Document 910EyQxd15nbnOekrYGnEDbkp

TOE DEGRADATION OP POLYCHLORINATED BIPHENYLS BY MICRO-ORGANISMS R. A. Baxter, P. E. Gilbert, R. A. Lidgett J. H. Mainprlze, H. A. Vodden MONSANTO EUROPE SA Monsanto Technical Center Rue du Laid Burniat Louvain-la-Neuve, Belgium SUBMITTED FOR PUBLICATION SUBJECT TO REVIEW DSW 032886 STLCOPCB4016848 The Drgrodotion of Polychlorinated Biphenyls by Micro-Organisms Multicomponent mixtures of polychlorinated biphenyls (PCBs) have been used In a variety of industrial applications and some of these components have been found in the environment. This subject has been reviewed extensively - for example, Edwards (1971). The persistence of some PCBs has been a cause for concern and it is therefore important to establish possible fates of PCBs in the environment. The metabolism of PCBs in pigeons and quail have been studied by Bailey and Burtyan (1972) who found that compounds with a relatively low degree of c lor (nation were fairly rapidly metabolised and are not as persistent as the more highly chlorinated compounds. Similar results had been obtained by Grant et al (1971) in metabolic studies in the rat where it was found that tissue residues of the lower chlorinated components were significantly less than the residues of the more highly chlorinated ones. Of the various natural mechanisms for degrading organic materials, micro-organisms would be expected to make a significant contribution. We have therefore studied microbial degradation of some PCB compounds and commercial mixtures in the laboratory. Early experiments were made using activated sludge inocula from the biological effluent treatment plant of Monsanto Chamicals Ltd., at Ruabon, North Wales, pre-adapted to degrade biphenyl. During this phase of the work the experimental techniaues were developed and some degradation of PCBs was observed. Meanwhile, work by Lunt and Evans (1970) on microbial degradation of biphenyl was brought to our attention ; ws obtained from Professor Evans some of the culture, classified as a Nocardia spp. (NCIB 10603), and studied its effects on PCBs. later we obtained another organism. Pseudonomas spp. (NCIB 10643), also described by Lunt and Evans 0972) and studied the effects of. this culture on selected PCBs. Since this work was completed Ahmed and Focht (1973) have reported on the biodegradation PCBs by a species of Achromo bacter. and Gibson et al (1973) have confirmed the metabolic pathway from biphenyl oxidation which was postulated by Lunt and Evans. DSW 032887 STLCOPCB4016849 EXPERIMENTAL 2 Essentially two series of experiments have been carried out, the firat (Series a) dealing with single substances or simple two or three compound mixture* and the second (Series B) investigating the behaviour of complex multicompound mixtures obtained commercially by chlorination of biphenyl. The basic experimental procedure consisted of shaking prepared substrates comprising PCBs, inocula, mineral solutions, inhibitors, etc., for various periods of time and determining the loss of PCB by gas chromatographic analysis of extracts. In this work only primary degradation was measured and the products of metabolism have not been identified. Preliminary experiments showed that the rates of degradation were increased by the addition of biphenyl* in many of the experiments, therefore, biphenyl was added initially and subsequently, at intervals, during extended periods of exposure. Stocks of the active organisms received from Professor Evans were prepared and maintained according to his recommendation as follows:* A prepared growth medium (Table l) was inoculated with the culture and the mixture was continuously aerated in the dark at 25C using filtered and scrubbed air. Inocula for subsequent experimental substrates were withdrawn after stopping the air flow for a short time to allow the biphenyl to settle. The media were prepared in 15 litre quantities and, as prepared, had a pll of approximately 5*8* this was adjusted to pH 7*0 with sodium hydroxide DSW 032888 STLCOPCB4016850 solution before introducing the inoculum. When small volumes 5 (1-2 litres) of stock culture solution were needed the bottle was replenished with an equal volume of growth medium. For larger quanti ties a fresh culture was started. ^ TABLE I Media for Culture of Biphenyl-Degrading Organisms Constituent Ammonium Sulphate Potassium Dihydrogen Phosphate Magnesium Sulphate (71^0) FerrouB Sulphate v Biphenyl Concentration 1.0 1.0 0.5 0.5 1.0 The following procedure was used to prepare samples for bio degradation :- A solution (5 ml) of diethyl ether containing 100 ppm of PCB wbb pipetted into a 250 ml glass stoppered conical flask. To this was added (for those samples subjected to biodegradation in the presence of biphenyl) a second diethyl ether solution (5 ml) containing 1000 ppm biphenyl. The ether was completely removed by evaporation using dry nitrogen at ambient temperature and this was followed by addition of the oulture solution (100 rale). '. Two agitation systems were used in this study, A Monsanto built shaker (120 cyclec/minute, ambient temperature) wa3 used for all the (series a ) single compounds and simple mixtures^while a Callenkomp orbital incubator (lJO cyoles/min., 25C) was used for oomraercial PCB mixtures. (Series B) DSW 032889 STLCOPCB4016851 V-/. 4. Flasks were vented at 7 day intervals to ensure that an adequate supply of air was available in the system and, for those flasks containing exoess biphenyl, this was maintained by adding solid biphenyl during the venting procedure. For each experiment with a single compound in which biodegradation was attempted( either in the presence of biphenyl or alone) a control sample duplicating the test sample but containing 100 ppm of mercuric chloride was run simultaneously. The mercurio chloride prevented bacterial aotion and enabled a check to be made on any FCB losses arising from the experimental method, e.g. from evaporation, adsorption etc. In praotically all cases it was found that more than 90$ of the PCB was recovered in the procedure described here; later experiments dispensed with the mercurio chloride controls, giving greater shaker capacity for teBt samples. On removal from the shaker or incubator the total contents of each shake-flask were transferred to a separating funnel (2^0 ml), the flask was washed with pentane (2 x 10 ml) and the waohings added to the oontents of the separating funnel. The aqueous phase was then extracted with pen tane (4 x 25 mla){ the pentane extracts were bulked ready for concentra tion and gas chromatographic analysis. In all experiments the total oontents of each shake-flask were used for analysis, oinoc it was suspected that adsorption effects might invali date the results if aliquqts were taken. This suspicion was verified sub sequently by direct experiments, whioh indicated |hat up to 90$ of the PCB DSW 032890 STLCOPCB4016852 V 5. could remain on the flask wall, although virtually 100% reoovery was aohieved using the solvent extraction method described. Thus the study of each compound or mixture required several flasks as follows:- PCB pluB culture PCB plus culture plus biphenyl PCB plus culture pluB biphenyl plus HgCl^ When studying single compounds and simple mixtures three sets of flasks were used. Concentrations of PCB, initially and after several days shakeflask exposure were determined and significant biological degradation assessed by comparison with the inhibited controls. If substantial de gradation was observed the experiment was terminated, but, where necessary, exposure was continued for a further period to establish whether any degradation could be observed. When commercial PCB mix tures were studied, however, five sets of flasks were used so that residual concentrations of PCB components could be determined initially and after 10, 20, JO and 60 days exposure. Concentration and Gas Chromatography Two methods for concentration and gas chromatography were ueed for the samples as followst- SBRIKS A Single Compounds and Simple Mixtures - For these samples simple ooncentr&tion and gas ohromatographio methods were used because the PCBb were present in relatively large amounts and were easily measured using a packed column and electron oapture detector:- The pentane was reduced to a volume of approximately 2-3 mis using a stream of dry nitrogen and a warm water bath. The concentrate was then DSW 032891 STLCOPCB4016853 \s 6. analysed for PCB using the following gas chromatogTaphio con- ditionsi Instrument Column Temperature Sample size Internal Standard Detector Carrier Gas - Hewlett Packard 402 - Silicone Oil 0V101 on Gas Chrom Q 60/80 mesh. - Between 150C and 250C depending upon oompound. - 5 pl - Usually a second PCB oompound - Ni^ Eleotron capture operated at 280C. - Argon/Methane 95/5 SERIES B. Complex Commercial Samples - For these samples special concentration and gas chromatographic methods were needed. V Because of the complexity of the samples (up to 70 components) a method capable of separating as many components as possible prior to measurement was required and a capillary column system was therefore used. Since each component could be as little as 1> in the original PCB (i.e. 0.05 ppm) and upon degradation this could drop to l^C of that concentration (i.e. 0.0005 ppm) a method with a high concentration factor was essential. The pentane solution was therefore initially reduced to approximately 5 min when a known weight (O.lg) of deoane wsb added. The solution was further concentrated using dry nitrogen and a water bath until the pentane had been completely removed (checked by reweighing). For an initial so lution of 100 mis containing 5 PP of PCB this was equivalent to con centrating to 5000 ppm and for a residual PCB of 0.1 ppm a final level of 100 ppm oould be obtained. DStt 032892 STLCOPCB4016854 7. The PCB concentrate in decane vas then analysed by gas chromatography using the following conditions Instrument Column Temperature Sample Size Detector Cartier Gas r A M Model 8l0 300' x 0.030" I.D. Stainless Steel capillary coated with Apiezon L according to the method of E.J. Malec (I97l) 220WC 2 ul Split 30:1 Ni^ Electron Capture operated at 28oD Argon/Methane 95/5 As the biodegradation study was concerned with relative losses of PCB isomers the known variability in response of the electron capture detector to components Of different chlorine level and structure was not a problem. Reduction in any component was determined by comparison of the component peak area in the chromatogram of the degraded sample with the peak area in the original samplef correction being made if necessary for the use of different sample sizes or concentrations. PCB samplos were either obtained from commercial laboratory suppliers or were synthesised by tho method of Bain et al (1962) which is particularly well suited for laboratory preparation of specific chlorinated biphenyls. Tho purity of all samples was checked by g.l.e. examination and shown to be 3 99* in all cases. OSW 032893 I STLCOPCB4016855 RESULTS Series A Six single compounds, one 3" component and two 2-component mixtures hare been exposed to the micro-organism cultures described by Lunt and Evans. The loss of PCB was measured in each case by packed column g.l.c. with the results shown in Table II which gives the %age loss after a given period of time. Results are also shown in which biphenyl was added as additional substrate. Table II Compound 2,4 *- dichlorobi phenyl " * biphenyl 4,4 <-dichlorobiphenyl M + biphenyl 2,3"dichlorobiphenyl " 3 4- dichiorobiphenyl " biphenyl + biphenyl 2*3*2 -trichlorobiphenyl M * biphenyl 2,3 '|4 *- trichlorobiphenyl " + biphenyl 2,5*4,-tri chlorobi phenyl " + biphenyl 3,4,3 -trichlorobiphenyl M biphenyl % Degradation/Days NC1B 10603 70/7 100/7 nil/121 nil/121 67/8 64/8 80/8 100/8 50/7 95/7 94/7 100/7 nil/73 60/73 76/12 70/12 ncib io643 60/73 100/73 50/15 50/10 - - - - 15/73 60/73 - OSW 0 3 2 8 9 4 STLCOPCB4016856 Compound 2*4,6-trichiorobiphenyl " 4 biphenyl 2*4*2 ,4 *-tetrachlorobiphenyl n 4 biphenyl 2*4,6,2 -tetrachlorobiphenyl > 4 biphenyl 2,3,4,5,2 *3 -hexachlorobiphenyl 4 2,3,2 *- and 2*3 '4*-trichlorophenyl " 4 biphenyl w 9 % Degradation/Days NC1B 10603 nil/12 nil/12 nil/9 nil/9 nil/9 nil/9 nil NC1B 10643 nil/84 nil/84 - - - 50/n > - Series B Two multicomponent PC0mixtures have also been exposed to these cultures. The first of these* Aroclor 1242, is a chlorination product of biphenyl containing 42% chlorine. The second, Aroclor 1016, is a similar mixture from which the high boiling components have been removed. The %age degradation wan determined after periods of 52 and 100 days using capillary column g.l.c. with the results shown in Table III. Time (days) 52 10O Table III 1242 10603 10643 88 76 95 85 1016 10603 10643 96 >98 91 >96 DSW 032895 STLCOPCB4016857 W 10. The components spearated by capillary g.l.c. hare been identified by the ethoda described by Sissons and Welli (1971) and the percentage degradation for each component calculated from comparative peak heights. Analyses were made after 0, 10, 20, 30 end 60 days'exposure and the change in percentage refiidue with time for a ntraber of significant components comprising approx. 90^ of the total starting material for Aroclor 1242 exposed to NC1B 10603 are shown in figs. 3 and 4 Typical chromatograms of Aroclor 1242 and the same material after degradation are shown in fig* 1 &2. Similar results were obtained with $4.C1B 10643 but are not given in detail here. v V DSW 032896 STLCOPCB4016858 DISCUSSION The work reported here shows oulte clearly and In agreement with Ahmed and Focht, that many PCBs can be degraded by enzyme hydroxylation Induced by micro organisms. The results horn Series A on single compounds and simple mixtures showthat under the experimental conditions many of the lower chlorinated biphenyls (^3 chlorine atoms /molecule are readily degraded and that even certain compounds containing as many as 6 Cl atoms could be degraded in favourable circumstances. There is some evidence that the pattern of chlorination will influence the ease of biodegradation butjnsufficient specific compounds were available to explore this fully. Hrwever, it is clear that under the experimental conditions some compounds are much more readily degraded than others. When the results from Series B on the complex mixtures are examined a different pattern emerges which would not be expected from the single compound work. Some of the lower chlorinated compounds which appear to be only slowly degraded when alone, degrade quite readily when present in the mixture. For example, 4,4'-dichlorobiphenyl degrades to 50 % In approximately 2 days when exposed to Nl CB 10603 as a component of Aroclor 1242 whereas it is virtually unaffected after 12 days when exposed alone. Mutual solubilisation may play some part in the effect and this conclusion is supported by the observation that 2, 3, 4, 5, 2' 3' - hexachlorobiphenyl can be degraded in a three component mixture but not when it is exposed alone. However, 4'4 -dichlorobiphenyI could not be similarly induced to degrade when mixed wi th 3, 4, 2' and 2, 3, 2'-trich!orobiphenylsand a more complex mechnism seems likely. The curves showing the pattern of degradation for some of the significant components in Aroclor 1242 (figs. 3 and 4) tend to fall into three groups. Those compounds containing only one or two chlorine atoms degrade rapidly. In less than 10 days. Another group, consisting mainly of trichlorobiphenyls , seems to OSW 032897 STLCOPCB4016859 12 degrade rapidly after an initial induction period, but then tends to a slower rate when the level has dropped to about 109#, The third group, primarily of tetrachlorobiphenyls, degrades sore slowly. This pattern of degradation can be explained in terms of adaptation of the organisms. Mono- and dichiorobiphenyls can probably be degraded with little or no adaptation of the biphenyl-degrading organisms. For more highly chlorinated components, however, adaptation may be necessary and the ease with which this may be accomplished will depend on the concentrations of precursors available. The induction times observed for many of the components probably indicate `these adaptation periods. There are a number of possible explanations for the apparent decrease in rate of degradation later on. The viable cell population may have dropped considerably, the g.l.c. analysis may not give adequate isomer resolution or, simply that the accuracy available at such low concentrations of individual components may not reflect the true shapes of the degradation curves in this region. The observation of enhanced biodegradation of certain PCBs by addition of biphenyl to the substrate may indicate a co-metabolic mechanism (Horvath 1972). Similarly orgnnisms which have become adapted to the simple PCBs may then become capable of metabolising the more complex structures. More detailed study of the nature of the metabolic products would be necessary to elucidate these mechanisms but it seems clear from the present study that examination of the behaviour of single PCBs does not enable predictions to be made on the biodegradability of complex mixtures. DSW 032898 STLCOPCB4016860 In the natural environment It can be expected that there will be a number of organisms capable of degrading the PCBs and a mixed culture oeting on a multicomponent mixture of PCBs mfghfoe expected to give more complete degradation than do single organism.*. Further work in a natural environmeM? Is intended to investigate this possibility. DSW 032899 STLCOPCB4016861 'w 'sS 14 ACKNOWLEDGEMENTS It is a pleasure to reoord our thanks to Professor W.C. Evans, Mr. D. Lunt and Mr. C. Harrison of University College, Bangor, for supplies of mioro-organiBniB and useful advice. We are also indebted to Dr. J.P. Brown of Monsanto Chemioala Ltd. for stimulating disouaeion. R.A. Baxter P.E. Gilbert R.A. Lidgett J.H. Mainprize H.A, Vodden Nickell Laboratories, Monsanto Chemicals Ltd*, Cefn Mawr, Wrexham, Denbighshire. DSW 032900 STLCOPCB4016862 15. REFERENCES 1. Ahmed M. and Focht D.D. (1973) Bull. Environmental -Contam and Toxicology. 10, 70 2. Anon (1966) - Now Scientist, 612 3. Bailey ond Bunyan (1972). Nature 236, 34 4. Bain etal. (1962) Proc Chem. Soc. 186 5. Edwards, R. (1971) -Chem. &ind., 1340 6. Gibson. D.T, etal. (1973). Biochemical & Biophysical Research Comm. 50, 211 7. Grant et al. (1971) Bull. Environmental Contam & Toxicol. 6, 102 8. Horvath R.5. (1972) Bacteriological Reviews, 36, 146, 155 9. Lunt, D. and Evans, W.C. (1970)- Biochem. J., 120 10. Lunt, D. and Evans, W.C. (1972) - Biochem. J. , In Press 11. Mnlec, E.J. (1971)- J. Chromatographic Sci. 12. Sissons, D. and Welti, D. (1971)- J. Chromatogr. 60, 15. / DSW 03290L STLCOPCB4016863 STLCOPCB4016864 STLCOPCB4016865 STLCOPCB4016866 ERCENT RESIDUE 0-R P0 <o \ \ DSW 032905 1 A ... .A 4> ... A to ao 30 lfX> 60 iO TVie :* STLCOPCB4016867