_w—_——-——‘ w— -‘— THE TRANSLOCAHQN CF DDT FROM HYWD‘SOELES MS EYE AfiCEéWLATION AND DEGRADATION IN THE BIOTA flint: for flu Dogma 6-5 M. 5. MBCHlG-AN STATE UNIVERSE? Ronaid Cliffcrd Waybrant I969 ABSTRACT THE TRANSLOCATION OF DDT FROM HYDROSOILS AND ITS ACCUMULATION AND DEGRADATION IN THE BIOTA BY Ronald Clifford Waybrant The manner and degree of DDT translocation from a pond bottom material into the pond biota was studied. Three levels of DDT in the hydrosoil were prepared and examined, each in a separate pond and each one representing a level of DDT which can be found in the natural environment. The amount of DDT translocated from the hydrosoil was dependent upon the insecticide concentration in the hydrosoil. A logrithmic relationship between the concen- tration of DDT in the hydrosoil and the accumulation of DDT in the fish, microcrustaceans, and periphyton was found. DDD became the major degradation product of DDT and was transported throughout the aquatic environment. DDE was relatively unimportant as a breakdown product of DDT. The initial displacement of DDT from the bottom ma- terial was into the water, which resulted in an accumulation of DDT by the aquatic organisms as they removed DDT from the water. The insecticide accumulation in the periphyton Ronald Clifford Waybrant depends upon the concentration present in the water. It was found that a DDT concentration of ten ppm in the hydrosoil will cause constant mortality of the fish. DDT and its metabolites were continuously recycled in the aquatic environment and were not inactivated after three months. THE TRANSLOCATION OF DDT FROM HYDROSOILS AND ITS ACCUMULATION AND DEGRADATION IN THE BIOTA BY Ronald Clifford Waybrant A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Fisheries and Wildlife 1969 ACKNOWLEDGMENTS I wish to express my appreciation to Dr. Robert C. Ball for his advice and guidance during my graduate study, and for the opportunity he gave me to learn when I was hired while still an undergraduate to assist in the research pro— jects at the Lake City Experiment Station. I also wish to thank Jerry Hamelink, who taught me much about the biology of pesticides while I was learning the mechanics of pesticide analysis from him. Thanks are also due to Will Gross and other graduate students whose comments and ideas were helpful in interpreting the results of this study. .I am grateful for the financial assistance from the Michigan State University Agricultural Experiment Station Funds. This project was also supported in part by the Federal Water Pollution Control Administration Training ii INTRODUCTION . TABLE OF CONTENTS PREPARATION OF THE STUDY PONDS Artificial Ponds . . . . . Bottom Material . . . . . Water . . . . . . . . . Periphyton . . . . . . . Fish and Microcrustaceans Temperature . . . . . . . SAMPLING SCHEDULE . . . . . . ' WTHODOLWY o o o o o o o o 0 water 9 O O O O O O O O O FiSh . O O O O O O O O O Microcrustaceans . . . . . Periphyton . Hydrosoil . . RESULTS AND DISCUSSION . . . Water . . . . Fish . . . . Microcrustaceans Periphyton Hydrosoil . General Results SUMMARY . . . . LITERATURE CITED . iii Page 10 11 12 13 14 15 15 16 17 18 19 22 22 27 52 61 73 80 85 87 Table LIST OF TABLES The average concentrations of insecticide in the water of all ponds at each sampling period . . . . . . . . . . The average insecticide content of the fish samples taken at each sampling period from each pond . . . . . . . . . . . . . . . The average insecticide content of the re- cycled fish at each sampling period from each pond . . . . . . . . . . . . . . . . The insecticide content in the micro- crustaceans from all ponds . . . . . . . . The average insecticide content of the peri- phyton samples taken at each sampling period from all ponds (parts per million). The average insecticide content of the peri— phyton samples taken at each sampling period from all ponds (micrograms per square meter) . . . . . . . . . . . The insecticide content of the recycled periphyton from all ponds . . . . . . . . The insecticide concentrations in parts per million, based on the dry weight of the bottom material, in each of the artificial ponds . . . . . . . . . . . . . . . . . The linear regression data for the log-log plots with the parts per million total insecticide in the hydrosoil on the x axis, and the parts per million total insecticide of the fish, periphyton, and microcrustaceans on the y axis . . . . iv Page 25 29 48 53 63 64 72 74 81 Figure LIST OF FIGURES A photograph, looking to the east, showing the four farm ponds at the Lake City Experimental Station and the four arti- ficial pools set up in Pond A . . The average concentrations of insecticide in the water showing and comparing the fluctuations and changes with time in each pond . . . . . . . . . . . . . The insecticide makeup of the fish in the study ponds based on the individual per- centages of the total insecticide that DDT, DDD, and DDE comprise at each sampling period . . . . . . . . . . The semi-log plot of the average total insecticide concentrations in the fish at each sampling period of each study pond The semi-log plot of the average DDT concen- trations in the fish at each sampling period of each study pond . . . . . . . The semi-log plot of the average DDD concen- trations in the fish at each sampling period of each study pond . . . . . . . The semi-log plot of the average DDE concen- trations in the fish at each sampling period of each study pond . . . . . . . . The plot of the water temperatures in pond H in degrees F. from July 13 to July 20 is compared to the numbers of dead fish and fish with DDT induced convulsions . . The initial uptake of insecticides that oc— curred in the recycled fish is compared to the initial uptake of insecticides by the original fish . . . . . . . . Page 24 32 35 37 39 41 46 51 Figure Page 10. The change and fluctuation of insecticides in the microcrustaceans of study pond H With time 0 0 O O O O O O O O O O O O O O O 56 11. The change and fluctuation of insecticides in the microcrustaceans of study pond M With time 0 O O O O O O O O O O O I O O O O 58 12. The percentage of the total insecticide that DDT and each of its metabolites com- prise in the microcrustaceans at each sampling period . . . . . . . . . . . . . . 6O 13. The composition and changes with time of insecticides in the periphyton of pond L on a parts per million basis and on a total micrograms per square meter basis . . 66 14. The composition and changes with time of insecticides in the periphyton of pond M on a parts per million basis and on a total micrograms per square meter basis . . 68 15. The composition and changes with time of the insecticides in the periphyton of pond H on a parts per million basis and on a total micrograms per square meter basis . . 7O 16. The insecticide concentrations, in parts per million, based on the dry weight of the bottom material in each of the arti- ficial ponds are compared on a semi—log scale . . . . . . . . . . . . . . . . . . . 76 17. The percentage of the total insecticide that DDT and each of its metabolites com- prise in the bottom material of the treated ponds . . . . . . . . . . . . . . . 79 18. The log-log plots of the parts per million total insecticide of the fish, periphyton, and microcrustaceans, against the parts per million total insecticide in the bottom material . . . . . . . . . . . . . . 83 vi INTRODUCT ION Since the introduction of DDT as an insecticide during WOrld War II, the use of organic pesticides has in- creased enormously. Such compounds when applied to foliage, soil, or water courses may be expected to move with rainfall and runoff into lakes and streams, provided the compounds are sufficiently resistant to degradation by physical and biochemical action. The toxicity of organic pesticides to fish and other aquatic organisms has been documented in the literature. Graham (1960), Warner and Fenderson (1962), and Kirswell and Edwards (1967) reported fish kills caused by forest spraying with DDT. Young and Nicholson (1951) cite stream fish kills and relate them to applications of organic insecticides. Sublethal amounts of pesticides may result in egg or fry mortality (Burdick_gt-al., 1964). Hitchcock (1965), Schoenthal (1964), and Jones and Moyle (1963) indicated popu- lation changes of aquatic invertebrates resulting from en- vironmental treatments with DDT. Croker and Wilson (1965) studied the kinetics and effects of DDT in a tidal marsh ditch and found that DDT was dispersed to all aspects of the environment. DDT was removed from the water in five days, yet it was still trans- ported to and from the vegetation, fish, and sediments for the next ten weeks. In the water DDT is attracted to surfaces due to its hydrophobic nature (Bowman,l964), and this attraction can result in an accumulation of DDT on pond and stream bottoms. Hindin, May, and Dunstan (1964) demonstrated that the Entiat River contained 18,000 times more DDT in the bottom Isoil than in the flowing water. When the bottom is sandy or pervious the pesticide may penetrate into or through the substrate. If the bottom is rich in organic material the pesticides can leave the water and adsorb on the organic surface or in the organic matter. DDT may attach to silt and suspended solids, organic or inorganic, in the water (Fredeen, Arnason, and Berck,l953). DDT in water is taken up by living organisms and is effectively removed from the water. Microscopic plants and higher aquatic vegetation accumulate large amounts of DDT from water (Meeks and Peterle,l965). Aquatic animals both large and small, similarly tend to remove DDT from water as has been described by Cope (1965) in his work with C-14 labeled DDT. DDT is strongly adsorbed from an aqueous dispersion by soils. Weidhass, Bowman, and Schmidt (1961) showed that from two different volumes of water the same percentage loss of C-14 labeled DDT, 78%, occurred in twenty-four hours. The distribution changes, with time, of DDT at 0.02 ppm in the water was not affected by large differences in pH, total solids, or chloride content. Harris (1959) found that DDT was biologically active in wet clay or mineral soils and that wet muck or organic soils reduced the bioactivity of DDT. Bowman, Schecter, and Carter (1965) studied the behavior of DDT in various soil types and found that DDT was not leached from soils with water, whether the soils were mineral or organic. DDT has been found to persist in various soils at least one to three years after application (Edwards, 1964). Since DDT has been found to be biologically active in wet soils, to persist for long periods of time in all soils, and since it is not readily leached from the soil by water, the hydrosoils of lakes and streams could essentially be an important reservoir of DDT. The bioactivity of DDT in the hydrosoils was indi— cated by Hickey, Keith, and Coon (1966) in a study of pesti- cides in a Lake Michigan ecosystem. They found DDT in the bottom sediments of Green Bay averaging about 0.014 ppm and a composite Lake Michigan bottom sample to have 0.05 ppm DDT. Crustaceans had 0.41 ppm and alewives had 3.4 ppm of DDT. The concentrations in the organisms were believed to arise from the DDT levels in the bottom sediments. Hence, bottom sediments of an aquatic ecosystem may be an important reservoir of active pesticides. This study was designed to investigate the potential and degree of DDT transport to the biota from a wide range of hydrosoil concentrations. The available information indi- cates that DDT is accumulated in the bottom sediments, and presents a potential hazard to aquatic organisms. Three levels of DDT in the hydrosoil were used, each in a separate pond and each representing a level of DDT which may be found in the hydrosoils of a natural environment. This required the use of four ponds which would be insecti- cide free with the exception of the hydrosoil, which would be homogeneous in composition and insecticide distribution. Four artificial ponds were used, one as a control and three with prepared DDT concentrations in the hydrosoil. Three levels of DDT were chosen, 0.05 ppm, 1.0 ppm, and 10.0 ppm. These were picked to span a wide range of insecticide contamination of natural soils and to check the possibility of differential transport of DDT to the pond biota which is dependent on the DDT concentration in the hydrosoil. Fish, periphyton, microcrustaceans, and water were added to the four artificial ponds and sampled along with the hydrosoil at regular intervals for a period of twelve weeks. The artificial ponds were located at the Fisheries and Wildlife field laboratory on the Michigan State Uni- versity Agricultural Experiment Station at Lake City, Michi- gan. A small creek, Mosquito Creek, has been dammed and is used to maintain water levels in four farm ponds which have been previously designated as Ponds A, B, C, and D (Sohacki, 1968). The artificial ponds were placed in Pond A and the water for these ponds was taken from the adjacent pond, Pond B. All fish, periphyton, and microcrustaceans used in this study were taken from these four farm ponds. The analyses for DDT and its metabolites in all samples were made by gas chromatography, using a MicroTek 220/DP floor model, dual column gas-liquid chromatograph. The instrument was equipped with two columns packed with 3% SE-30 on 60-80 mesh Gas Chrom Q. The column operating temperature was 1800 C and the carrier gas flow was 70 ml/minute of nitrogen. One column is connected to a Dohrman microcoulometric unit which was operated at a range of 200 ohms, an oxygen flow of 25 ml/minute, and a combustion tube temperature of 830° c. The other column leads to a parallel plate electron capture detector. This detector utilizes a pulse mode power supply, which has a pulse rate of 100, a pulse width of one microsecond and a power supply of 50 volts. The argon- methane scavenger gas flow through the detector is 60 ml/minute. Two Honeywell Brown Electronik recorders, equipped with disc chart integraters were used with both analysis systems. PREPARATION OF THE STUDY PONDS Artificial Ponds The artificial ponds were circular plastic lined swimming pools, ten feet in diameter and four feet deep. These four pools were placed off shore in a large farm pond (Figure l). The farm pond kept the artificial ponds the same temperature and the artificial ponds received an equal amount of light and rain, yet each pond remained a closed system within itself. The four ponds had been used to study the tranSport of DDT the previous summer which necessitated a cleanup of these ponds before they could be used again. This was done by removing all the water, the top layers of sand and organic matter from the bottom, and scrubbing down the sides of the pond. No detergent was used as this could leave residues that interfere with operating a gas chromatograph. It was assumed that any DDT that had adsorbed onto the sides of the ponds the previous summer would be tightly held to the plastic and would not interfere with this study. However, because the ponds had been used before, the pond that was the control the previous summer was used as a control the second summer. Figure l. A photograph, looking to the east, showing the four farm ponds at the Lake City Experiment Station and the four artificial pools set up in Pond A. . .. 4 . r. . .. 0| '0 .. II |. .4 . i u .4: . . . 1‘1: 9. . l t a, '. ‘ a: . {I . . 0’1“}!!! . .. d. v 0 VI! 0‘ 0 I. t, o O n- -34.!) .I 1.. y} I A wooden hexagonal frame was built for the ponds to keep the periphyton sheets (described in the methods section) suspended in the water. When the sheets were attached, a crisscrossed mesh was formed which had a grid size of one foot and nearly filled the pond. This grid was numerically designated as rows and columns to facilitate taking random water and bottom samples. Bottom Material Three levels of DDT based on a dry weight ratio of DDT to sand were used: 0.05 ppm, 1.0 ppm, 10.0 ppm of DDT. The lowest level, 0.05 ppm, was selected to approximate some of the low contamination levels found in natural hydrosoils. The two remaining levels are considerably higher, but are still reasonable levels to study as DDT concentrations with- in this range and higher have been reported in the literature. It was thought that a ten fold difference in DDT concen- trations between the two high level ponds would be high enough to show different transfer rates of DDT to the biota of a farm pond. The formulation and the addition of the coated hydro- soils was approached ina similar manner for each pond. Each pond received two inches of bottom sand which amounted to six hundred kilograms of dry sand. Ungraded mortar sand was graded just prior to adding the insecticide to give a more uniform bottom material and to dry the sand. The sand was 10 assumed dry when it flowed easily and showed no tendency to stick together. The addition of DDT to six hundred kilograms of sand posed a real problem. A homogeneous distribution of DDT was needed and six hundred kilograms of sand had too large a volume to be mixed all at once. To overcome this problem a base mixture was prepared. The required amount of DDT was dissolved in one liter of acetone and a few milliliters of food-coloring, and mixed with forty kilOgrams of sand. The food-coloring dyed the mixture a dark green and gave a good indication of how well the acetone mixture had mixed with the sand. The base mixture was then added to the rest of the sand in a mortar box and mixed well with rakes and a hoe. The food-coloring in the base mix indicated the homogeneity of the final mixture. Because of the large volume of sand only one third of the sand was mixed at a time. The final mixture was added to the ponds with a wheelbarrow and shovels. Water A plastic sheet was laid on the sand to prevent ex- cessive stirring of the sand when the water was added. The water was taken from an adjacent farm pond and pumped into the artificial ponds using a gasoline powered Homart pump. The water was not filtered in any way to remove plankton or 11 algae because a natural pond situation was being sought. The adjacent farm pond had never been treated with DDT and pretreatment samples showed it to be free of insecticides. No turbidity was noticed in the ponds after a few hours. The water levels in the artificial ponds were main- tained three inches above the water level of the pond in which they were setting. Twice during the summer the water levels in the artificial ponds evaporated down to nearly equal the water level of the outside farm pond, and each time about three inches of water from the adjacent farm pond was added. The extra water in the artificial ponds was deemed necessary to keep them stable. Periphyton Periphyton was added to the ponds as the primary pro— ducer utilizing the available solar radiation and dissolved nutrients. It can easily be grown on artificial substrates and removed for measurement (Kevern, 1962). In this study, plastic sheets with a large surface area were used to obtain the large amounts of periphyton required for analysis of its DDT content. The periphyton sheets were added immediately after filling the ponds with water. The periphyton sheets were clear vinyl plastic strips, thirty-one inches long and ten inches wide. They had one half cup of clean sand stapled in a fold of the lower three inches of the sheet to hold it 12 vertical in the water. These sheets were seeded in the adjacent farm pond for ten days prior to adding them to the study ponds. A total of sixty periphyton sheets were added to each pond in a mesh like pattern, one foot by two feet, which encompassed the whole pond. The sheets were numbered consecutively and then randomly sampled. Midway through the summer ten more periphyton sheets were set in the study ponds, without seeding them first. This was done to check the possibility of DDT recycling through the ponds. Fish and Microcrustaceans The addition of fish and microcrustaceans completed the preparation of the study ponds. The fish were added fourteen hours after the water was added to the ponds. Four hundred green sunfish, Lepomis cyanellus Rafinesque, between one and three inches long, were seined from the adjacent ponds and added to the artificial ponds, one hundred fish per pond. About five hours after the water had been added to the ponds, several tows with a plankton net were made and the microcrustaceans were added to the ponds. That same evening microcrustacean light traps (Baylor and Smith, 1953), had been set out in the four large farm ponds and the captured microcrustaceans added in the morning. 13 Midway through the summer on August 20, fifty pumpkinseeds, Lepomis gibbosus (Linnaeus), ranging from one to three inches long, were added to each pond to check for recycling of DDT and its metabolites. Temperature A Taylor Water Thermograph was installed in one of the ponds to record a daily water temperature. Because the artificial ponds were placed in the same farm pond all ponds were assumed to have the same water temperature. SAMPLING SCHEDULE The artificial ponds were designated by letters. The control pond was designated as pond C, the pond with 0.05 ppm DDT in the bottom as pond L (Low), the pond with 1.0 ppm DDT in the bottom as pond M (Medium), and the pond with 10.0 ppm DDT in the bottom as pond H (High). The first samples were taken from all ponds twelve to twenty hours after the ponds were completely set up. The control pond samples were used as the initial treatment samples. This first sample was taken on July 9 and was designated as the 7/9 sample. The next three sample periods were spaced one week apart and were designated by the date they were taken, as the August 1 sample was recorded as the 8/1 sample. From August 1 to September 9, the sampling periods were ten days apart, and the last sampling period, on September 30, was twenty—one days after the previous sample. All samples are designated by the date of the day they were taken. 14 -METHODOLOGY Water Column water samples were taken from each of the four ponds during a sampling period. The water sampler was a glass tube which could be stoppered at either end with corks. The glass rod was lowered into the water, a cork in- serted in the t0p of the column, and the tube was raised until the bottom of the tube was just beneath the water surface. Then a cork was placed in the bottom of the tube securing the column of water. The water thus attained was drained into a glass water sample bottle. Two l-liter water samples were taken at each sampling period. The locations of the water columns sampled were randomly picked. The water samples were refrigerated until they were extracted. The extraction procedure involved partitioning each liter of water with one hundred ml of purified 30-60O petroleum ether for five minutes. The aqueous portion was discarded, and the petroleum ether was dried with anhydrous sodium sulfate. The petroleum ether was concentrated on a rotary vacuum evaporator and transferred to a graduated centrifuge tube for analysis on the gas chromatograph. The 15 16 method was 77% efficient with a relative standard deviation of 8.5%. Fish Obtaining a fish sample from each of the four ponds proved to be the most difficult sample to get, as there was room for the fish to hide and they soon became trap shy. Overall, glass minnow traps, wire minnow traps, and a variety of hand nets were used to collect the fish. Two fish from each pond were kept at each sampling period, and were im- mediately frozen and stored for analysis. The fish were analyzed on a wet weight basis only. Each fish was weighed while still frozen, put into a glass mortar, and diced with a scalpel. It was ground and dried with granular anhydrous sodium sulfate and a little sand. When the sample appeared to be dry, it was extracted three times with a 20 mls portion of 6% ethyl ether in petroleum ether, for a total of sixty mls. The total extractant was then added to a prewet standard Mills Florisil column (Mills, 1959), and eluted with 250 mls of 6% ethyl ether in petroleum ether. All fractions were collected in a round bottom flask, evaporated to a workable volume, and transferred to a graduated centri- fuge tube for analysis on the gas chromatograph. The efficiency of extraction was 87%_i 1.5% for DDE, 9I%.i 3% for DDD, and 81% i 2.5% for DDT. 17 Microcrustaceans The microcrustaceans were captured with a light trap at night (Baylor and Smith, 1953). These traps were hand- made light traps powered by a six volt car battery, and uti- lized the principles that amber light attracts certain micro- crustaceans and blue light repels them. A plankton net could not be used because of the small size of the ponds and the periphyton sheets hanging in the ponds. The traps gener— ally caught enough microcrustaceans to allow analysis for DDT. The samples were concentrated with a Foerst plankton centrifuge, placed in a plastic vial and frozen until analysis. The microcrustacean extraction procedure was es— sentially the same as the procedure used with the fish. Due to the small size and amounts of microcrustaceans, a pro- cedure for obtaining an equivalent wet weight was developed. The microcrustaceans were centrifuged in a graduated centri- fuge tube and an estimate of the wet weight was obtained by assuming that one cubic centimeter of microcrustaceans was equal to one gram of microcrustaceans. The microcrustaceans were ground and extracted with 6% ethyl ether in petroleum ether, and the ether was eluted through a Florisil column in the same manner, and with the same efficiencies of extraction as the clean up procedure for the fish. 18 Periphyton The periphyton was sampled by removing two of the sixty periphyton sheets present. Each of the sheets in a pond was given a number from one to sixty and the samples were randomly choSen by number. The sheets were removed from the water, placed in a plastic bag and frozen until analysis. The extraction of DDT and its metabolites from peri- phyton was complex and inefficient; recoveries were: DDE, 69.04% i 2.05%; DDD, 91.97% i 3.61%: and DDT, 81.18% i 3.48%. The sheets were scraped with rubber scrapers and rinsed with distilled water. The excess water was filtered off in a Buchner funnel, and the periphyton weighed. The weighed periphyton was placed in a glass mortar with granular sodium sulfate and sand, and extracted three times with acetonitrile. The volume of acetonitrile used was dependent upon the amount of periphyton. Ten ml of acetonitrile was used for the first gram or less and five ml for each ad- ditional gram. After extraction the acetonitrile was placed into a separatory funnel. The total volume of acetonitrile was partitioned with one half its volume of petroleum ether for one minute and drained into a second separatory funnel. The acetoni- trile was again partitioned with one half its volume of petroleum ether and allowed to stand. The petroleum ether 19 in the first funnel and its rinse were added to the second separatory funnel. The acetonitrile was drained back into the first separatory funnel. A recovery partition with acetonitrile against the petroleum either was made and this was added to the acetonitrile in the first separatory funnel. The petroleum ether was then discarded. The acetonitrile was then solivated with ten times its volume of 1% sodium sulfate in water. Petroleum ether, equal to the total volume of acetonitrile used, was added and partitioned for five minutes. The aqueous layer was dis- carded and the petroleum ether dried with anhydrous sodium sulfate. NuChar Attaclay was added to the petroleum ether, approximately one tenth of a gram for every gram of peri- phyton to remove the phytopigments. The liquid was filtered through a filter bed of anhydrous sodium sulfate into a round bottom flask, evaporated to five m1, and transferred to a graduated centrifuge tube for analysis. Hydrosoil The hydrosoil was sampled with pre-placed, randomly distributed bottom samplers. These samplers, seventy-five per pond, had been placed according to random numbers, flat on the pond bottom before the sand was added. A sampler consisted of a plastic bag attached to a circular steel band, 20 ten centimeters in diameter. A wire 100p protruded above the sand and was attached to the steel band so the sampler could be located and removed. When sampled, the wire 100p pulled the steel band up through the hydrosoil cutting a core sample into the plastic bag as it came up. These samples were easy and fast to take, and the samplers pro— tected the plastic bottoms of the artificial ponds from be— ing punctured. Three samples were taken from each pond at a sampling period, and were immediately frozen and stored. The procedure for extracting DDT and its metabolites from the hydrosoil required first a dry weight, so the samples were dried in an oven at fifty degrees Centigrade. When they were dry a weighed subsample was placed in a one liter Erlenmeyer flask, and soaked in 150 mls of 20% ethyl ether in petroleum ether for twenty-four hours. The ether was drawn off into a separatory funnel and the sand rinsed with an additional 100 mls of 20% ethyl ether in petroleum ether. The ether left in the sand was washed out with 100 mls of distilled water and added to the separatory funnel. The water was discarded and the remain- ing ether dried with anhydrous sodium sulfate. The ether was concentrated with a rotary vacuum evaporator to ten milliliters and eluted through a Mills Florisil column with 250 mls of 6% ethyl ether in petroleum ether. The recovery 21 efficiencies were 86.0% i 1.0% for DDE, 92.0% i 3.0% for DDD, and 77.5% i 3.0% for DDT. RESULTS AND DISCUSSION Water The amount of DDT in the water varied considerably from pond to pond (Figure 2, and Table 1). In pond H, the highest level pond, the amount of dissolved DDT approached its saturation level of 1.2 ppb (Bowman, 1960). This was the highest concentration that any pond reached, and after reaching this peak the DDT concentration tapered off during the rest of the study period. DDT was detected in the water at every sampling period in pond H. DDD, a degradation product of DDT, was detected in trace amounts in pond H in the first two sampling periods. At the end of fifteen days the DDD concentration in the water was up to 0.14 ppb and by twenty-two days it had reached 0.6 ppb. For the rest of the study period the DDD concen- tration fluctuated about a mean of 0.5243 1 0.0234 ppb. This did not decline in the fall as the DDT concentration did. After the first week DDE was detected in trace amounts in all water samples. The medium level pond, pond M, had its highest level of DDT in the first days sample. It maintained this ap- proximate level until the cold water temperature set in near 22 23 Figure 2. The average concentrations of insecticide in the water showing and comparing the fluctuations and changes with time in each pond. 24 N wusmam up: 023.21.. SA cum 5..» :1» 3..» 7» 3A 2A a; _IIIIII||IIIII ll- ‘E ... a. link-.- ‘.I.I'II...I..'II.I'II.'."II'.I‘-£ a u Gun...— _....... a .23.. EIIIIIIE ! 'coa ‘llllll‘ I ”can .ll. .— Icon EIE E 1:01 ‘I‘ I ‘Cou hon N... m... e.— N.— NOH'I II I“ 91 I'd 25 Table l. The average concentration of insecticide in the water of all the ponds at each sampling period. sampling DDT DDD DDE Total Dates Insecticide (parts per billion) No detectable insecticides were Pond C found in this pond. Pond L 7/9 0.054 N.D. N.D 0.054 7/17 Tr N.D. NQD Tr 7/24 Tr N.D. N.D Tr 8/1 Tr N.D. N.D. Tr 8/11 Tr N.D. N.D. Tr 8/21 0.0285 N.D. N.D. 0.0285 8/31 N.D. N.D. N.D. N.D. 9/9 N.D. N.D. N.D. N.D. 9/30 N.D. N.D. N.D. N.D. Pond M 7/9 0.1593 N.D. N.D. 0.1593 7/17 0.0663 N.D. N.D. 0.0663 7/24 0.1573 0.0950 Tr 0.2523 8/1 0.1053 0.1442 Tr 0.2495 8/11 N.C. 0.1250 Tr N.C. 8/21 0.1296 0.1244 Tr 0.2540 8/31 Tr 0.1140 Tr 0.1140 9/9 Tr 0.1510 Tr 0.1510 9/30 Tr 0.0765 Tr 0.0765 Pond H 7/9 0.231 Tr N.D. 0.231 7/17 N.C. Tr N.D. N.C. 7/24 0.8196 0.140 Tr 0.9596 8/1 1.0813 0.602 Tr 1.6833 8/11 1.016 0.504 Tr 1.5200 8/21 0.528 0.572 Tr 1.100 8/31 N.C. 0.440 Tr n.c. 9/9 0.4514 0.500 Tr 0.9514 9/30 0.1825 0.528 Tr 0.7105 Tr - trace amount N.D. - not detected N.C. - not calculated 26 the end of August, about fifty days after the experiment was started. In the colder water the DDT was detected only in trace amounts. DDD was first detected in the water of pond M after fifteen days, at nearly 0.10 ppb. The DDD level then re— mained constant at this level, 0.1218 1 0.0099 ppb, in all following water samples. Again the DDD levels did not de- cline with the onset of cold water temperatures as the DDT concentrations did. The lowest level pond, pond L, had water with a DDT concentration of 0.054 ppb in the first days sample. DDT was then detected in trace amounts for most of the summer with the exception of the forty-second day sample, where 0.0285 ppb was found. However, this is just within the range that DDT can be detected in the water and quantified. ‘With the occurrence of cold water in the fall no DDT was detected in the water of this pond. DDD or DDE was not detected in this pond. The control water samples from pond C never con- tained detectable insecticides. Column water samples were taken and compared to water-soil interface samples, and no differences were found. This is probably due to the shallow depth of the ponds where winds and temperature changes could easily mix the water. The analysis of water for DDT and its metabolities was hindered by the presence of an artifact which interfered 27 with DDT. Because of this the water data were not complete, and the data recorded have been checked on the microcoulo- metric gas chromatograph when possible or on another gas chromatograph which had a column containing QF-l as its liquid phase. Some of the samples were saponified with alcoholic potassium hydroxide and the resulting DDE peak was quantified as DDT. In general the water data seems to indicate that the amount of DDT translocated to the water is temperature de- pendent, because the DDT concentrations decline when the water gets cold. DDD concentrations did not decrease as the water temperatures decreased so DDD in water may not be temperature dependent. DDD can be expected to occur in these pond waters, as Miskus, Blair, and Cassida (1965) found that natural lake waters can degrade DDT to DDD. So in this case the DDD could have come from the degradation of the DDT dissolved in the water or it may have come from another source, such as being translocated to the water from the bottom where DDD was found to be present. However, sampling was not extensive enough to show this to occur one way or the other. Fish With one hundred fish in a small system like the artificial ponds, the food supply became a problem. After the first week, at the eight day sample, no microcrustaceans 28 were trapped in any of the artificial ponds. It was believed that the small size of the fish allowed them to feed heavily on the microcrustaceans and they had virtually eliminated their food supply. Because of this the fish were then fed ground food pellets, two to three grams per pond. By feeding the fish, transport and concentration of DDT by the food chain was probably reduced. The majority of the fish re- mained healthy for the study period. Throughout the summer six fish were observed to have died in the control pond and four of these died during the first week. All fish analyzed contained DDT and its two main metabolites, DDD and DDE (Table 2). The level of these insecticides in the control fish remained about the same, 0.2018 3 0.0102 ppm total insecticide, on a wet weight basis, throughout the study period. The metabolite and DDT to total insecticide ratios remained nearly constant in the control with only a slight increase in the DDD ratio at the end of the summer. The slight fluctuations in the total insecticide con- tent in the control fish on a part per million basis can be explained by virtue of growth and weight changes. As the summer progressed and growth occurred, the parts per million of insecticide in the fish decreased because they gained weight faster than they could accumulate the DDT that was available only in trace amounts in their environment. When the cold weather occurred in the fall and they were no longer 29 Table 2. The average insecticide content of the fish samples taken at all sampling periods from all ponds. Sampling DDT DDD DDE Total Dates Insecticide (parts per million) Pond C 7/9 0.0706 0.0180 0.0955 0.1841 7/17 0.0756 0.0242 0.0970 0.1968 8/21 0.0536 0.0275 0.0675 0.1486 8/31 0.0754 0.0385 0.1087 0.2226 9/30 0.0881 0.0446 0.0857 0.2184 Pond L 7/9 0.0806 0.0127 0.0870 0.1804 7/17 0.1146 0.0385 0.0906 0.2437 7/24 0.0928 0.0275 0.0903 0.2106 8/1 0.0865 0.0509 0.0853 0.2228 8/21 0.1045 0.1295 0.1043 0.3383 8/31 0.0967 0.1519 0.0997 0.3483 9/9 0.2636 0.2875 0.2347 0.7858 9/30 0.0881 0.3005 0.1021 0.4907 Pond M 7/9 0.3725 N.D. 0.2505 0.6230 7/17 0.4058 0.1173 0.3408 0.8639 7/24 1.0077 0.2755 0.5252 1.8084 8/1 1.2525 0.5175 0.3500 2.1192 8/11 2.0307 1.1300 0.7501 3.9108 8/21 3.5490 2.9415 1.3088 7.7993 8/31 1.1741 1.7159 0.8883 3.7783 9/9 3.9543 3.3637 2.3244 9.6424 9/30 2.3785 8.3565 1.4268 12.1618 Pond H 7/9 0.1936 0.1071 0.5534 0.8541 7/17 1.0472 0.4816 0.2750 1.8038 7/24 2.1313 0.3444 0.3204 2.7961 8/1 11.5348 1.1595 1.2137 13.9080 8/11 15.3679 4.0453 2.9798 22.3929 8/21 9.7812 4.6605 1.9465 16.3883 8/31 13.0416 7.4071 2.6384 23.0871 9/9 9.9403 10.6777 2.9705 23.5884 9/30 All fish had died. N.D. - no insecticide detected 30 fed, the fish may have had a weight loss which would raise the ratio of insecticide in the fish. The fish in the treated ponds contained more insecti- cide than the control fish. The differences in levels of DDT and its metabolites on a part per million basis with the fish‘s wet weight were tested with the Mann-Whitney U Test to find the probability of the distribution. The null hy- pothesis was: there are no differences in insecticide levels between the control and the treated ponds. The Mann—Whitney U Test was used because the samples were from different ponds and they did not have homogeneity of variances. The two high level ponds, ponds H and M, were com- pletely different from the control and had a probability of 0.001 or less that they were not different from the control. When pond L was tested with the control, DDT and DDD levels were significantly different at a 0.95 level of significance. The DDE distribution of ponds L and C had a probability of occurrence of 0.311 which is not significant. DDT and its metabolites in the fish were compared on an interpond basis (Figure 3) by plotting the percentage of the total insecticide that DDT and each of its metabolites comprised in all ponds with time. All the treated ponds ap- peared to follow the same trends while the control pond, pond C, remained relatively constant. Initially DDT accounted for the largest portion of the total insecticide present, but as time went on DDD Figure 3. 31 The insecticide makeup of the fish in the study ponds based on the individual percentages of the total insecticide that DDT, DDD, and DDE com- prise at each sampling period. 52 DDD POfld M C--. DOHd l .—-—. . Pond c gnu"... /:. //’ 50 4‘ ,/_/.':~ ' l' 46?" >63}, . ”(9 0’. / My / .' {fin-on." “en-IIIIIIII..--IIlll-...----IIIIIII ----------- III-Inn... (on / m e 7.9 7.17 7-24 8" 8'" 8-21 8‘31 9-9 9-30 3100 .- 3 DD: Pond H O O a find c .IIIIIIII. OF THE TOTAL 0: O .’.\. -.I.IIl-II..-. . I ~ I - . — - ~ \\ .—--.—-—:~.~.~.~ U ‘3 '2' 7-9 7-‘I7 7-24 I" 3'“ 8'21 8-31 9-9 9-30 In 2100 3 DOT Pond H ._' Pond M 0-‘0 POfld L .—-—. Pond c .IIIIIIIII. .\ .”O‘\ \. 50 fi/ ‘.~‘~ \ ." "'.u..."""'fie’mlwg't'll-II.IIDI}QI .. c‘nl’flhlc'III-IIII-IIII. l‘.—.—¥.-I.~.‘\\ I ‘ . 7-9 7-‘I7 7-24 8'] 8'" 0-21 8-31 9-9 9-30 SAMPLING DATE Figure 3 33 replaced DDT as the most abundant compound present. DDD in- creased from approximately 10% to 55% of the total insecti— cide present. The percentage of DDE decreased as uptake of DDT occurred and thereafter remained as a nearly constant percentage of the total insecticide. This shows clearly that insecticide changes do occur with time, however DDE did not become a major factor as a degradation product of DDT. All the treated ponds followed the same general pattern of change when compared to the control pond, even though there were large differences between the hydrosoil insecticide concentrations in the ponds. The main effect of increased hydrosoil concentrations appears to be in the de- gree of change when compared to the control rather than the type of change. This is shown in Figure 3 when the changes in the low level pond lie closer to the control than the changes in the high level pond. The uptake, concentration, and change of DDT and its metabolites in the fish of each individual pond was studied (Figures 4, 5, 6, and 7) by plotting DDT, DDD, DDE, and total insecticide in the fish of each pond on the same graph. In all treated ponds DDT increased and plateaued faster than its metabolites. DDE seemed to follow the DDT changes in the treated ponds and the control pond. By the end of the study DDD had become the most abundant metabolite present and, after sixty days, all ponds showed it to have equalled or surpassed the DDT concentrations in the fish. Initially, 34 Figure 4. The semi-log plot of the average total insecti- cide concentrations in the fish at each sampling period of each study pond. 55 e wusmam wh(n 023A51n cmna To 2?» uNnn 3:» Ta ¢Nn~ :uu mn~ ..... c ........................... ---- . ............................................... . ........ .I|.....H.h.u..\...\.. O “\\\|\\ \O 0” \\ ""l \ u\ 'R. 0‘ 9.. .\ \ ‘ \I . O . \\t\.o 0\\\\\ \‘ \Olo/ \.\O .\. .II \‘ \ I O\ \.\... o ......... o o \ O'O\ /. U VB: 0 nnnnnnnn O a icon 0". ¢¢18£. Ollili :— 2: NOI‘ITIW lid Sll'd 36 Figure 5. The semi-log plot of the average DDT concen- trations in the fish at each sampling period of each study pond. 37 m muzmflm whlunlO 30 main ~nns e e w musmam c—Ih m e m—uh e ¢~|h mo e~ IIHLVUIGWSL l I lVM 47 decrease in the temperature of the pond water could induce DDT poisoning of the fish. This appears to be the case at hand, as the DDT was actively present because uptake was still occurring and there was a probable food shortage at the time of the mortality. While other temperature drops occurred over the rest of the study period they were not accompanied with a food shortage and no major mortalities occurred. Also the insecticides present later in the summer were not pre- dominately DDT, but included a higher percentage of DDT degradation products. By midsummer theiinsecticides were probably stored in the fats and lipids where they would be somewhat protected during times of stress. Hence, the insecticides later in the summer were not easily accessible to cause poisoning of the central nervous system. On August 20, forty-five days after the study was initiated, small pumpkinseeds, Lepomis gibbosus, one to three inches long were added to the ponds to check for con— tinuous cycling or recycling of DDT and its metabolites. In pond L only DDD differed significantly from insecticide levels in the control fish. The DDD concentration in pond L fish was approximately ten times the DDD concentration in the control fish (Table 3). In ponds M and H the DDT, DDD, and the DDE concentrations were higher than the concen- trations in the control fish. Again as in pond L, the DDD 48 Table 3. The average insecticide content of the recycled fish at each sampling period from each pond. Sampling DDT DDD DDE Total Dates Insecticide (parts per million) Pond 8/31 0.0274 0.0253 0.0744 0.1271 9/30 0.0572 0.0369 0.0795 0.1736 Pond 8/31 0.0340 0.0865 0.1301 0.2505 9/9 0.0513 0.0946 0.1465 0.2924 9/30 0.1697 0.4123 N.C. 0.5820 Pond 8/31 0.1787 0.7341 0.1741 1.0869 9/9 0.2971 1.2140 0.2600 1.7711 9/30 0.1536 1.7860 0.2577 2.1973 Pond 8/31 0.3095 3.4480 1.2096 4.9671 9/9 2.6160 7.5060 1.0326 11.1546 N.C. - not calculated 49 concentrations in ponds M and H increased faster than DDT or DDE. The uptake of total insecticide that occurred.in the pumpkinseeds with time was compared with the uptake that oc- curred in the original fish with time (Figure 9). The up- take patterns were quite similar for each pond, which is sig- nificant when the fact that a different species of fish was used and that the recycling experiment took place in late summer and early fall when the weather conditions were different. The water temperatures were colder during the 7! recycling experiment and the insecticide levels in the water were lower. Thus, because the initial uptake of insecticides by the pumpkinseeds was equal to or greater than the initial uptake of the original fish it appears that the uptake of DDT and its metabolites by these fish is not completely de- pendent upon the insecticide concentrations in the water. In general it was found that a DDT concentration in a sandy hydrosoil of 10.0 micrograms per gram will cause constant fish mortality. There were no effects noticed on the fish of the other treated ponds, although analysis showed that DDT and its metabolites were found to be con— tinuously recycled in the aquatic environment and it was not inactivated with time. 50 Figure 9. The initial uptake of insecticide that occurred in the recycled fish is compared to the initial uptake of insecticide by the original fish. ORIGINAL FISH lNSEOTlCl DE IOTA 1 PPM 51 7-9 7-17 7-24 8-1 8'11 8-21 100 SAMPLING DATE FISH RECYCLED H c 8-21 8-31 9-9 9-30 SAMPLING DATE Figure 9 52 Microcrustaceans All the microcrustaceans of the treated ponds con- tained DDT and each of its metabolites, while the micro- crustaceans from the control pond contained no detectable insecticides (Table 4). The lowest level pond, pond L, con— tained DDT, DDD, and DDE in concentrations that were de— tectable but not always measureable. This is a function of the small amounts of microcrustaceans that were caught which could not give enough insecticide to measure when they were extracted. The study of microcrustaceans is not complete due to the fact that the microcrustacean population was kept very low by the large numbers of fish in the ponds. The micro- crustaceans were caught with light traps at the first sample period. When the traps were set in the experimental ponds for the second sample one week later, no microcrustaceans were captured. So more microcrustaceans were trapped and netted from the adjacent ponds and added to the study pools. After seven days there were still only small amounts of microcrustaceans captured so more microcrustaceans were added to the ponds. In addition the fish were fed ground food pellets. During the rest of the study period micro- crustaceans were always captured, but not always in amounts large enough to measure the DDT content. The controls never contained detectable levels of insecticides so it logically 53 Table 4. The insecticide content in the microcrustaceans from all ponds. _—‘ *— Sampling DDT DDD DDE Total Dates Insecticide (parts per million) Pond L 7/9 tr tr tr tr 8/11 0.0792 0.0752 0.0779 0.2323 8/31 tr tr 0.1410 0.1410 9/9 tr tr tr tr Pond M 7/9 0.5610 0.9815 0.5359 2.0784 8/11 1.0736 0.8648 0.4683 2.4067 8/31 0.1514 1.1920 0.3608 1.6968 9/9 0.2726 0.7405 0.2870 1.3001 Pond H 7/9 2.3570 1.4340 0.8972 4.6882 8/11 2.8901 1.2910 1.3760 5.5591 8/31 3.2800 5.0372 1.0540 9.3712 9/9 1.5800 2.3629 0.6905 4.6324 Pond C All samples contained no detectable levels of insecticide tr — trace of insecticide 54 follows that none of the three additions of microcrustaceans contained any insecticides. The samples analyzed do not represent a pOpulation that has lived in the ponds the duration of the study period because of the many additions of microcrustaceans to the ponds at different times. The low microcrustacean popu- lations and hand feeding the fish reduced the accumulative effect of food chain transport of DDT to the fish. However, some of the initial uptake of DDT would be a result of the fish consuming all the available microcrustaceans in the first week, as the microcrustaceans had DDT concentrations of 2.357 ppm in pond H and 0.5610 ppm in pond M after one day in the ponds (Table 4). Overall there was not much change in the insecticide levels in the microcrustaceans with time (Figures 10 and 11). A graph of the percent of the total insecticide that either DDT, DDD, or.DDE makes up with time (Figure 12) shows that the DDE comprised a nearly constant percentage of the total insecticide, about 20%. When the DDT percentage decreased, the DDD percentage increased and compensated for the DDT decrease. This could indicate that DDD is the main degra— dation product of the microcrustaceans or it could indicate the available insecticide ratio in the bottom material be- cause the microcrustaceans do spend the daylight hours on the bottom. Hunt (1960) indicated that zooplankton or micro- crustaceans might have the ability to accumulate DDT from 55 Figure 10. The change and fluctuation of insecticides in the microcrustaceans of study pond H with time. 56 OH THDUHW :2. 62.2.2: ala "mun =Iw o. ...... ....... 6................ fl . ........... X. n: o/ \ \ o/ / I“ IIIII o / o. .. I .V\ / \ O/h..\\\0 “no . IIIIIIII . 6...: .32.. .28 6|. NOI'I'IIW lid SLIV‘ 57 Figure 11. The change and fluctuation of insecticides in the microcrustaceans of study pond M with time. 58 mua nmnu 5.09 039.302.. .20.— Hd mhflwflm up: 02:62: .1 IIVJIIOII-“f‘ l-"-'|. :- W 3 8— NOI'I'IIW lid SLlVl 59 Figure 12. The percentage of the total insectide that DDT and each of its metabolites comprise in the microcrustaceans at each sampling period. 60 “an . llllllll . 2:. 6| . Io mmnn NH musmflm ~59 0236s.: 2.» mfi .3 IOIDILDISNI 1'10]. 3H]. ‘0 ROVLNIDlll 61 the bottom, and to distribute it to higher organisms. Jones and Mbyle (1962) showed population changes of ZOOplankton as a result of farm pond treatment with DDT so the zooplankton are affected by concentrations of DDT. It seems likely that the insecticide makeup of the hydrosoil and the ability of the microcrustaceans to de- chlorinate DDT to DDD will be reflected in the DDD accumu- lation in the microcrustaceans, but more extensive research will have to be done to separate these two factors of DDT and DDD accumulation. Periphyton The periphyton from each pond showed considerable variation in DDT content, but the DDT concentration in the periphyton of each pond was found to be significantly differ- ent from all other ponds at a 0.95 level of significance. The Mann-Whitney U Test was used to calculate these probabilities. The variable results of the periphyton study are due to the large weight differences of the periphyton samples. The large samples were consistently lower in their DDT con- tent (per unit weight) than the small samples. The weights of the samples were influenced by the large amounts of float- ing algae that accumulated in the study ponds, which were abundant enough to shield out one half to three fourths of the available light. The algae did not float continuously 62 so the amount of area shaded was not constant and this would have its effect on the periphyton growth rates. The amounts of insecticide found in the periphyton were quite variable so two insecticide to periphyton ratios were used. These were based on the premises that-DDT may be adsorbed as a function of growth which would be measured by weight, or that DDT may be adsorbed as a function of the exposed surface area available for periphyton growth which would be measured in square meters. The parts per million of insecticide in the peri- phyton was based on a wet weight of the periphyton (Table 5). The total micrograms of insecticide present was calculated to give the value, total micrograms per square meter of available surface area (Table 6). These two values were compared for ponds H, M, and L (Figures l3, 14, 15). It can be seen in ponds H and M that initially the total amount of insecticide increased while at the same time the parts per million based on the wet weight decreased. This is due to the DDT not being adsorbed proportionally to an increase in weight of the periphyton, nor is it adsorbed strictly as a function of the available surface area. Thus DDT uptake by the periphyton appears to be a function of both growth and exposed surface area. When the insecticides in each individual pond were compared (Figures l3, 14, 15) showing the uptake of DDT and its metabolites by the periphyton with time, it can be seen 63 Table 5. The average insecticide content of the periphyton samples taken at each sampling period from all ponds. Sampling DDT DDD DDE Total Date (parts per million) Insectic1de Pond C 7/9 0.0533 N.D. N.D 0.0533 7/24 0.0832 N.D. N.D 0.0832 8/11 0.0392 N.D. N.D. 0.0392 9/9 0.0152 N.D. N.D 0.0152 9/30 0.0195 N.D. N.D 0.0195 Pond L 7/9 0.0534 N.D. N.D. 0.0534 7/17 0.1266 N.D. N.D. 0.1266 7/24 0.1021 N.D. N.D. 0.1021 8/l 0.0741 N.D. N.D. 0.0741 8/11 0.1004 N.D. N.D. 0.1004 8/21 0.1371 Tr Tr 0.1371 8/31 0.0455 Tr Tr 0.0455 9/9 0.0221 Tr Tr 0.0221 9/30 0.0387 Tr Tr 0.0387 Pond M 7/9 0.4341 N.D. N.D 0.4341 7/17 0.2118 Tr Tr 0.2118 7/24 0.1255 Tr Tr 0.1255 8/1 0.5339 Tr Tr 0.5339 8/11 0.4481 0.1557 Tr 0.6038 8/21 0.1109 0.0731 Tr 0.1840 8/31 0.1210 0.1138 Tr 0.2348 9/9 0.0294 0.0461 Tr 0.0755 9/30 0.1174 0.1420 Tr 0.2598 . Pond H 7/9 0.5463 Tr N.D. 0.5463 7/17 0.2735 N.D. N.D. 0.2735 7/24 0.8259 Tr N.D. 0.8259 8/1 1.0760 Tr Tr 1.0760 8/11 0.7259 0.2554 Tr 0.9813 8/21 0.2605 0.1571 Tr 0.4176 8/31 0.5787 0.4396 Tr 1.0183 9/9 0.6173 0.7366 Tr 1.3539 9/30 0.2317 0.3534 Tr 0.5851 Tr - trace of insecticide N.D. - no insecticide detected 64 Table 6. The average insecticide content of the periphyton samples taken at each sampling period from all N.D. - no insecticide detected ponds. Sampling DDT DDD DDE Total Dates (micrograms per square meter) Insect1c1de Pond C 7/9 0.1126 N.D. N.D. 0.1126 7/24 0.2270 N.D. N.D. 0.2270 8/11 0.1022 N.D. N.D. 0.1022 9/9 0.0844 N.D. N.D 0.0844 9/30 0.1916 N.D. N.D 0.1916 Pond L 7/9 0.3551 N.D. N.D 0.3551 7/17 0.2233 N.D. N.D 0.2233 7/24 0.2272 N.D. N.D. 0.2272 8/1 0.1510 N.D. N.D. 0.1510 8/11 0.2324 N.D. N.D. 0.2324 8/21 0.7657 Tr Tr 0.7657 8/31 0.3473 Tr Tr 0.3473 9/9 0.2742 Tr Tr 0.2742 9/30 0.3228 Tr Tr 0.3228 Pond M 7/9 0.9168 N.D. N.D. 0.9168 7/17 1.8116 Tr Tr 1.8116 7/24 1.5508 Tr Tr 1.5508 8/1 1.9375 Tr Tr 1.9375 8/11 0.9943 0.2931 Tr 1.2874 8/21 1.3917 0.9175 Tr 2.3093 8/31 0.6041 0.5684 Tr 1.1725 9/9 0.4274 0.6701 Tr 1.0976 9/30 0.4865 0.5903 Tr 1.0769 Pond H 7/9 0.7205 Tr N.D. 0.7205 7/17 2.6045 N.D. N.D. 2.6045 7/24 4.1179 Tr N.D. 4.1179 8/1 2.5516 Tr Tr 2.5516 8/11 1.7716 0.6234 Tr 2.3950 8/21 2.2277 1.3460 Tr 3.5689 8/31 1.6252 1.2347 Tr 2.8599 9/9 1.6637 1.9855 Tr 3.6492 9/30 1.5077 2.3114 Tr 3.8075 Tr - trace of insecticide 65 Figure 13. The composition and changes with time of insecticides in the periphyton of pond L on a part per million basis and on a total micro- grams per square meter basis. 66 .o MICROGRAMS PER SQUARE METER P -: 7-9 7-17 7-24 8‘1 t—II 8-21 8-31 9.9 9-30 2 SAMPLING on: 0. PER MILLION PARTS 7-9 7-I7 7-24 B-I 8'" 8-21 8’31 9-9 9-30 SAMPLING DATE Figure 15 67 Figure 14. The composition and changes with time of insecticides in the periphyton of pond M on a part per million basis and on a total micro- grams per square meter basis. 68 2.0 MICROGRAMS PER SQUARE METER I 7-9 7.17 7-24 3-1 8-11 8-21 8-81 9-9 SAMPLING on: .75 DDT . DDD 3-50 \ : 2 z I m I. a '5 2 O E 7.9 7.17 7-24 3-] B-II 8-21 8-31 9 9 SAMPLING DATE Figure 14 9-30 69 Figure 15. The composition and changes with time of the insecticides in the periphyton of pond H on a part per million basis and one a total micro— grams per square meter basis. MICROGRAMS PER SQUARE METER PARTS PE R MIL L ION 7O DDT .~. I. 20 .\ .I’."’-.’ ——* .——o ’O —-—.l I /° ’0 7-9 747 7-24 8-I B-II 8-2I 8-3I 9—9 9-30 Id SAMPLING DATE DDT 0—0 .2 7-9 7-l7 7'24 l-I B-II 8'2] SAMPING DATE 8' 3 I 9-9 9- 30 Figure 15 71 that all ponds did not contain meaSurable amounts of DDD until the 8/11 sampling period, thirty-two days after the study was started. Since this was well after DDD became prevalent in the water it is not likely that the algae is a basic bactor in the DDD transformation in the ponds, and that the DDD occurring in the periphyton is not a result of the periphyton actively degrading the DDT to DDD. Only trace amounts of DDE were detected in any of the ponds so the route of DDT degradation, if it occurs, is not towards DDE. Recycling of DDT and its metabolites was checked by adding new unseeded periphyton sheets to all ponds on the forty-fifth day of the study, on August 20. Because this was the late summer, the algae did not grow fast or become very abundant. The final day of sampling, two sheets from each pond were taken and combined into one sample for analysis. The results (Table 7), show that DDT and DDD were recycled to the periphyton in the two high concentration ponds. Pond L periphyton differed from the control only be— cause a trace of DDD was detected. These results indicate that DDT and its metabolites are not readily recycled in the periphyton. The original periphyton accumulated DDT quite readily and by the end of one week it had relatively large quantities of DDT present. During the period when the original periphyton was accumu- lating DDT there was also an abundant amount of DDT in the water. However, the recycling experiment was made in the 72 Table 7. The insecticide concentrations of the recycled periphyton from all ponds. Sampling DDT DDD DDE Total Dates Insecticide (parts per million) Pond C 9/30 0.0454 N.D. Tr 0.0454 Pond L 9/30 0.0540 Tr Tr 0.0540 Pond M 9/30 0.1313 0.2598 Tr 0.3911 Pond H 9/30 0.1973 0.6044 Tr 0.5029 (micrograms per square meter) Pond C 9/30 0.0813 N.D. Tr 0.0813 Pond L 9/30 0.0870 Tr Tr 0.0870 Pond M 9/30 0.3582 0.7086 Tr 1.0668 Pond H 9/30 0.3197 0.9790 Tr 1.2957 Tr - trace of insecticide N.D. — no insecticide detected 73 late summer after the DDT levels in the water had decreased. Because the recycled periphyton did not accumulate DDT where- as the original periphyton did, it appears that the trans- port of DDT or DDD is dependent upon the insecticide concen- trations in the water. Meeks and Pterle (1965), using Cl-36 labeled DDT, found that there is rapid uptake of DDT from the water by algae. Since there was DDT in the water of the experimental ponds, the route of transport to the algae was probably by way of the water. Hydrosoil The prepared bottom concentrations of DDT in sand were not as accurate as expected (Table 8). This ruled out budget analyses, and short term changes could not be seen. The bottom samples were analyzed at four regular spaced intervals of the study period, which would show the major changes in the hydrosoil insecticide concentration and in the DDT and metabolite composition of the hydrosoil. In ponds M and L, there was a definite decrease in the amount of measured insecticide in the bottom (Figure 16). The highest level pond, pond H, did not show this tendency nearly as well, but this can be explained by virtue of the larger amounts of insecticide present in pond H masking the changes presented by displacement and loss of DDT and its metabolites. 74 Table 8. The insecticide concentrations in parts per million, based on the dry weight of the bottom material in each of the artificial ponds. Sampling DDT DDD DDE Total Dates Insecticide (parts per million) Pond C 7/9 0.00168 N.D. N.D. 0.00168 8/31 0.00256 N.D. N.D. 0.00256 Pond L 7/9 0.0468 N.D. 0.0017 0.0485 8/11 0.0268 0.0084 0.0016 0.0368 8/31 0.0252 0.0082 0.0015 0.0349 9/30 0.0068 0.0075 0.0003 0.0146 Pond M 7/9 0.9676 N.D. N.D. 0.9676 8/11 0.2821 0.0656 0.0190 0.3667 8/31 0.2721 0.1039 0.0022 0.3782 9/30 0.2212 0.1372 0.0016 0.3600 Pond H 7/9 8.5710 N.D. N.D. 8.5710 8/11 6.1500 0.2709 0.2152 6.6525 8/31 9.6649 0.1418 0.2415 10.0482 9/30 5.5851 0.4717 0.1055 6.0623 N.D. - no insecticide detected 75 Figure 16. The insecticide concentrations, in parts per million, based on the dry weight of the bottom material in each of the artificial ponds, are compared on a semi-log scale. On :0 ma wnsmam uh‘fl OZ.._A 11¢. Zulu :Im 76 ban Olu-IIO 622.32.. .63» all!- —0.0 NOIT'IIW lid SLlVl O— 77 All ponds indicated that DDD was the major degra— dation product of DDT. The two low level ponds showed a de- crease in the DDT percentage of the total insecticide which corresponded to a similar increase in the DDD percentage of the total insecticide (Figure 17). Pond H again did not show this tendency as clearly as the two low ponds, but by the end of the summer the DDD level was four times that of the DDE. While pond H did not show the differences in per- centage units, the amount of DDT actually converted to DDD in pond H was much higher than it was in the other ponds. The reason this change did not show up in the percentage units equal to the other ponds, could be a saturation of the mechanism of degradation, whether it is by bacteria (wedemeyer, 1967) or by reduced porphorins (Miskus_g£‘al., 1965). While DDE was present and comprised a nearly con— stant percentage of the total insecticide in all ponds, it did not assume the importance that the DDD did as a break- down product of DDT. I I The fact that DDD is a major breakdown product of -DDT, and increases with time, and that it is transported to and concentrated in the biota of an aquatic environment is significant.2 Hunt (1960) showed that DDD was mobile in an aquatic environment and it is harmful to aquatic organisms. DDT was detected in all the bottom samples. The Lake Michigan study by Hickey, Keith, and Coon (1966) found DDT in Lake Michigan bottom materials and indicated that the DDT 78 Figure 17. The percentage of the total insecticide that DDT and each of its metabolites comprise in the bottom material of the treated ponds. 79 amua . llllllllllllll Hull-in: .. nun-Inflam- léI-IIIIH HHHHH —‘E gfiafifiafififllfifll‘fllfllfll‘u'i'g'l III-a a? p. ‘ ‘I‘I‘I — as musmflm uu