The effect of DDT on the growth and respiration of pseudomonads under aerobic and anaerobic conditions Donald A. Paul Hopkins Marine Station of Stanford University Pacific Grove, California 93950 INTRODUCTION The widespread use of insecticides and their accumula- tion in the environment has prompted several studies on the effects of these pesticides on the activity of soil micro- organisms. Jones(3) reported no inhibition of ammonification, nitrifi- cation, or the oxidation of reduced sulfur compounds by DDT, 1,1,1-trichloro-2,2-bis(p-chlorophenylethane), at levels between 0.O1 and 0.001 per cent. Inhibition was observed at levels of 0.1 per cent. These studies were carried out using soil samples incubated in open containers for periods up to three years. Similarly, he found no effect upon nitrogen fixation even at DDT levels as high as 1.0 per cent. A more recent study by Bartha et al.(1) indicated no in- hibition of aerobic oxidation of glucose in soil samples con- taining 150 parts per million DDT. Nor was an effect observed on the process of nitrification at this concentration of DDT, although DDE was observed to have a stimulatory effect at the same concentration. In neither the studies of Jones nor Eartha was an attempt made to account for the loss of DDT from the preparations by codistillation during the lengthy incubation periods The present study was made using elective cultures, with 2 -2- sodium acetate as a carbon source and either oxygen or nitrate as a terminal electron acceptor. The use of such preparations allowed a study of growth as well as metabolic activity, and a more precise control of DDT concentration during experiments. In addition, the range of concentrations of DDT used in this study is comparable to that of the environment. In areas not subjected to the repeated application of insecticides, residues as high as 20 ppm have been found(7); while in orchard soils subjected to regular application of pesticides, residues have been reported as high as 60 ppm(5). METHODS The medium used elects from soil innocula organisms cor- responding to aerobic pseudomonads and, with the addition of nitrate, similar bacteria capable of denitrification. The medium contained 0.036% NahpPO H2o, 0.07% KpHPOL, 0.05% Mgson. 0.005% Cacl», 0.005% Fecl3, and 0.5% NacH302. For aerobic cultures 0.12 NHLCI was added to the above, the pH adjusted to 7.0; for anaerobic cultures, 0.2% KNO3 was added, the pH ad- justed to 7.5. Garden soil was used as an initial innoculum, and in- cubation was carried out at 28 C.. Sub-culturing was necessary to maintain actively growing cultures. Growth experiments were carried out in sidearm Erlenmeyer flasks containing the medium and DDT in concentrations ranging from ,02 to 200 ppm. Cultures were innoculated, and turbidity -3- was measured in a Klett-Summerson photoelectric colorimeter. using a green filter. Sterile blanks were prepared similarly, A stock solution of DDT--obtained from the Aldrich Chemical Co., Inc--was prepared by dissolving 1.0 gram in 100 ml. of 958 ethanol. Necessary dilutions were carried out using distilled water in most cases. The highest final concentration of ethanol in any of the cultures was 2.0%; no effect upon growth was noticed at this level. Respiration of aerobic cultures was measured manometrically at 30'C. in Warburg flasks containing 0.036% NaH»PO'H,O, 0.07% KoHPO, and 0.5% NaCH0, as a carbon source. RESULTS The elective cultures developed rapidly within 24 hours and produced a water soluble, green, flourescent pigment, charac- teristic of members of the genus Pseudomonas. The appearance of aerobic and anaerobic cultures was identical. Figure 1 presents the growth of the aerobic cultures in the presence of 0, 0.02, 0.2, 2, 20, and 200 ppm DDT. A lower growth rate and less total growth was observed with increasing concentrations of DDT. Anaerobic cultures, Fig, 2, exhibited no growth inhibition by DDT, the growth rates and total growth being comparable in the three concentrations used. The measurement of growth by turbidity was made diffscult by the turbidity of DDT at concentrations of 20 ppm and above, The effect upon growth should therefore be confirmed by additional -4. methods of measurement. No effect upon respiration was detected over this same range of DDT concentrations in the aerobic cultures. DISCUSSION This study is only preliminary. Earlier investigations in- dicated an effect by DDT only at much higher concentrations, e.g. exceeding 1 per cent. DDT may affect the growth of bacteria at much lower concentrations which are not uncommon in unsprayed areas, e.g. 20 and 200 ppm, or .002 and .02 per cent. The absence of any effect on anaerobic pseudomonads requires further investigation. Wedemeyer(6) has found that Aerobacter aerogenes can dechlorinate DDT under anaerobic conditions, but che process is hindered greatly in the presence of oxygen. tenersen obtained similar results with E. coli(4), and a study by Guenzi and Beard(2) showed that C+ DDT incubated anaerobically in soil was broken down to DDD, DDE, and other derivatives. Whether the pseudomonads, during the process of denitrification, are also capable of altering the structure of DDT has yet to be determined. SUMMARY Growth of aerobic pseudomonads in elective cultures was significantly inhibited by DDT in concentrations as low as 2 ppm. Anaerobic cultures showed no inhibition by DDT even at 200 ppm. No effect upon aerobic respiration was detected. 2 O ACKNOWLEDGEMEN" This work was supported in part by Grant GY-5878 from the Undergraduate Research Participation Program of the National Science Foundation. I would like to express my thanks to Dr. J.H. Phillips and Ray Markel for their advice and assistance. Figure 1: Figure 2: FIGURE LEGENDS Growth of aerobic culture of pseudomonads in the presence of DDT, as measured by turbidity. —0—0- ho DDT; -0—0-.02ppm DDT;-A—/-.2ppm DDT;-A—A-2ppm DDT; ++ 20ppm DDT;-E— 200ppm DDT. 100 ml. cultures were incubated at 30 C. in sidearm flasks without shaking; turbidity was measured in Klett-Summerson photoelectric colorimeter. Growth of anaerobic culture of pseudomonads in the presence of DDT, again measured in the Klett- Summerson colorimeter. -o-9-.02ppm DDT;-A—A 2ppm DDT; -E— 200ppm DDT. Same conditions as for aerobi¬ cultures. . 4 4. O CE D 8 E E 8 9 + + O 5. REFERENCES CITED 1. Bartha, R., R.P. Lanzilotta, and David Pramer, Appl Micro- biol 15(1): 67-75, 1967. 2. Guenzi, W.D. and W.D. Beard, Science 156(3778): 1116-1117, 1967. 3. Jones, L.W., Soil Science 73(3): 237-241, 1952. 4. Stenersen, J.H.V., Nature 207(4997): 660-661, 1965. 5. Terriere, L.C., Ulo Kiigemagi, R.W. Zwick, and P.H. Westegar in Organic Pesticides in the Environment. American Chemical Society, 1967. pp. 263-270. 6. Wedemeyer, G., Science 152(3722): 647, 1966. 7. Woodwell, G.M., C.F. Wurster, Jr., and P.A. Isaacson, Science 156: 821-824, 1967.