I 1 WI 1 1 l Ml 1m H‘I‘MW l \ ‘ ‘ UIHHWIW 114 SHINE 3N THE ROLE 03"” mm“ ffifimfiifi» m meusm 3E” SELEC‘FEQ (‘ZHLGRWA‘E’EQ MYQFJCAEEGK ENSSECE’ECmEfi “E‘Easzs Foe! é-‘Em {)0ng at} ‘E‘Aa S. E’EECEEGAR ”TATE UXWEKSEN Erik Kirk Kia‘kneE i968 '1‘! _."~ u’ AO-M‘h“ ’an L [BRA R Y . Michigan State lein;-r.sLty v—T‘ ur.-v—v.— ‘. -4 ..—H—v-m..._-—.. . .-.- ”70' w-ao .vu—w-‘. ....4_ W . I ABSTRACT STUDIES ON THE ROLE OF FOLSOMIA FIMETARIA IN METABOLISM OF SELECTED CHLORINATED HYDROCARBON INSECTICIDES 6 By Erik Kirk Kirknel Folsomia fimetaria has been exposed to pp' DDT, pp' TDE and op' TDE. The experiments have been analyzed for pp' DDE, Op' DOE and the methyl ester of pp' DDA. Previously described metabolism of pp' DDT to pp' DDE was confirmed. No metabolism of pp' DDT to the methyl ester of pp‘ DDA could be detected. There was no metabolism of pp' TDE or pp' TDE to pp' DDE or to the methyl ester of pp' DDA. An unidentified compound in extracts of Collembola showed up in the GLC analysis but did not appear on TLC, as the expected compound op' DDE. STUDIES ON THE ROLE OF FOLSOMIA FIMETARIA IN METABOLISM OF SELECTED CHLORINATED HYDROCARBON INSECTICIDES BY Erik Kirk Kirknel A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Entomology 1968 ACKNOWLEDGEMENTS Q I wish to thank the following members of my graduate committee for their help and assistance during the study; Dr. James Butcher, Committee Chairman, whose guidance and encouragement was most valuable to me; as was the generous aid and counsel provided by Dr. Matthew Zabik, Dr. James M. Tiedje and Dr. Gordon Guyer. Also I wish to thank the Danish government, especially Director, Ingvard Petersen, Statens plantepatologiske Forsdg, Chairman, IIc. agro. Knud Lindhardt, Department of Zoology, Staten plante- patologiske Forsdg, Danmark and the Danish Planteavlsudvalg, who provided economic and other support for this study. Further financial assistance and facilities was given to me by the Department of Entomology through arrangements made by Dr. Gordon Guyer and Dr. James Butcher. This assistance is gratefully acknowledged. Without support provided by the Michigan Agricultural Experiment Station and the National Institute of ' Health (Grant No. CC-002h6-03) this study would not have been possible. Finally I am pleased to acknowledge the assistance of Michael Guthrie an undergraduate student at Michigan State university who assisted with culture preparation and analyses. TABLE OF CONTENTS Page ACKNOWLEDGEMENTS . . . . . ‘. . . . . . . . . . . II LIST OF TABLES . . . . . . . . . . . . . i. . . iv “LIST OF FIGURES . . . . . . . . . . . . . . . . v INTRODUCTION . . . . . . . . . . . . . . . . . 'I METHODS . . . . . . . . . . . . . . . . . . . i 3 RESULTS '. . . . . . . . . . . . . . . . . . . 8 DISCUSSION AND CONCLUSION . . . . . . . . . . . . . II LITERATURE CITED . . . . g. . . . . . . . . . _. . I2 LIST OF TABLES Table I. Experiment extracted after 1 day. 2. Experiment extracted after 6 days. . . . . . 3. Experiment extracted after IZ days. A. Experiment extracted after 22 days. 5. Collembola exposed to pp' DDT for I2 days and then transferred to non-pp'DDI_agar. . . . Page IN IS l6 l7 l8 IO. LIST OF FIGURES Concentrations of pp' TDE in Collembola extracts in relation to length of exposure to pp' TDE with and without antibiotic. . . . .Concentrations of op' TDE in relation to length of exposure to 0p' TDE with and without antibiotic. . . . . . . . . . Concentration of pp' DDT in Collembola extracts in relation to length of exposure to pp' DDT in presence of antibiotic. . . . . . Concentration of pp' TDE in Collembola extracts in relation to length of exposure to pp' DDT in presence of antibiotic. . . . . Concentration of pp' DDE in Collembola extracts in relation to length of exposure to .pp' DDT in presence of antibiotic. . . Concentration of pp' DDT in Collembola extracts in relation to length of exposure to pp' DDT without antibiotic. Concentration of pp' TDE in Collembola extracts in relation to length of exposure to pp' DDT without antibiotic, . . . . . . . . Concentration of pp' DDE in Collembola extracts in relation to length of exposure to pp' DDT without antibiotic. . . . . . . . Page 19 '9 20 20 20 2] 2i 2l INTRODUCTION Q Since it became available for insecticide use, metabolism of DDT and related compounds has been a subject of investigation for several reasons. First, it has been an insecticide used in relatively large amounts against a wide variety of insects, and second, its half life is relatively long. ‘ It has been shown in numerous papers (I, 6, 9, IO and II), that different systems are capable of decomposing DDT to different compounds and in different degree. Five different routes of DDT metabolism have been described including, l) DDA, 2) Kelthane, 3) dichlorobenzophenone, A) DDE, and 5) TDE. In insects the most common described route is probably metabolism of DDT to DDE. To the authors knowledge, metabolism of TDE has not been reported in insects, although it is widely used as an insecticide, and is frequently reported as a contaminant. It is known that lake water is capable of degrading DDT to TDE (II) in the presence of reduced porphyrins and it is reported (2) that porphyrins in many milieus have a half life of billions of years. TDE was found to be common in samples of water, soil plants and animal tissue, in areas where only DDT had been sprayed and was shown to be able to degrade DDT to TDE (l0). Metabolism of DDT to DDA has been reported for the body louse Pediculus humanus humanus, which was able to degrade DDT to three meta- bolites, DDE, DBP and DDA in the ratio l:2:2 (l2). / I It has recently been shown by Butcher, Kirknel and Zabik I969,I that in the presence of Folsomia fimetaria Linn., pp' DDT is metabolized to pp' DDE, in significant quantities; but the mechanism of metabolism is not defined. Folsomia fimetaria is a commonly distributed Collembolan in soils, particularly those rich in organic matter. It could be potentially important in soil decontamination since it has been shown that Collembola can survive heavy application of DDT. Folsomia fimetaria itself appears to be highly resistant to DDT (l3). Although large quantities of pp' DDE were produced by Collembolans in culture in previous experiments (Butcher, Kirknel and Zabik I969), large quantities of TDE were also observed in these same cultures. This raised the question: is TDE also metabolized by Folsomia fimetaria? ' In trying to answer this question It seemed logical to investigate two metabolic routes for both pp' TDE and op' TDE; especially metabolism to pp' DDE, op' DDE and pp' DDA; and 0p' DDA; and pp' DDT to pp' DDA. I Manuscript (in press). METHODS The Folsomia fimetaria used in these experiments had been kept at 2l C° and saturated atmosphere in glass jars half filled with a mixture of plaster of paris and charcoal substrate to which powdered Brewers yeast was added as a food supply. The Folsomia strain had been cultured in the Michigan State University Laboratory under these conditions for about 3 years. The metabolism experiments reported here were carried out in pyrex culture tubes ISO mm long, with an inside diameter of_lh mm. The sub- strate for the insecticide to be tested for metabolism was a l-l/2% Difco bacto-agar, control No. Sll79l from Difco Laboratories, Detroit, Michigan. The agar was poured into cold water and heated up to 90 C°. After being cooled down to 60 C°, a #00 ppm concentration of DDT in glass distilled hexane was slowly poured into the agar, while stirring with a magnetic stirrer. The ratio between the water agar and the solvent in which the pesticide was dissolved was l9:l. The hexane had a boiling point of 68-69°F. The final concentration of the insecticide in the agar was 20 ppm. The insecticides used were obtained from Pesticides Repository, Pesticides Research Laboratory, Perrine, Florida. The insecticide purity was represented as follows: pp' DDT, 99%; pp' TDE, 99+% and op' TDE, 98%. Gas-liquid chromatography analyses of the insecticides showed that pp' DDT contained about Il.l% pp' TDE. No other compounds could be detected with GLC in the samples of pp' TDE and op'TDE; The agar with the insecticide (presumably now in a microcrystal- line phase), was pipetted into culture tubes. Each culture tube received 7 ml and the culture tubes were plugged with cotton. In experiments with- out insecticides, only hexane was poured into the agar: Culture tubes with DDT were cooled at room temperatUre with the tube tilted to give the agar a 40 mm long surface. Culture tubes containing TDE were autoclaved for 20 min. at l2l C°, l atmosphere pressure and cooled as described above for DDT. - - DDT was not autoclaved, because, for unknown reasons, conversion of DDT to TDE in the agar proceeded more rapidly in the autoclaved experi- ments. Twenty-four hours after the agar was poured, an antibiotic was added to the culture tubes in a Microvoid inoculation chamber with a syringe attached to a Swinnex millipore filter holder equipped with a 0.h5 micron l3 mm millipore filter. Antibiotics were used in these experiments to see what metabolism, if any, was suppressed or eliminated. Dihydrostrepto- mycin was used because of its reputation as a wide spectrum antibiotic. The particular antibiotic was a crystalline sulfate from Pfizer Laboratory (Control No. 709Ah; exp. date January, I973). One gram, containing approxi- mately l06 units was dissolved in 50 ml distilled water, and 0.08 ml was injected (I600 units/culture tube or #00 Units/cm2 agar surface) into the culture tubes. After 2% hours, Folsomia were added to the culture tubes. Approximately 30 mg (live weight) were added to each culture tube. The age of the Collembola was I week at this time. They were not supplied with any food except for the agar in the culture tubes. The culture tubes_ were placed in a constant temperature cabinet at 2l C° without light. After exposing the Collembola for predetermined periods of time, the culture tubes were removed from the cabinet and analyzed. Three culture components were analyzed for metabolites: l) macerated Collembola, 2) surface agar, and 3) agar in the bottom of the culture tube. The GLC detected compounds were quantitatively determined in the Collembola, and qualitatively determined in both of the agar samples. For purposes of extraction the Collembola were all removed from the culture tubes and while still alive were transferred to a 125 mm long, IA mm inside diameter test tube, and closed with a Teflon lined screw cap. In order to prepare Collembola for analysis, a cottom plug saturated with ethylacetate was inserted into each test tube opening. After a one minute exposure, the Collembola were weighed on a Cahn electro balance, returned to the test tube and macerated with a glass rod. One milliliter of diethyl ether was added immediately and the test tube was capped. After 2A hours the Collembola extract was injected directly into the GLC without any clean-up. Concentration was calculated on a weight by weight basis. i In addition to the Collembola, a 2 mm thick layer of the surface agar was removed from the culture tubes with a spatula and transferred to a test tube, to which l.S ml diethyl ether was added. After 2h hours in the test tube, the extract was injected in the GLC without any clean-up. Concentration was determined in the extract and relationships between the observed metabolites were calculated. The GLC used was a Beckmann GC-A with an electron capture detector. The column was a l/8”, 3' copper column vibrator packed with 7.3% 0V-l7 + 3% QF-l on a-Cas-Chrom Q. column: temperature was 220 C°. The helium flow through the column was A0 mI/min. Concentrations were calculated using peak height. The recorder was running at l/2”/min. and the retention times for the insecticides were: op' DDE . l.77 min.' pp' DDE 2.20 min.. Methyl ester of pp' DDA 2.20 min. op' TDE ‘ 2.60 min. pp' TDE 3.39 min. op' DDT 3.23 min. pp' DDT 4.09 min. I The results obtained from GLC analysis were checked on thin layer using glass plates covered with aluminum oxide, F(25h) type T. These plates show flourescence when illuminated with a light source with a 2537 Angstrom wavelength. The solvent system used was 85% glass distilled hexane with a boiling point of 68-69 C°, and IS% Diethyl ether with a boiling point of 3A.6 C°. Rf values for pp' DDT OJA63 pp' TDE 0.362 pp' DDE 0.558 8 Methyl ester of pp' DDA 0.290 The pp' DDA was methylated so that it could be detected in an elec- tron capture detector. The samples were first injected in GLC, and then methylated with diazomethane. A new injection was then done to determine ~if pp' DDA had been in the sample. Diazomethane was prepared in the fol- lowing way: Diazald in ether was slowly dropped down into a l00 ml reaction bottle containing an alcholic solution of potassium hydroxide (25 ml ethyl alchol, plus 5 gram potassium hydroxide, plus 8 ml distilled water). The reaction bottle was in a water bath heated up to 65 C°. The diazomethane and ether vapor were condensed in 2 receiving flasks and cooled down to 0 C°. The second receiving flask was filled to l/A” with ether. This was just enough to reach the glass tube coming from the first receiving flask. Care had to be taken when preparing because of the explosion. hazard. RESULTS Analyses for pp' DDT metabolites. In this experiment the earlier observations made by Butcher, Kirknel and Zabik I969, that pp"DDT.was metabolized to pp' DDE, were confirmed (Table 2). 'Analysis was made I, 6, l2, and 22 days after the Collembola were introduced into the agar. After 22 days the Collembola extract contained between 23% and 33% pp' DDE compared to pp' DDT. The metabolism ' did not seem to be affected by the antibiotic added to the culture tube, Tables I, 2, 3 and A, and pp' DDA could not be found in any of the extracts. A compound with the same retention time as the methylated acid did show up In all 3 extracts but could not be identified on thin layer plates as the methyl ester of pp' DDA. As Table 3 shows, the concentration of pp' DDT in the Collembola in some cases reached l000 ppm. After 22 days the con- centration of pp' DDE was higher. The cultures after 22 days did not have the same viability as they had in the beginning. An explanation for the decreaSed concentration of pp' DDT in the Collembola might be because they stopped feeding. Support for this argument may be derived from the follow- ing observations. After l2 days the Collembola in the culture tube were split into two groups. One group remained in the culture tube with 20 ppm pp' DDT and the other group was transferred to a culture tube with l-l/2% agar but no pp' DDT. Table 5 shows these Collembola have almost the same relationship between pp' DDE and pp' DDT as the first group. Concentrations of DDT in the Collembola are shown in the Tables I, 2, 3, A and 5 and the Figures 5, 6, 7, 8, 9 and lo. Analysis for pp' TDE metabolites Neither the pp' DDE nor pp' DDA methyl ester metabolites of pp' TDE were found. ‘However, an unidentified compound appeared in the Collembola extract in the GLC analysis, with the same retention time as op' DDE (l.77 min.), and the concentration increased slightly with time. It could not be identified on thin layer plates as op' DDE. Measured in GLC as Op' DDE the relationship between pp' DDT and the compound in Collembola - extracts after 3 days was l00: 3.6 and l00: l.8 in experiments with anti- biotic. In experiments without the antibiotic the relationship was IOO: l.5 and ICC: l.l. After l6 days, in experiments with the antibiotic, the rela- tionship was l00: 6.5 and loo: 3.]. Without the antibiotic it was l00: A.5 and l00: h.5. The antibiotic did not seem to affect the presence of this compound. In the experiment designed to observe metabolism of pp' DDT to pp' DDE, pp' TDE was present. The experiment described here with pp' TDE shows that metabolism to pp' DDE started out with pp' DDT. Evidently pp' TDE was not metabolized to pp' DDE in the Collembola. As in the previously described experiment with pp' DDT, a compound with the same retention time as the methyl ester of pp' DDA showed up in the GLC analysis after methyl- ation of the extracts. It was found in all three extracts, but it was not possible to confirm the finding on TLC. Concentrations of pp' TDE in the Collembola are shown in Table 6 and the Figures l and 2. Analysis for op' TDE No op' TDE metabolites were detectable with the above described analyti- cal tools, not even after methylation with diazomethane. Concentrations of .l0 op' TDE in the Collembola are shown in Table 7 and in Figures 3 and A. DISCUSSION AND CONCLUSION This study confirms that in the presence of Folsomia fimetaria, pp' DDT is metabolized to pp' DDE rapidly and in relatively large quan- tities. This substantiates earlier work by Butcher, Kirknel and Zabik I969. However, it has not been possible to Show metabolism of pp' DDT to pp' DDA. Neither was it possible to show any metabolism of either pp' TDE or op' TDE to pp' DDE, op' DDE, methyl ester of pp' DDA or the methylester of op' DDA." The data presented here appear sufficient to justify these conclusions. 0n the other hand had the technique been different, further information could have been obtained. A ring labelled isotope would have given better Information about the real rate of metabolism. Furthermore, variations from culture tube to culture tUbe were in some cases quite large. It would have been better for example if the Collembola which were extracted at different times had been taken from the same culture. Although these and earlier studies do not deal with the mechanism of pp' DDT conversion to pp' DDE, it is felt that Collembola gut flora or those associated with the media substrate may be involved. Collembola reared from surface sterilized eggs in sterile culture, with sufficient nutrition, may provide answers to this question. Some preliminary work has been done on this problem, but results are inconclusive. Also, consideration has been given to the possibility that detoxifying Collembdlan enzymes are involved. This work is continuing. ll DISCUSSION AND CONCLUSION This study confirms that in the presence of Folsomia fimetaria, pp"DDT is metabolized to pp' DDE rapidly and in relatively large quan- tities. This substantiates earlier work by Butcher, Kirknel and Zabik I969. However, it has not been possible to show metabolism of pp' DDT to pp' DDA. Neither was it possible to show any metabolism of either pp' TDE or op' TDE to pp' DDE, op' DDE, methyl ester of pp' DDA or the methylester of op' DDA.’ The data presented here appear sufficient to justify these conclusions. 0n the other hand had the technique been different, further information could have been obtained. A ring labelled isotope would have given better information about the real rate of metabolism. Furthermore, variations from culture tube to culture tUbe were in some cases quite large. It would have been better for example if the Collembola which were extracted at different times had been taken from the same culture. Although these and earlier studies do not deal with the mechanism of pp' DDT conversion to pp' DDE, it is felt that Collembola gut flora or those associated with the media substrate may be involved. Collembola reared from surface sterilized eggs in sterile culture, with sufficient nutrition, may provide answers to this question. Some preliminary work has been done on this problem, but results are inconclusive. Also, consideration has been given to the possibility that detoxifying Collembolan enzymes are involved. This work is continuing. ll LITERATURE CITED Abedl, Z.H., J.R. Duffy and A.w.A. Brown, 1963. Dehydrochlorination and DDT-resistance in Aedes aegypti. J. Econ. Entomol. 56: 5Il. Abelson, P.H., 1957. Some aspects of paleobiochemistry. Ann. N.Y. Acad. Sci.-69: 276. Agosin, M., D. Michaell, R. Miskus, S. Nagasawa and w.N. Hoskins, 1961. A new DDT metabolizing enzyme in the German cockroach. J. Econ. Entomol. 5A: 3A0. Brown, A.W.A., 1960. Mechanisms of resistance against inSecticides. Ann. Rev. Entomol. 5: 301-326. Bull, D.L. and P.L. Adkisson, I963. Adsorption and metabolism of CIA-labeled DDT by DDT-susceptible and DDT-resistant pink bollworm. J. Econ. Entomol. 56: 6A1. ' Chacko, C.I., J.L. Lockwood and M. Zabik, I967. Chlorinated hydrocarbon pesticides: Degradation by microbes. Science, ISA, No. 3750, 893-895. Edwards, C.A. and K.A. Jeffs, 196A. The persistance of some insecticides in soil and their effects on soil animals. Proc. Xllth Int. Cong. Entomol. 559-560. Finley, R.B. and R.E. Pillmore, 1963. Conversion of DDT to TDE in animal tissue. Am. Inst. Biol. Sci. Bull. 13: Al. Guenzi, w.D. and W.E. Beard, 1967. Anaerobic biodegradation of DDT to 000 in soil. Science, 156, No. 3778. lil6-Ill7. Kallmann, B.J. and A.K. Andrews, I963. Reductive dechlorination of DDT to 000 by yeast. Science, IAI: 1050. ' Miskus, R.P., 0.P. Blair, and J.E. Casida, 1965. Conversion of DDT to 000 by bovine rumen fluid, lake water and reduced porphrins. J. Agr. Food Chem. 13: A81. Perry, A.S., S. Miller and A.J. Buckner, 1963. Theenzymatic in vitro degradation of DDT by susceptible and DDT-resistant body lice. J. Agr. Food. Chem. 11: A57. 12 l3 Scopes, N.E.A. and E.P. Lichtenstein, I967. The use of Folsomia fimetaria L and Drosophila melaanaster as test insects for the detection of insecticide residues. J. Econ. Ent. V. 60, No. 6, pp. 1539'15A1. Sternburg, J., c.w. Kearns and H. Moorefield, 195A. DDT-dehydrochlorinase, an enzyme found in DDT-resistant houseflies. J..Agr. Food Chem. 2: I125. i ' 1A / I Table 1. Experiment extracted after 1 day. ppm in the . Collembola x 100 100 x 100 x 100 x Culture Culture pp'TDE pp'DDE ppIDDE Tubes Replic. Compound pp'DDT.pp'TDE.pp'DDE. pp'DDT pp'DDT pp'TDE With I) Collembola 2.60 0.36 0.08 I3 3 20 Collembola ' surface 16 2 IO agar 18 I 6 With ‘ 2) Collembola 3.00 0.56 0.08 IA 3 19 antibiotic . surface 20 0 0 agar IA 1 6 'With 1) Collembola 1.5A 0.22 0.0A 1A '2 l7 Collembola surface 12 l 7 agar - IA 1 7 Without 2) Collembola 2.3A 0.5A 0.06 18 3 15 antibiotic . surface 1A 1 7 agar I2 1 8 Without 1) surface 16 2 12 Collembola agar 15, l 7 With 2) surface 16 2 , 10 antibiotic agar 15 0.5 3 Without 1) surface 12 I 7 Collembola agar l2 1 7 Without .2) surface Analysis not made. antibiotic agar 17 0 0 15 Table 2. Experiment extracted after 6 days. ppm in the Collembola x 100 ' 100 x 100 x 100 x Culture Culture pp'TDE pp'DD§_ pp'DDE Tubes Replic. Compound pp'DDT.pp'TDE.pp'DDE. pp'DDT pp'DDT pp'TDE With - I) Collembola 1.62 0.22 0.08 13 5 38 Collembola ' . I surface 1A 1 8 agar 11 0.A A With ' ' 2) Collembola 1.27 0.16 0.07 13 15 A3 antibiotic surface 12 ' 0.3 3 agar 12 0.A 3 With 1) Collembola 2.22 0.30 0.13 IA 6 A2 Collembola surface . l6 1 A agar I6 0 0 Without 2) Collembola 1.76 0.23 0.09 13 5 38 antibiotic surface 1A 0 0 agar i 10 0.3 3 Without 1) surface 18 1 3 Collembola agar 15 0.A 3 With 2) surface 12 l 5 antibiotic agar 12 1 A Without 1) surface 13 0.2 ' 2 Collembola . . agar 12 0.A 3 Without 2) surface 12 1 5 antibiotic agar 12 0.A A J6 Table 3. Experiment extracted after 12 days. ppm in the Collembola x 100 100 x 100 x 100 x Culture Culture ' pp'TDE pp'DDE pprDE Tubes Replic. Compound pp'DDT.pp'TDE.pp'DDE. pp'DDT pp'DDT pp'TDE With 1) Collembola 7.25 0.97 1.09 13 15 112 Collembola ' surface 12 1 12 agar 15 0 0 With ‘ ° 2) Collembola 11.A 1.37 0.93 12 I 8 68 antibiotic surface 10 1 12 agar 11 0 0 With ' 1) Collembola n.35 0.72 0.A5 17 10 63 Collembola - surface 15 2 13 agar . 1A. 0 0 Without 2) Collembola 8.32 1.08 1.32 13 16 122 antibiotic surface 12 1 6 agar l6 0 0 Without 1) surface . 10 i 0.3 3 Collembola agar 13 0 0 With 2) surface 11 0 0 antibiotic . agar 12 0 0 Without . 1) surface 11 0 O Collembola agar 13_, 0-3 3 Without 2) surface 1A 0 antibiotic agar 13 0 0 17 Table A! Experiment extracted after 22 days. ppm in the Collembola x 100 100 x 100 x 100 x Culture Culture pp'TDE pp'DDE pp'DDE Tubes Replic. Compound pp'DDT.pp'TDE.pp'DDE. pp'DDT pp'DDT pp'TDE With 1) Collembola 5.A0 0.69 0.97 13 19 IA] Collembola ' ' surface . 15 ' 2 12 agar ' 10 l 8 With 2) Collembola 5.35 0.90 1.50 17 28 167 antibiotic - ~ ' surface 16 3 l9 agar 12 1 11 With 1) Collembola 3.21 0.72 1.18 22 37 165 COllembola . surface 11 2 22 agar 9 l 9 Without 2) Collembola A.76 0.80 1.36 17 28 1A5 antibiotic surface 10 3 30 agar 12 1 8 Without‘ 1) surface 11 1 9 Collembola agar 9 I 6...... With 2) surface 12 2 17 antibiotic agar 10 l 7 “7 Without 1) surface 11 0 0 Collembola agar - 13 0.3 3 Without 2) surface ' 22 0 0 Antibiotic agar 21 0 0 18 Table 5. Collembola exposed to pp' DDT for 12 days and then transferred to non-pp' DDT agar. Culture Culture Tubes Replic. Compound ppm in the Collembola x 100 100 x 100 x 100 x pp'TDE pp'DDE pp'DDE pp'DDT.pp'TDE.pp'DDE ' p DDT pp DDT pp TDE p ,With 1) , Collembola A.60 0.39 0.7A 8 16 190 antibiotic - 2) Collembola 2.87 0.32 ‘ 0.8A‘ ‘ '11' ‘ 29 ' 266 Without _ 1) ' Collembola 2.51 0.26 0.80 12 32 '257 antibiotic ’ ‘ 2) Collembola 2.91 0.38 1.00 ‘ ‘ 13“ ‘ 3A‘ ' 26A Table 6. ppm pp' TDE x 100 in Collembola after exposure. Culture tubes Replic. 3 days 7 days" ' 12 days 16 days With 1) 2.19 2.70 1.A3 3.26 antibiotic 2) 2.90 1.80 15.00 ‘5.36 Without I) 1.90 6.20 3.72 12.00 antibiotic 2) A.70 7.A0 "‘l.80 1.90 Table 7. ppm Op' TDE x 100 in Collembola after exposure. Culture tubes Replic. 3 days 7 days 12 days 16 days With 1) 7.80 18.00 11.20 .. 6.211 antibiotic 2) ‘6.60 10.00 ' 8.15 57.00 Without 1) no analysis made 12.60 10.80 A.AA - antibiotic 2) A.80 8.10 __10.00 _ A1.2 19' FIGURE I '5; J 12. ppm pp' T01: InCoIIam- x 102 O . FIGURE 2 3’8 .' I .1 01.: .' 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