168 9 -170 9-177e-134e - - 162.8 with the females. Of the females which Mean 135.4 grew to sexual maturity in concentrations of endrin, most contained well-developed *Fish were killed at the end of the test. **Fish which were living at the end of the test are not included eggs when they died. (Death presumably in the calculation of mean survival. was due to the disease.) pecially noticeable in 0.5 p.p.b. of endrin. Fish were not measured during the tests, An inspection of the individual survival so their growth rates are not known. times in table 7 reveals that, in general, fish However, the two highest concentrations which survived 30 days were likely to sur- definitely retarded or stopped growth. vive much longer. This pattern would be Several fish in 0.5 p.p.b. grew very little. even more definite if the disease had not One was 126 days old when killed and been present. A discussion of the cumu- measured only 8 mm. standard length, this lative effects of endrin will be found in the size being no larger than that of many section beginning on page 29. normal guppies at birth. Since I have Such evidence, plus data on sublethal noted similar stunting in the past under effects presented, in a later chapter, indi- normal aquarium conditions, this may not cate that the threshold falls in a rather have been caused by endrin. narrow range of concentrations near 0.5 The symptoms of poisoning were very 17 similar to those described for bluntnose from observations of fishes put into test minnows, with one notable exception. solutions. Without exception, the fish Guppies went to the bottom, whereas that survived for long periods in 0.5 p.p.b. bluntnose minnows went to the surface. endrin went through a period of poisoning This was unusual, because bluntnose min- with symptoms like those described on nows are bottom feeders and guppies are page 12, for the females in TB-7. Like surface feeders. the female bluntnose, the guppies returned No data were obtained on increased re- to normal appearance and behavior after sistance in offspring of fish chronically ex- a period of time. Hyperactivity and posed to endrin. I planned to run tests on slight hypersensitivity were the only ab- offspring of fish raised in endrin to deter- normal types of behavior noticed. If after mine whether there was increased toler- the initial period endrin's effect is all or ance, but unfortunately no young were none, as I have postulated earlier, then obtained for these tests. The only avail- one would not expect tolerance to increase able data on this aspect of the subject come to any great extent.

ACUTE-TOXICITY TESTS ON BLUNTNOSE MINNOWS

Objective tinuous-flow systems hold these factors to The objective of this part of the inves- a constant minimum, and while these fac- tigation was to determine whether there tors are not eliminated in absolute terms, were significant differences between the they are eliminated as variables. If * various size groups in their acute toler- aeration is necessary, the continuous-flow ances to endrin. system has a distinct advantage.

Methods and equipment

Continuous-flow tests were used to deter- RESERVOIRS mine the endrin-toxicity values for various size groups of bluntnose minnows. The necessity of using a continuous-flow sys- tem as opposed to a static or nonflowing system is often questioned. Henderson and Tarzwell (1957) suggest a static test, probably because it is quicker and easier. When testing volatile substances there is a loss due to evaporation. If aeration is necessary (as it often is if dissolved oxy- gen is to be maintained at 5 p.p.m. or higher) the loss may become appreciable. PRESSURE HEAD 4, II TO When aeration is needed, the above-men- DRAIN tioned authors suggest a slow stream of TEST pure oxygen, but the use of oxygen is very CHAMBER bothersome. Other problems arising in static testing are the accumulation of excretory products, especially carbon di- FIGURE 4.—Diagram of continuous-flow system oxide, and the growth of bacteria. Con- used for acute testing. 18 Sometimes the amount of a toxic sub- bers were used, one as a control and four stance absorbed by the test animals in as test chambers. static tests becomes significant, and the A stock solution of endrin in alcohol continuous flow will offset this effect also. was made for each test chamber, using In this test I suspected that the differences the initial stock solution of 0.1 mg. of en- between size groups would be slight, if drin per milliliter of 95-percent alcohol. they occured at all. The continuous-flow Additional alcohol was added so that equal system was used, therefore, to minimize the volumes of the stock solutions added to lowering of the endrin concentration by each reservoir would give the desired con- absorption. centration. This volume was 0.00001 ml. The test chambers were 5-gallon glass of stock solution per milliliter of water. jugs cut off at a height of 12 inches. An This gave a uniform concentration of al- overflow hole was drilled 6 inches from cohol in water in each chamber. The the bottom ; the capacity at overflow level measured amount of solution was placed was approximately 9 liters. These test on a glass stirring rod, and lowered into chambers were placed in a large water the reservoir. The same volume of pure bath which was maintained at 22.2° C., 95-percent ethyl alcohol was added to the plus or minus 1° C. These chambers were control. Five fish were assigned to each supplied from a 5-gallon glass jug reser- testing chamber by the use of a table of voir placed on a rack above them. Water random numbers. was siphoned from the reservoir, and the The tests followed the basic routine flow was controlled by means of a small described by Henderson and Tarzwell glass jet. Constant pressure was main- (1957). Fish were held for at least 2 tained in the siphon by stoppering the res- weeks at 22.2° C. before testing. They ervoir and running the air tube to the were not fed for 48 hours prior to testing bottom of the jug. In this way the pres- nor during the test. Alkalinity, pH, dis- sure on the siphon was a head of water solved oxygen, and free carbon dioxide equal to the vertical distance from the end were measured at the beginning of the test, of the air tube to the end of the siphon. again when the fish began to die, and Pressure was independent of the water finally at the end of the test. The 96-hour level so long as the level was above the TL E values were determined by straight- end of the air tube. This system gave a line graphic interpolation. The concen- very constant pressure as the jug emptied. tration of endrin was plotted on a log scale All water lines were made of glass, with and percent survival on a linear scale. A flexible joints made of tygon tubing. line was drawn between the two points When the flow was 10 ml. per minute, one bracketing the 50-percent survival concen- 5-gallon jug held a 32-hour supply. tration, and the point on the concentration There were two jugs for each test cham- scale at which this line crossed the 50- ber, the siphon and air tube being trans- percent survival level was read as the ferred from one to the other. This made 96-hour TL,T, value. Percent survival was it possible to fill one jug 24 hours in ad- calculated as the percent living in each vance of its use, which was sufficient time chamber after exactly 96 hours. In all for the water to warm to room temperature cases fish were clearly alive or dead, and and for the free chlorine to escape. Each there was no problem with borderline test chamber was aerated at a uniform rate cases. with a glass air jet. This aeration also en- The chemical properties of the water sured adequate mixing. Five test cham- are listed in tables 1 and 8. All determi- 19

TABLE 8.-Chemical characteristics of the water TABLE 9.-Data and analysis of variance for in the acute-toxicity test chambers acute-toxicity tests on bluntnose minnows kill values, except pH, are expressed in p.p.m. These values A. 96 hour TL,7 values are based on 27 determinations] (Values expressed in p.p.b. endrin in water] Characteristic Range Mean Size Size Size group 1 group 2 group 3 Dissolved oxygen ------6. 4-9.1 7.5 (29.8 mm. (40.6 mm. (59.8 mm. Sum Mean Free carbon dioxide ------1.0-0 .5 mean mean mean Phenolphthalein alkalinity ------0-14.0 4. 1 S.L.) S.L.) S.L.) Methyl orange alkalinity ------11. 0-32. 0 23.2 Total alkalinity ------17. 0-33. 0 27.3 PH ------*7. 5-9.0 8.4 Test 1 ------.26 .35 .81 1.12 .37 Test 2 ------.28 .24 .43 .95 .32 Test 3 ------.28 .28 .46 1.02 .34 The high pH was present only for the first few minutes of the test until aeration reduced it. Sum ------.82 .87 1.40 3.09 - - - - - Mean ------.27 .29 .47 - - - - - 34 nations were made by use of techniques listed in Welch (1948). The unmodified B. Summary table for analysis of variance

Winkler method was used for dissolved Source Degrees of Sum of Mean oxygen, and the pH determinations were freedom squares square made colorimetrically as described earlier. Total ------8 0.0786 ------Group ------2 .0689 0.0345 All fish used were collected from the Test ------2 .0049 .0025 same pool in Rocky Fork Creek as were Discrepancy ------4 .0048 .0012 the animals used in the chronic-toxicity value for groups= 28.75. study. With few exceptions, all fish were Fol value with 2 and 4 degrees freedom =18.00. heavily parasitized by tapeworms ( Plero- cercoids of Proteoceph,a1u8) in the mesen- is based on five fish in each of four con- teries. These parasites did not noticeably centrations. Eight preliminary tests were affect the fish, all of which appeared very run before a suitable range of concentra- healthy. On four occasions large num- tions was found. bers of fish (200 to 500) were brought into The F value for between groups exceeds the laboratory, and each time not more the value of F at the 0.01-percent level, than 10 died during the acclimation pe- with two and four degrees of freedom, in- riod. These died within the first few days, dicating a probable difference in the toler- probably as a result of injury when cap- ance of various sizes. This difference, tured. No deaths occurred in the control while detectable, is not large enough to be chamber during the testing. The fish were of importance in setting safe levels for fed ground dog-chow checkers during the natural bodies of water. acclimation period before testing. Only No statistical test is available to deter- three size groups were available, with mine the effect of acclimation period on mean sizes of 29.8, 40.6, and 59.8 mm. the toxicity in this case. An inspection of standard length ; these are designated table 9 indicates that the variation within size groups 1, 2, and 3 in table 9. Fish any group is not nearly as great as between were preserved in 10-percent formalin and certain groups. As an example, the first were measured to the nearest millimeter, Tka- value listed under size group 3 is for standard length. fish held 14 days before testing. The third value in the same group is for fish Results and discussion held 57 days before testing. Both groups Table 9 summarizes the data and analy- were from the same collection of Janu- sis of variance for the acute tests ( Snede- ary 1, 1960. Reference to appendix table cor, 1946). Appendix 1 lists the raw data 1, tests S-11 and S-17, will show that the from the tests. Each 96-hour TL,T, value mean sizes of these two groups were 63.4 20 and 58.9 mm., standard length. The dif- periods. This does not appear to be the ference in TLin- values could be due. in part case, as mentioned above. to size differences. Since the maximum There are three other possible causes for variation of TL- values within any group this difference: (1) The fish used in the is not more than 0.09 p.p.b., the length of chronic-toxicity tests were not parasitized the acclimation period beyond, 14 days and may have been in better physical con- (recommended by Henderson and Tarz- dition. (2) The fish used, in the chronic- well, 1957) does not introduce variation of toxicity tests were from smaller popula- practical significance. tions in less crowded conditions. The The effect of temperature on toxicity stream population was extraordinarily was investigated by Iyatomi et al. (1958), large when the fish used in the acute- who found that a decrease of 20° C., from toxicity tests were collected. Langlois 27° C. to 7° C., caused the tolerance of 7- (1951) reported that bluegills living in to 8-centimeter carp to increase by a factor crowded conditions were less hardy. (3) of 28. It is doubtful whether this differ- The toxic effect of endrin may be affected ence would be as great in longer exposures. by the reproductive physiology. What- The effect of temperature should be in- ever the cause of the reduced tolerance, the vestigated further. effect should have been the same for all From table 4 and appendix table 1 it groups, and therefore the difference found is clear that the fish used in the chronic- between size groups should be a true one. toxicity test were more tolerant to endrin Since bluntnose minnows cannot be than those used in the acute-toxicity tests. sexed by external characters, except dur- At first it seemed most probable, that the ing the breeding season, any sexual dif- lower tolerances of the fish in the acute ferences in the tolerance to endrin must be obtained from the acute toxicity tests. tests were caused by physiological changes Sexually mature fish were used in five in the fish as a result of the colder weather, acute-toxicity tests, and the mean survival since all fish used, in the acute tests were time of both the 56 females and the 44 collected on December 6, 1959, and Jan- males used in these tests was 3.2 days. uary 1, 1960. If this were the case, one Treon, Cleveland, and Cappel (1955) and would expect the acute Thin values to others found that in mammals there is a change as the fish were held for longer sexual difference in tolerance to endrin.

EFFECT OF ENDRIN ON OXYGEN CONSUMPTION

Objective these oxygen-consumption tests. The basic plan was to measure the difference The objective of this part of the inves- in the dissolved oxygen in the effluent of tigation was to determine whether endrin two closed chambers, one containing a fish has any effect on the rate of oxygen con- and one without a fish. A reservoir and sumption. The large increase in the ven- constant-head chamber were used as a tilation rate indicated that the fish might water supply for the oxygen tests (fig. be using more oxygen. 5). The water was aerated with com- pressed air in the constant-head cham- Methods and equipment ber. Two 1-quart glass jars with stoppers A system similar to the one described served as test chambers. They were sub- by Keys (1930) was modified and used for merged in a water bath which was main- 21 FROM CONSTANT HEAD

" i TEST zi CONTROL CHAMBER =-1 CHAMBER II II Uii CLEAN -OUT —

EFFLUENT LINE

GLASS WOOL FILTER SAMPLE BOTTLE

FIGURE 5.—Diagram of apparatus used to measure oxygen consumption. tamed at 2,4° C. by a commercial aquarium the flow was measured for 30 seconds three heater. Temperature stayed within the consecutive times and the average of the range of 23° to 24° C. Each jar was placed three readings was used. If a flow of 6 on its side, and water was introduced into ml. a minute was used, the flow was the stoppered end through a siphon and re- measured one time for a period of 1 min- moved by another syphon leading from ute. Measurements of flow rate were made the top of the closed end. The test-cham- immediately after the sample for oxygen ber effluent was filtered through a glass- determination was taken. The dissolved- wool filter which was cleaned every 2 days oxygen concentration of the effluent from to prevent bacterial growths. Flow rates the test chamber was approximately 4 were regulated by means of small glass p.p.m. On three occasions it went down jets, and were measured with a 10-mi.- to 3 p.p.m. capacity graduathd cylinder. A flow rate Sample bottles, 125-ml. capacity, were of 6 ml. or 12 ml. a minute was used, de- placed under the glass jets at such a level pending on the size of the fish being tested. that when the bottle was overflowing only 1 If a flow rate of 12 ml. a minute was used, inch to -5 2. inch of the glass jet was be- 22 neath the surface of the water in the bot- The fish were weighed in water on a tle. In this way the sample bottles could beam balance to the nearest tenth of a be filled and left under the jets all the time gram before the test and were measured without reducing the flow or admitting and sexed after the test. All fish used for oxygen to the water. Sample bottles were oxygen-consumption tests were collected left under the water jets for a minimum of from the same location as those used in 4 hours before the dissolved oxygen was the other testing. They were held at 18° determined. This method was also advan- to 20° C. before being tested. tageous in that the sample could be re- After a fish had been weighed, it was moved before my presence disturbed the placed in one test chamber and left for a fish and caused its oxygen consumption to few hours, usually overnight. Then serial increase. The 125-ml. sample was repre- measurements of its oxygen consumption sentative of the preceeding 10 minutes, or were begun. Measurements were made 20 minutes, depending on the flow rate, and every 24 hours in tests 0-1, 0-2, 0-4, and therefore small increases of oxygen con- 0-5, and at about 12 hour intervals in sumption for short periods did not disrupt tests 0-6, 0-7, and 0-8. Disturbances the determinations. Methylene blue was were at a minimum in the basement where used several times to determine whether the tests were conducted. There were few the water in the sample bottles was being vibrations, because the floor was concrete. completely mixed by the water stream. It The actual formula used for determining was found that the stream of water from oxygen consumption was as follows: the jet went to the bottom of the sample Cx0.8xFx0.698 =ml. bottle and then moved outward and up- TV of oxygen per gram ward. By the same method it was found per hour. that good mixing was also taking place C is the difference in titration values in the test chambers. between test and control samples. The Winkler method, with a few minor 0.8 is a factor for converting titration changes, was used for dissolved-oxygen values to mg. determinations. All volumes of the solu- F is the flow rate in liters per hour. tions used were reduced by half, and N/100 0.698 is the factor to convert milligrams of sodium thiosulfate was used instead of oxygen to milliliters of oxygen un- N/40, in order to obtain greater accuracy. der standard conditions. One hundred milliliters of the sample were W is the weight of the fish in grams. measured with a volumetric pipette and titrated. Since oxygen consumption was Results based on the difference between the two Table 10 lists the mean consumption sample concentrations, no correction for rates for each fish tested. In the first four loss of oxygen due to addition of reagents tests, the fish were held in the test chamber was necessary. for 5 to 15 days in an attempt to get a In the first tests, the fish were fed constant consumption reading. There was ground dog-chow checkers daily through little change in consumption after the first a T-joint in the water line. In later tests, few hours, however, so in later tests the feeding was discontinued because it was period of time before endrin was intro- felt that it introduced too much variation duced was shortened. into the consumption rate. This was true Although with one exception the mean even in readings taken 24 hours after the oxygen consumption is higher in the pres- fish were fed. See figures 6 through 12. ence of endrin than under normal condi-

23

1.0 10 A PPB. ENDRIN ADDED ML. .8 ML 8 PER PER .4 PPB. ENDRIN ADDED GRAM .6 GRAM PER PER HOUR .4 HOUR .4

.2 2

4 8 12 16 20 4 8 12 16 20 DAYS DAYS

FIGURE 6.-Test 0-1. Oxygen consumption of a FIGURE 8.-Test 0-4. Oxygen consumption of a 0.7-gram female bluntnose minnow, 38 mm. 5-gram male bluntnose minnow, 73 mm. stand- standard length. Fish was fed in the evening ard length. Fish was fed approximately 24 and consumption was determined at 7 a.m. hours before consumption was determined at 7 a.m. 1.0 1.0 ML. .8 5 PPB. ENDRIN ADDED PER ML. .8 GRAM .6 PE R PPB. ENDRIN ADDED PER GRAM .6 HOUR .4 PER HOUR .4 2 .2 4 8 12 16 20 DAYS 4 8 12 16 20 DAYS FIGURE 7.-Test 0-2. Oxygen consumption of a 4.2-gram male bluntnose minnow, 70 mm. FIGURE 9.-Test 0-5. Oxygen consumption of a standard length. Fish was fed in the evening 2-gram female bluntnose minnow, 56 mm. and consumption was determined at 7 a.m. standard length. Fish was not fed. Consump- tion was determined at 7 a.m. tions, the difference is not over 0.06 ml. per gram per hour in any case. Usually 10 the difference between means is less than ML 8 3 PPB. ENDRIN ADDED PER the daily variation. GRAM 6 Figures 6 through 12 represent graph- PER HOUR 4 ically the oxygen consumption for all fish . tested. In test 0-8, figure 12, oxygen con- .2 sumption was measured every 4 hours for 4 8 12 16 20 16 hours after endrin was added, but no DAYS variation was found. FIGURE 10.-Test 0-6. Oxygen consumption of a Discussion 2.6-gram female bluntnose minnow, 60 mm. standard length. Fish was not fed. Consump- Harvey and Brown (1951) report that tion was determined at 7 a.m. and 6 p.m. compounds related to endrin (aldrin and dieldrin) cause a fivefold increase in increases as occurred were 25 percent or oxygen consumption when injected into less above normal. Fry, in The Physiology the cockroach. They found a latent period of Fishes (Brown, 1957), gives data on of 200 to 300 minutes before oxygen con- goldfish which show a fourfold increase in sumption reached the peak and then ta- oxygen consumption due to activity. The pered off until death. No such increase slight increase found here in fish exposed was noted in the bluntnose minnows; such to endrin would be most logically attrib- 24

I0 TABLE 10.—Mean oxygen-consumption rates of bluntnose minnows ML. .6 PER .5 PPB. ENDRIN ADDED Tests 0-1 and 0-2: Fed 12 hours before readings were taken. Test 0-4: Fed 24 hours before readings were taken. GRAM .6 Test 0-5: No feeding for 12 days before endrin was added. PER Test 0-6: No feeding, killed after 6 days in endrin. HOUR .4 Test 0-7: No feeding for 1 day before endrin was added. Test 0-8: No feeding for 9 days before endrin was added. .2 Normal Con- Weight Standard oonsump- sump- 4 8 12 16 20 Test No. in grams length in Sex tion Son in mm. ml/g/hr. endrin DAYS ml/g/hr. FIGURE 11.—Test 0-7. Oxygen consumption of a

38 F 0.35 0.37 B.)

3-gram male bluntnose minnow, 61 mm. stand- 70 M .22 .25 73 M .23 .28

ard length. Fish was not fed. Consumption 00 56 F .11 .11

C was determined at 7 a.m. and 6 p.m. 60 M .18 .19 0

61 M .20 .21

c=, 61 M .13 .16

ML. .8 PER sumption is surprising in view of the great GRAM .6 .3 PPB. ENDRIN ADDED PER increase in ventilation rate. No counts of HOUR .4 ventilation rates were feasible on small .2 fish with such high rates, but it appeared that the fish were ventilating at a maxi- 4 8 12 16 20 DAYS mum rate. Since the ventilation rate in- creased greatly, while the rate of oxygen FIGURE 12.—Test 0-8. Oxygen consumption of a consumption did not increase significantly, 3-gram male bluntnose minnow, 61 mm. stand- ard length. Fish was not fed. Consumption and since the ability of the fish to work was determined at 7 a.m. and 6 p.m. was not affected, it seems that the apparent respiratory distress must be due to a uted to the increased movement due to stimulus other than a deficiency of oxygen tremors and convulsions. in the tissues. The effect could be attrib- The insignificant increase in oxygen con- uted to accumulation of metabolites.

EFFECT OF ENDRIN ON SWIMMING OF BLUNTNOSE MINNOWS

Objective and method under them, so that the fish could not rest The object of this experiment was to de- in currentless water. termine whether endrin reduces the ability Test fish were specimens from the of fish to carry on muscular exercise. The highest concentrations of the acute-toxic- fish were forced to swim against a current ity tests in which four or five of the fish in a current tray (Mount, 1959) . The tray had survived. Control fish were taken was 2 feet wide by 4 feet long by 6 inches from the control chamber. Both groups deep, with a divider in the center. A of fish had not been fed for 6 days before paddle wheel powered by a pump motor testing. All testing on both sexes was at propelled the water around the tray. The 22.2° C. The test fish were distinguished tray was modified slightly in that the cor- from the control fish by clipping opposite ners were rounded, thereby increasing the pelvic fins on each group. The paddle current to approximately 9 inches per sec- wheel was started and a periodic check ond. Eddies and slow areas were elimi- was made to remove any dead or exhausted nated by placing bricks on the bottom fish. 25 Results convulsions. One died after 24 hours, but The first two tests were run on fish 30 the remaining four test fish and the con- to 43 mm. standard length, which had been trols lived for 48 hours. These fish ranged in a concentration of 0.2 p.p.b. endrin for in size from 56 to 59 mm. standard length. 4 days. In both tests the test fish were Discussion tremulous. In the first test one fish died after 3.5 hours. The other four test fish, It is my opinion that the three fish which the five controls, and all five test fish and died in the last two test runs would have the five controls in the second test swam died if they had been in quiet water. One against the current for 48 hours, at the fish which died in the first test may have end of which the test was discontinued. died from exhaustion or may have been in- In the third test, four fish from the test jured while being handled. In tests 3 and chamber containing 0.4 p.p.b. endrin were 4, the fish stopped having convulsions as used. They were at the surface and in the soon as the current was started. In test 3 latter part of the convulsion stage when I turned the current off momentarily after the test was begun. They ranged in size about 30 minutes, and they began having convulsions again. from 49 to 75 mm. standard length. Five The only conclusion which can be drawn hours after the current was started one is that in acute poisoning endrin has little died, and after 8 hours another died. The effect on the ability of fish to swim against other two "test fish and the five controls a current. However, it is possible that if were alive and normal after 48 hours. the fish could have been tested in a much The last test was run on five fish from swifter current in which even normal ani- the test chamber containing 0.4 p.p.b. en- mals became exhausted, the test fish would drin. The fish were in the early stages of have become exhausted first.

PORT OF ENTRY INTO AND DISTRIBUTION OF ENDRIN IN THE FISH'S BODY Objective steam is condensed and collected, most of the endrin is found in the first 200 ml. of The extreme toxicity of endrin to fish causes one to wonder what the specific ac- distillate. tion of endrin might be. The symptoms With slight modification, this method appearing during the period of poisoning was employed successfully. A 1,500-ml. indicate that the effect is mediated through Erlenmeyer flask was connected to a Lie- the nervous system. In an effort to gain big condenser. The material or solution to insight into this problem, an experiment be extracted was placed in the flask. A was conducted to determine the point of fraction of the volume was collected as dis- entry and the movement of endrin through tillate and the bioassay was run in the the body. distillate. After experimenting with solu- tions of known concentrations, it was Methods found that collection of 250 ml. of dis- Davidow and Sabatino (1954) describe tillate, regardless of the original volume, a method of steam distillation by which gave satisfactory recovery. By collecting very low concentrations of endrin were re- 250 ml. of distillate from 1,000 ml. of solu- coverable. When a solution or material tion known to contain 0.5 p.p.b. endrin, it containing an endrin residue is boiled in was possible to get a concentration of 1.5 an aqueous solution or slurry, and the p.p.b. or more in the distillate, as deter- 26 mined by a fish bioassay. This represents "of a different chemical nature" occurred a recovery of 75 percent or more. in the kidneys. Several bluntnose minnows (with a total For the study of endrin's entry and dis- weight of 8.1 grams) which had died in tribution within the fish, large carp of ap- 0.5 p.p.b. endrin in 5 days were cut into proximately 40 cm. standard length were small pieces and put into a flask with 500 used. They were "snagged" from the nil, of water. Two hundred and fifty Scioto River at Columbus with treble milliliters of distillate were collected, and hooks. Only those not seriously injured a small bluntnose minnow was put into were used for testing. They were main- this solution. At the same time, another tained for 2 or 3 days before being put into small bluntnose minnow was placed in 250 endrin. In the test, a 30-gallon porcelain ml. of a solution containing a known con- tub was used for each fish. Twice daily centration of 2.0 p.p.b. endrin. The fish the tub was partially drained and 10 gal- in the distillate died in 11 hours and the lons of fresh solution were added. The one in the known concentration in 15 temperature was approximately 20° C.; no hours, which indicated a concentration of attempt was made to control it. The test over 2.0 p.p.b. in the distillate. Another numbers in table 11 represent the follow- fish was added to the distillate, and it died ing exposure times to endrin : in 10 hours. Other distillations were made CONCENTRATION EXPOEURE TIME with similar results. Fish not exposed to IN p.p.b. IN DAYS endrin were also distilled, but no deaths Test 1 0.0 0.0 occurred when the distillates were assayed. Test 2__ ------10. 0 2.5 From this evidence it was concluded that Test 3 5.0 4.0 Test 4 5.0 5.0 the technique was reliable. It was also Test 5 2.5 28.0 more sensitive than the best chemical test, which is only accurate to 5 p.p.b. Bio- Test 1 served as a control to establish assay also has the advantage of being a that no toxic substances other than endrin test of toxicity, which would therefore be were carried over into the distillate. This positive even if the endrin were slightly fish was killed and distilled immediately altered chemically within the fish or by after being captured. The fish in tests 2, the distillation. A chemical test might 3, and 4 died ; the fish in tests 1 and 5 were give negative results if the endrin were killed. chemically altered (Klein et al., 1956). Immediately after death the fish were No attempt was made to determine opened. The various organs were re- whether the endrin had been chemically moved, weighed in water, mascerated, and altered. One purpose of this testing was placed one at a time in the flask. Five hundred milliliters of tap water were to estimate how much endrin accumulates added to the flask and distilled until 250 in the body, as an index to the potential ml. of distillate had been collected. The danger of such fish in the food chains and distillate was clear but had a smell of fish to man. So long as the compound is toxic, oil which disappeared after 5 to 10 hours it makes little difference what its specific at room temperature. All distillates were form may be. Some insecticides are left at room temperature for approximate- changed into different compounds. For ly 12 hours before being assayed. example, aldrin is changed to dieldrin and Each distillate was diluted with tap stored in the fat (Bann et al., 1956). water so that a 250-ml. aliquot represented Kunze and Laug (1953) found that endrin the extract from 10 grams of tissue. Thus 27 if there were 100 grams of tissue, 2,250 ml. found to be much more variable. The lat- of tap water would be added to the 250 ml. ter took longer for some fish in 10 p.p.b. of distillate, the solution mixed, and 250 than for others in 5 p.p.b. Davidow and ml. aliquot used for the bioassay. In this Schwartzman (1955) found the same vari- way errors in weighing tissue were mini- ation in their bioassays with goldfish, and mized, and since each 250 ml. sample rep- they too used convulsions as the end point. resented the extract from 10 grams of tis- Tarzwell and Henderson (1956) used loss sue, the times of exposure to the extract of equilibrium as an end point. Klein et until convulsions set in ( and therefore the al. (1956) used both convulsions and loss concentrations) could be compared di- of equilibrium as end points. In this in- rectly. Ten grams was selected as the vestigation, loss of equilibrium was more basic weight because this was the usual variable than onset of convulsions. minimum weight of the smaller organs. The assay was carried out in 1-quart Results glass jars at room temperature with very Table 11 summarizes the results of the gentle aeration, for a 48-hour period. Two bioassays on the corp organs. In tests 2, hundred and fifty milliliters of various 3, and 4, heart-spleen-blood (combined), known concentrations were placed in sim- digestive tract, kidney, and liver ranked ilar containers for comparison. In the highest in endrin concentrations. In ad- fourth test, enough endrin was added to dition, in test 5 the brain contained a each distillate and to the known concen- measurable amount of endrin, as did the trations to make a 0.5 p.p.b. solution. In fat. In test 5, blood was tested sepa- the fifth test, enough was added to make a rately and contained measurable amounts 1.0 p.p.b. solution. Endrin was added for of endrin, but heart-spleen ranked higher. two reasons : (1) It speeded the test but did not affect the results, since concentra- TABLE 11.-Bioct88ay of extracts of carp tissues tions were determined by comparison. using bluntnose minnows [Tests were conducted for a 48-hour period in the extract from 10 (2) It minimized the effect of individual grams of tissue, except as noted. "Neg." indicates that no con- variability which is less significant at vulsion occurred in 48 hours] higher concentrations. All fish used in Hours until the onset of convulsions of the tests were bluntnose minnows, 20 to bluntnose minnows

30 mm. standard length, from the lot col- Test 1 Test 2 Test 3 Test 4 1 Test 5 lected on January 1, 1960, for use in the Known concentra- acute-toxicity tests. As shown by the tion of endrin: 15 p.p.b ------2.75 - - - - - acute-toxicity testing (appendix table 1), 10 p.p.b ------3.0 4. 5 3.0 3.25 5 p.p.b ------4.0 6. 5 4.0 6.25 fish in this size-range showed little varia- 2.5 p.p.b ------7. 5 7.0 10. 75 Carp tissue extracts: bility in tolerance. Daphnia and insect Blood ------8.25 3 ------Brain neg. neg. neg. 1 neg. 5 6.25 larvae were not sensitive enough for assay- Fat ------neg. 10. 3 neg. neg. 8.75 Gills ------neg. neg. neg. neg. 12.25 ing the concentrations. One fish was Heart, spleen, blood neg. 5. 25 25. 5 6 19. 0 7 3. 25 placed in each container for the test. Digestive tract_ _ _ _ neg. 7.25 13.0 40.0 10. 75 Kidney ------neg. 6.25 48.0 19.0 4.00 The onset of convulsions proved to be L iver ------neg. 7.0 48.0 19.0 7.75 Muscle ------neg. 9.0 neg. neg. neg. the most satisfactory end point. The Ovary ------neg. - - - - - neg. neg. neg. Testes ------10.25 - - - - - time from the beginning of exposure until 0.5 p.p.b. endrin added. the onset of convulsions was quite uniform 1 p.p.b. endrin added. 8 Includes some fat in tests 1, 2, 3, and 4. and predictable within a known concen- 4 8.5 g. tissue. 5 3.2 g. tissue. tration (see table 11), whereas that from 9.5 g. tissue. 7 6.2 g. tissue. the beginning of exposure until death was 8 6.5 g. tissue. 28 In previous tests, blood was included with and oral tissues, although some also these two tissues so that the total weight crosses the less permeable body sur- was approximately 15 grams; therefore, face . . . The only other water intake in the distillate had to be diluted by one-half. fresh-water fishes is that accompanying or This would tend to mask out a high con- bound in the food swallowed." There is centration in the spleen or heart. reason to doubt this statement, at least in Some error is introduced by the varia- the case of some species of fresh-water bility of the assay animals, and by the fish. Both Ellis (1937) and Allee and small size of the sample. The four tests Frank (1948) have reliable data to the do present a consistent picture, however, contrary, that fresh-water fish may drink and for this reason are considered reliable. sizable amounts of water. The latter workers showed this to be the case in gold- Discussion fish. Since the carp is closely related to On the basis of the data in table 11, it the goldfish, it is not unreasonable to as- may be inferred that endrin is being car- sume that carp may also drink water, even ried away from the place of entry into the when not feeding. Allee and Frank give body by the blood. Of the organs assayed, a complete summary of the literature on excepting the gills, those having a rich water intake by fresh-water fish. blood supply contained the most endrin. Further evidence that the digestive tract The high concentrations found in the kid- may be the port of entry is furnished by ney suggest that endrin may be excreted Iyatomi et al. (1958) who found that the through that organ. Other investigators 2-day larvae of carp are drastically differ- (previously cited) have found significant ent from 6-day-old fish in their sensitivity damage in the kidney, further suggesting to endrin. They found that the 96-hour the importance of this organ in the excre- TL E value for carp decreased from 10.7 tion of endrin. p.p.m. of endrin at 2 days of age to 0.046 If the kidney is the organ excreting en- p.p.m. at 6 days of age. This represents drin from the body, the high concentra- a change of toxicity in the order of 250 tion found in the digestive tract presents times. It is quite probable that during a puzzling question. The gills gave this 4-day period the mouth became func- mostly negative results. (The one posi- tional and the larvae began to drink water tive value may have been due to the endrin containing endrin. added to the distillate.) It seems logical It is also possible that endrin is excreted that endrin would have been detected in by the intestine and so appeared in the dis- the gills if it were entering there. It is tillate of the digestive tract. Since the more probable, in view of the evidence, intestine was not emptied daily, there that the digestive tract was absorbing the could have been an accumulation of ex- endrin. This hypothesis accounts for the creted endrin in the lumen. negative results of the gill tissue and the The positive values for the brain in test positive results of the intestinal tissue. 5, and for fat in tests 2 and 5, are in agree- Since the carp were not fed during the ment with the known storage of chlori- exposure, endrin could not have been taken nated-hydrocarbon insecticides in those up from the water swallowed in connec- tissues (Sherman and Rosenberg, 1953; tion with food ingestion. The statement Kunze and Laug, 1953; Treon, Cleveland, appears in Brown's Physiology of the and Cappel, 1955; and Bann et al., 1956). Fishes (1957) that "Most of the water is Nothing has been said up to this point taken in through the semipermeable gill concerning the absolute concentration 29 present in the carp tissues. One must be dose of endrin. The hazard would be fur- very cautious in estimating concentrations ther reduced in the case of man, since he based on bioassay, especially with a small normally eats only the muscle tissue, sample. However, Klein et al. (1956) which gave negative results in three out and Davidow and Sabatino (1954) have of four tests. Most cooking methods predicted concentrations on the basis of would also evaporate much of the endrin, two goldfish in 250 ml. of distillate. although Hunt and Bischoff (1960) found They found the goldfish to give as reliable that baking fish in foil did not remove results as assays using 100 flies. DDD. Perhaps an idea of the order of magni- Muscle from a carp exposed to 2.5 p.p.b. tude can be obtained from the available endrin for 4 weeks was fed to two male data. Only in test 5 (table 11) is the time mice for 3 weeks. Neither mouse showed until convulsions in the extract close weight loss or any symptoms of poisoning, enough to that in a known solution to war- indicating that endrin was not present in rant an actual estimate of the concentra- concentrations high enough to be damag- tion of the extract. The time until con- ing. Of course, little is known about the vulsions in heart and spleen extract was effects of very minute amounts consumed the same as in 10 p.p.b. of endrin. If we by several generations of animals, and it is assume 1 ml. of water to weigh 1 gram, not safe to draw a conclusion based on this there must have been a total of approxi- small sample. mately 2.5 micrograms of endrin present. In summary, this experiment showed Since the tissue weight was 6.2 grams, this highest concentrations of endrin in the represents a concentration in the tissue of liver, heart-spleen, and kidney. These approximately 400 p.p.b. The concen- findings agree with work cited on page tration in the kidney would be slightly less, 4, in which experiments with rats as would the concentration in the brain- chronically exposed to endrin revealed that spinal cord tissue. This represents a level these organs were the ones evidencing 160 times more concentrated in the tissue tissue damage or storage of endrin. The than in the water in which the fish was liv- presence of endrin in the brain and fat in ing. The evidence indicates that endrin test 5 agrees with the work of other in- is actively taken up by some process at vestigators who found that chlorinated some point or points in the body. hydrocarbons are stored in the brain and It appears that if these estimates are fat. However, the cited investigation on even approximately reliable, there would mammals did not show any damage or ac- be little danger to mammals eating ex- cumulated endrin in the intestine, whereas posed fish. These fish, eaten in normal the carp intestine contained measurable quantities, would contain less than a lethal amounts.

MODE OF ACTION OF ENDRIN ON ANIMALS When a substance is as toxic to an ani- Henderson, Pickering, and Tarz well mal as endrin is to fish, the question arises (1959) and Iyatomi et al. (1958) that the what the specific effect might be. The con- range of the TIA,T, values among species of centration toxic to fish is usually a few fishes tested is not over 5 parts per billion. parts per billion, whereas the level toxic to Endrin's toxicity to mammals is also less insects is in the range of 100 or more parts than its toxicity to fish. Ely et al. (1957) per billion. It has been found also by reports 1.5 mg./kg. to be toxic to cattle, 30 and Treon, Cleveland, and Cappel (1955) mans, in which the optic disk swells as a determined that 43.4 parts per million of result of pressure in the central nervous endrin in food was toxic to rats. system, is used to detect diseases which Hartley and Brown (1955) reported cause an increase in pressure on the brain that endrin had no effect on the cholin- in man. The pressure is transmitted along esterase level in the American cockroach. the optic nerve, collapses the blood vessels, Morrison and Brown (1954) found that and causes the optic disk to swell because compounds related to endrin had no effect of the blocked circulation. on the cytochrome oxidase level in the I attempted to find papilledema in fish same animal. Nelson (1955) states that by fixing and measuring optic disks of serum alkaline phosphatase levels were normal fish and of fish which had died in higher in test than in control rats fed 100, 10 p.p.b. endrin. Four fish were placed in 50, 25, -5, and 1 p.p.m. of endrin in their 10 p.p.b. endrin and four of similar size food. (In human beings an increase in the were placed in tap water. As the fish in serum alkaline phosphatase levels may in- endrin lost equilibrium they were put into dicate liver disturbances.—Hawk, Oser, A. F. A. fixing solution. When the fourth and Summerson, 1954). All rats were hy- test fish was fixed, the control fish were persensitive to stimuli, and those fed 100, fixed also. After 12 hours in the fixative, 50, and 25 p.p.m. endrin in the food had the eyes were removed and imbedded in convulsive spasms. Roeder (1948) found paraffin. Serial cross-sections 15 microns that technical chlordane (related to endrin thick were made and stained with eosin. chemically) had no effect on synaptic Measurements were made with an ocular transmission of impulses in the nerve micrometer. preparation of Periplaneta and caused no The results of this experiment are shown neurologic disturbances in mammals. in table 12. Two measurements were Harvey and Brown (1951) stated that taken on each eye; the minimum thickness all insecticides which increase oxygen con- of the two middle sections was measured. sumption are known to be nerve poisons. No significant difference was found be- He used chlordane as an example of one tween the treated and the control groups. which is not a nerve poison and which It is possible that shrinkage due to fixa- does not increase oxygen consumption tion obliterated any swelling which may significantly. have been present. A clue to the possible mode of action was TABLE 12.—Optic-disk measurements of the blunt- found during the testing in this investiga- nose minnow tion. The symptoms—convulsions and [Two sections of each disk were measured. Numbers represent loss of equilibrium—indicate disturbances ocular-scale divisions in which 1 division equals 6.8 microns] in the central nervous system. There was Normal fish Fish in 10 p.p.b. endrin always an increased ventilation rate but, as was pointed out earlier, the oxygen con- Left eye Right eye Left eye Right eye sumption did not increase. In many of 22 28 33 24 20 26 31 25 the young guppies which died in endrin, 40 28 30 20 45 27 33 25 the skull burst and the brain protruded. 24 20 22 24 21 19 21 20 This indicated pressure on the central 28 25 35 25 nervous system. Such pressure would also 31 23 30 25 stimulate the respiratory center, produce .7, length of fish=48 mm. .7, length of fish=46.8- mm. standard length standard length .7, thickness of optic disk= i; thickness of optic disk= convulsions, and cause loss of equilibrium. 26.7 ocular divisions 26.4 ocular divisions A condition called papilledema in hu- 31 The carp used in the investigation of the were replaced with tap water. These five entry and distribution of endrin in the fish went through the symptoms of endrin body were examined with an ophthalmo- poisoning in a reverse order, and at the scope while convulsions were in progress, date of this writing, 3 months later, appear but it was very difficult to see into the eye normal and healthy. This type of reaction and I could not tell anything about the indicates that endrin does not cause irre- optic disk. Carp were difficult to hold versible damage. still, and the examination was unsatis- The available information concerning factory. the mode of action of endrin on animals When fish showing symptoms of poison- ing are placed in pure water, they usually does not permit any conclusion to be recover rapidly. In one instance, 10 blunt- drawn about the specific effects of endrin. nose minnows were placed into a contin- This present investigation furnishes little uously renewed solution of 0.3 p.p.b. new information other than that endrin When five fish had died and the other five does not appear to increase oxygen con- were having convulsions, the flow was sumption, nor does it appear to cause irre- stopped. Two days later approximately versible damage as a result of short half of the 9 gallons in the test chamber exposures.

SUMMARY

Investigations of the chronic toxicity of daily growth rates in 0.5, 0.4, 0.1, and 0 endrin to bluntnose minnows, Pinzephales p.p.b. endrin were 0.049, 0.047, 0.102 and notatu8, and guppies, Lebi,stes reticulatus, 0.069 mm., respectively. The toxic levels were conducted for periods of 10 and 6 for chronic exposures were not greatly months, respectively. Continuously re- different from those toxic in acute ex- newed solutions were employed. posures. Fish in 0.5 and 0.4 p.p.b. ex- Approximately 35 percent of the blunt- hibited much more activity than those in nose minnows survived for extended 0.1 p.p.b. and in the control aquariums. periods in 0.5 p.p.b. endrin in water, and The gonads in both sexes in all test cham- approximately 50 percent survived 0.4 bers showed increased activity and ap- p.p.b. All bluntnose minnows placed in peared to be ripening about as rapidly as 0.1 p.p.b. lived for extended periods; all those of fish taken from the stream. His- died in 0.75 and 0.9 p.p.b. within a 29-day tological examination showed fatty vacuo- period. lation of the liver cells in many of the fish, Symptoms of endrin poisoning in the including control fish. Such vacuolation bluntnose minnows were characteristic of is thought to be due to the high fat con- disturbances in the central nervous sys- tent of the diet fed. No tissue damage tem: hyperactivity, increased ventilation was found in the brain, heart, kidney, or rate, convulsions, loss of equilibrium, and spleen. death. The symptoms lasted for longer A chronic-toxicity test on guppies was periods at lower concentrations. Some conducted simultaneously with the blunt- fish were in a state of hyperactivity and nose minnow test. Because of a disease of increased ventilation rate for as long as the kidneys which was apparently unre- 2 months and then returned to normal lated to the presence of endrin, tests on condition while still in endrin. Mean the guppies were discontinued after 6 32 months. The effects of endrin on the gup- the acute-toxicity tests (collected in the pies were essentially the same as those on winter). This may have been due to one the bluntnose minnows. or more of the following : (1) the stage of Pregnant female guppies, with one ex- the reproductive cycle, (2) parasitism, ception, never had more than one litter of (3) seasonal temperatures and related young after being placed in 0.75 and 0.5 physiological conditions, (4) crowded p.p.b. endrin in water. In one case, a sec- conditions in the stream resulting in less ond litter of 12 was produced and included hardy fish. 6 young with large yolk sacs, presumably Endrin had no apparent effect on oxy- born prematurely. All young born from gen consumption of fed and nonfed fish of females in endrin, including the premature either sex. Endrin did not seem to affect ones, survived about as long as guppies the ability of fish to swim against a cur- born from parents not in endrin. No sex- rent of water. ual difference in survival was found. Evidence was found which indicates Adults were usually more tolerant than that endrin enters the bodies of carp young. through the intestines. It probably is One major difference in behavior was carried by the blood to other parts of the found during the period of poisoning; body. The liver, intestine, spleen, and guppies went to the bottom and bluntnose kidney of carp contained the highest con- minnows went to the surface. The nor- centrations of endrin in 2- to 5-day ex- mal resting and feeding positions of these posures. Blood, fat, and brain tissue also species are just the reverse. contained measurable amounts after a 28- In acute-toxicity testing of bluntnose day exposure. All concentrations in carp minnows, fish with a mean standard tissues were based on a fish bioassay of length of 59.8 mm. withstood 0.2 p.p.b. distillates collected by steam distillation. more endrin in 96 hours than did fish of The bursting of the skull and subsequent mean standard lengths of 29.8 and 40.6 protrusion of the brain of young guppies mm. The fish used in the chronic-toxicity which died in various concentrations of test (collected in March) proved to be endrin indicated that endrin may cause more tolerant to endrin than those used in pressure in the central nervous system.

CONCLUSIONS

Endrin does not appear to be cumulative It appears that the increase in activity in its effect on fish subjected to chronic ex- caused by endrin in concentrations of 0.5 posures. There was a narrow range from and 0.4 p.p.b. might be of greatest signifi- 0.4 to 0.5 p.p.b. in which fish showed some cance in natural populations through dis- ruption of spawning behavior and loss of weight and greatly increased activ- displacement from their normal habitat ity. No effect was found below this con- niche. centration. Although no reproduction On the basis of the data, endrin appears occurred in the bluntnose minnow, the to enter the body through the intestine as gonadal development indicated that repro- a result of the fish's drinking water. Very duction might have taken place if the test young fish are resistant to endrin, prob- had been continued. Concentrations as ably because their mouths are not func- low as 0.5 p.p.b. appeared to prevent re- tional and the endrin does not get into the production in guppies. intestine. 33 There is evidence of a slight accumula- secticide,s on all aquatic forms. Not one tion of endrin in carp tissue during a 28- such study was found in the literature. day exposure. The low concentration Investigations are needed to determine found in the muscle tissue would probably which organisms in the aquatic food not be harmful to human beings eating chains are most sensitive to various pol- exposed fish. Cooking would also reduce lutants. More work should be done to the concentration. determine the residual life of these pol- It appears on the basis of this investiga- lutants in the bottom sediments and in the tion that endrin would not harm fish seri- tissues of animals. Until this information ously so long as a concentration which is is known, the danger involved in con- not lethal in 30 days is not exceeded. tinuous use of insecticides cannot be de- Endrin is extremely toxic to fish, and ap- termined. There is little knowledge at plication rates would have to be very low. the present time to indicate what danger However, if endrin will effectively control there is to human beings, either directly pests at an application rate which would or indirectly, from continuous use of be safe for fish, its use would be preferable insecticides. to that of a compound less toxic to fish but Finally, it will be necessary to gain a with greater cumulative effects. better understanding of normal fish physi- There is a very serious need for chronic- ology before the true danger of insecti- toxicity studies of the effects of many in- cides to fish can be properly assessed.

LITERATURE CITED

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Ely, R. E., L. A. Moore, R. H. Carter, and B. A. Klein, A. K., E. P. Laug, J. F. Tighe, L. L. Ram- App. sey, L. C. Mitchell, Frieda M. Kunze. 1957. Excretion of endrin in the milk of cows 1956. Biological assay of endrin in leafy fed endrin-sprayed alfalfa and technical vegetables and its confirmation by paper endrin. Journal of Economic Entomology, chromatography. Journal of the Associa- vol. 50, No. 3, pp. 348-349. tion of Official Agricultural Chemists, vol. Fitzwater, W. D. 39, No. 1, pp. 242-253. 1958. Endrin for orchard mouse control. Kunze, Frieda M., and Edwin P. Laug. Mimeo prepared in office of District Agent, 1953. Toxicants in tissues of rats on diets U.S. Fish and Wildlife Service, Agricultural containing dieldrin, aldrin, endrin and i140- Experiment Station, Lafayette, Ind. drin. Federation of the American Society Harrington, Robert W., Jr., and William L. of Experimental Biologists, Federation Pro- Bidlingmayer. ceedings, vol. 12, No. 1, part 1, p. 339. 1958. Effects of dieldrin on fishes and inverte- Langlois, T. H. brates of a salt marsh. Journal of Wildlife 1951. Much ado about the sea lamprey. Ohio Management, vol. 22, No. 1, pp. 76-82. Conservation Bulletin, vol. 15, No. 5, pp. Hartley, J. B., and W. A. Brown. 29-32. 1955. The effects of certain insecticides on Laug, Edwin P. the cholinesterase of the American cock- 1946. A biological assay method for deter- roach. Journal of Economic Entomology, mining 2,2 Bis (p-cliloropheny1)-1,1,1 tri- vol. 48, No. 3, pp. 265-269. chloroethane (DDT). Journal of Pharma- Harvey, G. T., and A. W. A. Brown. cology and Experimental Therapeutics, vol. 1951. The effects of insecticides on the rate 86, No. 4, p. 324. of oxygen consumption in Blatella. Can- Morrison, P. E., and A. W. A. Brown. adian Journal of Zoology, vol. 29, pp. 42-43. 1954. The effects of insecticides on cyto- Hawk, Philip B., Bernard L. Oser, and William chrome oxidase obtained from the American H. Summerson. cockroach. Journal of Economic Entomol- 1954. Practical physiological chemistry. Mc- ogy, vol. 47, No. 5, pp. 723-730. Graw-Hill Book Company, New York, Mount, Donald L Toronto, London. 13th ed. 1439 pp. 1959. Spawning behavior of the bluebreast Henderson, Croswell, and Clarence M. Tarzwell. darter, Etheostoma camurum (Cope). 1957. Bioassays for control of industrial ef- Copeia, vol. 3, pp. 240-243. fluents. Sewage and Industrial Wastes, vol. Nelson, S. C. 29, No. 9, pp. 1002-1017. 1955. Effects of the insecticide endrin upon Henderson, Croswell, Q. H. Pickering, and C. M. the alkaline phosphatase level of the albino Tarzwell. rat. Utah Agricultural College Monograph 1959. Relative toxicity of ten chlorinated Series, vol. 3, No. 3, pp. 79-80. hydrocarbon insecticides to four species of Price, John W. fish. Transactions of the American Fish- 1931. Growth and gill development in the eries Society, vol. 88, No. 1, pp. 23-32. small-mouthed black bass, Micropterus Hunt, Eldridge G., and Arthur I. Bischoff. dolomieu, Lacepede. Contribution No. 4, 1960. Inimical effects on periodic DDD appli- Franz Theodore Stone Laboratory, Ohio cations to Clear Lake. California Fish and State University Press, Columbus, Ohio. Game, vol. 46, No. 1, pp. 91-106. Roeder, K. D. Iyatomi, Kisabu, Tamotsu Tamura, Yasuo I. 1948. Note on the effect of chlordane on syn- Tazawa, Isao Hanyu, and Syotaro Suriura. aptic transmission. Armed Services Re- 1958. Toxicity of endrin to fish. Progressive search Development Report on Rodent Fish-Culturist, vol. 20, No. 4, pp. 155-162. Control No. 1. Keys, Ancel B. Rosen, A. A., and F. M. Middleton. 1930. The measurement of the respiratory ex- 1959. Chlorinated insecticides in surface wa- change of aquatic animals. Biological ters. Analytical Chemistry, vol. 31, No. 10, Bulletin, vol. 59, No. 2, pp. 187-198. pp. 1729-1732. 35

Sherman, M., and M. M. Rosenberg. Treon, Joseph F., Frank P. Cleveland, and John 1953. Acute toxicity of four chlorinated di- Cappel. methanonaphthalene insecticides to chicks. 1955. Toxicity of endrin for laboratory ani- Journal of Economic Entomology, vol. 46. mals. Journal of Agricultural and Food No. 6, pp. 1067-1070. Chemistry, vol. 3, No. 10, pp. 842-848. Snedecor, George W. Welch, Paul S. 1946. Statistical methods. Iowa State Col- 1948. Limnological methods. Blakiston Com- lege Press, Ames, Iowa. 4th Ed. 485 pp. pany, Philadelphia and Toronto. 381 pp. Tarzwell, Clarence M., and Croswell Henderson. Young, L. A., and H. P. Nicholson. 1956. Toxicity of dieldrin to fish. Transac- 1951. Stream pollution resulting from the use tions of the American Fisheries Society, vol. of organic insecticides. Progressive Fish- 86, pp. 245-257. Culturist, vol. 13, No. 4, pp. 193-198. Appendix-RAW DATA FROM TESTS APPENDIX TABLE 1. -ReimIts of acute-toxicity testing on bluntnose minnows ("Juvenile" indicates fish too young to sex easily; "A" indicates fish living at end of test]

Standard Number Standard Number length of days length of days (mm.) lived (mm.) lived rest 8-9: Test 8-12: 0.8 p.p.b. endrin: 0.8 p.p.b. endrin: Juvenile ------26 1 Male ------37 1 Do ------27 1 Do ------37 1 Do ------30 1 Female ------39 1 Do - - - - 31 1 Do ------36 1 Do ------32 1 Do ------33 1 0.6 p.p.b. endrin: 0.6 p.p.b. endrin: Juvenile ------33 1 Female ------35 1 Do ------24 1 Do ------38 1 Do ------33 1 Do ------41 2 Do ------34 1 Do ------41 2 Do ------29 1 Male ------36 2 0.4 p.p.b. endrin: 0.4 p.p.b. endrin: Juvenile ------25 2 Male ------35 2 Do ------29 2 Female ------39 2 Do ------30 2 Do ------40 2 Do ------28 3 Do ------38 A Do ------34 3 Do ------38 A 0.2 p.p.b. endrin: 0.2 p.p.b. endrin: Juvenile ------32 3 Male ------30 A Do ------22 A Female ------31 A Do ------28 A Male ------40 A Do ------28 A Female ------43 A Do ------31 A Do ------43 A Mean size ------29.3 ------Mean size ------37.5 ------rest 8-10: Test 8-13: 0.8 p.p.b. endrin: 0.5 p.p.b. endrin: Juvenile ------27 1 Female ------45 2 Do ------1 Male ------46 2 Do ------3428 1 Female ------42 2 Do ------26 2 Male ------40 2 Do ------34 2 Female ------37 2 0.6 p.p.b. endrin: 0.4 p.p.b. endrin: Juvenile ------26 1 Female ------35 2 Do ------26 2 Male ------40 2 Do ------27 2 Do ------39 2 Do ------32 2 Female ------33 2 Do ------31 3 Male ------39 3 0.4 p.p.b. endrin: 0.3 p.p.b. endrin: Juvenile ------28 3 Male ------49 3 Do ------27 3 Female ------36 3 Do ------28 3 Male ------39 3 Do ------31 3 Female ------40 4 Do ------32 3 Do ------39 4 0.2 p.p.b. endrin: 0.2 p.p.b. endrin: Juvenile ------27 A Female ------42 A Do ------28 A Do ------42 A Do ------30 A Male ------47 A Do ------33 A Female ------41 A Do ------34 A Male ------39 A Mean size ------29.4 ------Mean size ------41.0 ------rest S-11: Test 8-14: 0.8 p.p.b. endrin: 0.5 p.p.b. endrin: Male ------2 Male ------66 3 Do ------6566 2 Female ------59 3 Female ------60 2 Do ------56 3 Male ------58 2 Male ------55 3 Do ------81 4 Do ------54 3 0.6 p.p.b. endrin: 0.4 p.p.b. endcrin: Male ------71 2 Female ------51 4 Do ------70 3 Male ------75 A Do ------64 3 Female ------53 A Do ------59 4 Male ------56 A Do ------70 A Female ------49 A 0.4 p.p.b. endrin: 0.3 p.p.b. endcrin: Male ------68 A Male ------71 A Do ------70 A Female ------63 A Do ------60 A Male ------59 A Female ------58 A Female ------57 A Male ------57 A Do ------51 A 0.2 p.p.b. endrin: 0.2 p.p.b. endrin: Male ------65 A Female ------53 A Do ------57 A Do ------53 A Female ------59 A Male ------61 A Do ------58 A Female ------55 A Do ------55 A Do ------52 A Mean size ------63.4 ------Mean size ------57. 1 36 37

APPENDIX TABLE 1.—Results of acute-toxicity testing on bluntnose minnows—Continued

Standard Standard Number length length of days (mm.) (mm.) lived

Test S-15: Test S-16—Continued

0.5 p.p.b. endrin: MNVDCOM N 1-- 0 0.3 p.p.b. endrin: Juvenile ------Male ------41 4 Do ------Do ------44 4 Do ------Female ------43 4 Do ------A Do ------C. I 40

Do ------Do ------52 A , 0.4 p.p.b. endrin: Ne0VDD,C0 0.2 p.p.b. endrin:

Juvenile ------0 Male ------40 A A Do ------00 Female ------36

Do ------. 0 CD Do ------41 A Do ------Male ------39 A

Do ------Do ------50 A

0.3 p.p.b. endrin: MMCVCOM Mean size ------Juvenile ------en 43. 2

Test Do ------0 S-17:

Do ------. N 0.5 p.p.b. endrin: Do ------Male ------57 3

Do ------Do ------62 3

. Do ------64 3 NVDCOMM

0.2 p.p.b. endrin: .,M

Juvenile ------C.- N Do ------60 4 Do ------Do ------65 A

Do ------. 0.4 p.p.b. endrin: .1 4

Male ------69 A Do ------..0

Do ------Do ------62 A 1 Female ------58 A Mean size ------30. 7 Unrecorded ------A Test S-16: 0.5 p.p.b. endrin: 0.3 p.p.b. endrin: Female ------53 Female ------53 A Do ------41 Do ------55 A Do ------49 Do - - - - 57 A Do ------36 Male ------65 A Do ------38 Female ------54 A 0.4 p.p.b. endrin: 0.2 p.p.b. endrin: Female ------50 Female ------55 A Male ------40 Male ------56 A Female ------42 Do ------65 A Do ------41 Female ------55 A Do ------52 Do ------52 A Mean size ------58. 9 ------

APPENDIX TABLE 2.—Results of tests on oxygen consumption in the bluntnose minnow [Consumption is expressed in ml. per gram per hour. All testing was done at temperatures between 23° and 24° C.] Test 0-1: A 0.7-gram female, 38 mm. standard Test 0-2: A 4.2-gram male, 70 mm. standard length. Consumption was measured at 7 lengh. Consumption was measured daily a.m., 12 hours after the fish was fed. at 7 a.m., 12 hours after feeding. Consumption in 0.4 Consumption in 0.5 Normal consumption p.p.b. endrin Normal consumption p.p.b. endrin 0. 366 0. 674 0. 210 0. 184 .320 .469 .215 .230 .320 .435 .223 .267 .366 .366 .209 .329 .366 .213 . 244 Fish died .352 .366 .220 . 366 .279 Mean__ .253 Mean__ .348 .366 . 215 . 160 . 215 . 320 . 188 . 275 . 310 Mean__ . 222 . 354 . 443 Test discontinued

Mean__ . 366

38

APPENDIX TABLE 2.-Results of tests on. oxygen consumption in the bluntnose minnow-Continued

Test 0-4: A 5.0-GRAM male, 73 MM. standard Test 0-6: A 2.6-grain female, 60 aim. standard length. Consumption was measured at 7 length. Consumption was measured twice a.m. daily, 24 hours after feeding. daily at 7 a.m. and at 6 p.m. The fish was Consumption in 0.4 not fed. Normal consumption p.p.b. endrin Consumption in 0.3 0. 135 0. 285 Normal consumption p.p.b. endrin .181 .219 0.176 0.217 .198 .305 .185 .209 .221 .316 .193 .217 . 272 Fish died . 217 . 266 Mean__ . 185 . 194 . 186 Mean__ . 281 . 180 .218 .173 .280 .184 .175 .184 .272 .147 .299 .137 .169 .249 Mean__ 0.187 . 327 . 266 Test 0-7: A 3.0-GRAM male, 61 MM. standard . 270 length. Consumption was measured twice . 150 daily, at 7 a.m. and 6 p.m. The fish was not fed. Consumption in 0.5 Mean__ . 230 Normal consumption p.p.b. endrin 0. 181 0. 219 .191 .207 Test 0-5: A 2.0-GRAM female, 56 MM. standard . 228 . 184 length. Consumption was measured daily . 197 . 233 at 7 a.m. The fish was not fed. . 224 Fish died Consumption in 0.4 Normal consumption p.p.b. endrin 0. 181 0. 073 Mean__ . 204 Mean__ . 211 .133 .104 . 182 . 151 Test 0-8: A 3.0-GRAM male, 61 MM. standard . 074 Fish died length. Consumption was measured twice . 044 daily, at 7 a.m. and 6 p.m. Fish was not fed . 236 Mean__ . 109 and was starved seven days before the test . 042 was begun. Consumption in 0.5 . 021 Normal consumption p.p.b. endrin . 127 0. 142 0. 116 .094 .132 .136 .106 .107 .144 .130 .130 .164 .134 .241 . 080 Mean__ . 128 Fish died

Mean__ . 113 Mean__ . 160