Arch Microbiol (1992) 158:282-286
Archives of
Microbiology
9 Springer-Verlag 1992
Transformation of synthetic pyrethroid insecticides by a thermophilic Bacillus sp. S. E. Maloney ~, A. Maule 1, and A. R. W. Smith 2 1 Divasion of Biotechnology, Public Health Laboratory Service, Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire, SP40JG0 UK z School of Biological and Chemical Sciences, Thames Polytechnic, Wellington Street, London, SE18 6PF, UK Received December 10, 1991/Accepted April 8, 1992
Abstract. Employing a mineral salts medium containing Tween 80 as the primary carbon source, a strain o f Bacillus s t e a r o t h e r m o p h i l u s was isolated which was able to hydrolyse selected second and third-generation pyrethroids to non-insecticidal products. Of a range of pyrethroid insecticides the t r a n s - i s o m e r of permethrin was the most readily transformed by this microbial isolate, whilst flumethrin was the least. 3-Phenoxybenzoic acid and the respective halovinyl or haloacid moieties were detected as the major hydrolytic products of the pyrethroids. It is believed that 3-phenoxybenzoic acid was formed from 3-phenoxybenzyl alcohol which was not however detected as an intermediate in these systems. 3-Phenoxybenzoic acid was further transformed to 4-hydroxy-3-phenoxybenzoic acid. A potential metabolic pathway has been described.
to suggest that the importance and use of these insecticides is likely to decline in the forseeable future. Previous studies (Kaufman et al. 1977; Chapman et al. 1981; Hill 1983), have demonstrated microbially-mediated pyrethroid breakdown in soil and pure culture (Maloney et al. 1988). However, the ability of a thermophilic isolate to hydrolytically cleave pyrethroid insecticides in axenic culture has not been previously demonstrated. We report here the isolation and characterization of a thermophilic Bacillus sp. able to transform a wide-range of pyrethroid insecticides so forming noninsecticidal products.
Key words: Bacillus s t e a r o t h e r m o p h i l u s insecticides - 3-Phenoxybenzoic acid
Chemicals, p y r e t h r o i d s and their m e t a b o l i t e s
-
Pyrethroid
The photostable synthetic pyrethroids are an economically important group of insecticides (Leahey 1985). They are widely used to control agricultural pests, and find increasing usage for the control of arthropods of medical and veterinary importance (Miller 1988). They have been developed over the last decade as replacements for the more toxic and environmentally persistent organochlorine, organophosphorus and methylcarbamate insecticides (Leahey 1985). The pyrethroids exhibit high insecticidal activity combined with low mammalian toxicity and although lipophilic are readily metabolised in biological systems (Elliott et al. 1978). These insecticides are all esters which possess both geometric and optical isomers. These isomers differ in their chemistry, biological activity and persistence (Elliott et al. 1978). Despite some toxic efects on certain non-target organisms (Jolly et al. 1977, 1978; Zabel et al. 1988), there is nothing Correspondence to: S. E. Maloney
Materials and methods
Permethrin (technical grade, 91% pure; analytical grade, 95.5% pure) (cis: trans, 40:60 [mol/mol] proportion by mass), Fastac (technical grade, 96.3% pure) (cis 1 : cis 2, 2.9:97.1 [tool/moll), cypermethrin (technical grade, 94% pure; analytical grade, 97% pure) (cis : trans, 45 : 55) [mol/mol],and fenvalerate (technical grade, 97% pure; analytical grade, 97.8% pure) were supplied by Shell Research Ltd., Sittingbourne, Kent, UK. Deltamethrin (technical grade, > 95 % pure), tralomethrin (technical grade, 96% pure) and tralocythrin (technical grade, 96% pure) was provided by Roussel Uclaf, Romainville, France. Fluvalinate (technical grade, 89% pure) was provided by Zoecon Corp., Surrey, UK. Flueythrinate (analytical grade, > 98% pure), cyfluthrin (technical grade, 96% pure) (cis: trans, 50:50 [mol/mol]), cyhalothrin (technical grade, 92.8% pure) (cis : trans, 50 : 50 [tool/tool]) and flumethrin (technical grade, > 95% pure) were obtained from Promochem Ltd., St. AIbans, Herts., UK. Dow Chemical Co., Midland, Michigan, USA supplied Fenpyrithrin (technical grade, > 98% pure). ICI, Jeallott's Hill, Berkshire, UK, supplied the single isomers of trans-permethrin (analytical grade, 98% pure) and cis-permethrin (analytical grade, 96% pure) and the pyrethroid transformation products 3-(2,2dibromovinyl)-2,2-dimethyleyclopropanecarboxylic acid (analytical grade, 95% pure) and 2- (4-chlorophenyl)butyric acid (analytical grade, 95% pure). Mitchell Cotts Chemicals, Mirfield, West Yorkshire, UK, and Rothamsted Experimental Station, Harpenden. Herts., UK, supplied the trans and cis isomers of 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid (DCVCA; analytical grade, 95% pure). The Wellcome Foundation Ltd., Temple Hill, Dartford, Kent, UK, provided samples of 4-hydroxy3-phenoxybenzoic acid and 2-hydroxy-3-phenoxybenzoic acid. 3-
283 Phenoxybenzyl alcohol, 3-phenoxybenzaldehyde, and 3-phenoxybenolc acid (analytical grade, 99% pure) were obtained from Aldrich Chemical Co. Ltd., Gillingham, Dorset, UK. The surfactant Tween 80 (technical grade) was obtained from BDH Ltd., Poole, Dorset, UK. All solvents used were of pesticide analysis or HPLC grade. Unless otherwise specified, all enrichments were established in the presence of technical grade pyrethroids. All the pyrethroids and their transformation products were routinely identified and quantified by HPLC, and their identities confirmed by gas chromatography-mass spectrometry (GC-MS) (Maloney et al. 1988). Culture growth was assessed by measuring the absorbance at 540 nm.
Media The mineral salts medium used was modified from the formulation of van den Tweel et al. (1986) by replacing 0.1 g yeast extract/1 with 10 ml/1 of the vitamin solution of Balch et al. (1979). The mineral salts medium, pH 7.0, was autoclaved (121 ~ 15 min) prior to the aseptic addition of filter-sterilized vitamin solution. For mineral salts agar, purified agar (1.5% w/v; Difco Laboratories, East Molesey, Surrey, UK) was added to the medium prior to autoclaving. After cooling to 50 ~ one of the following was aseptLcatly added, prior to pouring of plates: (1) 100 and 500 mg permethrin/1, final concentration, as a solution in hexane, (1% v/v); (2) aqueous 50 and 100 mg 3-phenoxybenzoate/1, final concentration (adjusted to pH 7.0 with 5 M KOH); (3) 100 mg trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid (DCVCA), final concentration, as a solution in hexane (1% v/v). For Tween 80 plates, purified agar (1.5% w/v), calcium chloride (0.01% w/v) and 10 g Tween 80/1 were added to the mineral salts medium prior to autoclaving. The above plates were incubated for 5 days a 60 ~ Viable counts, cell purity and separation of the members of the mixed population was achieved using Tryptone Soya Agar (TSA) plates (Oxoid Ltd., Baslngstoke, Hants., UK) after incubation at 60 ~ for 48 h.
colony type were Gram-stained and examined microscopically. Colonies were replated onto both non-selective and selective agar to ensure purity before subculture into Erlenmeyer flasks (250 ml) as described above. When growth became apparent as shown by the development of turbidity, an aliquot of the culture (5 or 10 ml) was used to inoculate mineral salts medium (100 ml) in a fresh flask. The culture was also replated on non-selective and selective agar to confirm purity.
Results
A thermophilic, pyrethroid-hydrolysing isolate designated s t r a i n S M 4 ( D i v i s i o n o f B i o t e c h n o l o g y , P H L S , C A M R , P o r t o n D o w n ) , was i s o l a t e d as the m a j o r c o m p o n e n t m e m b e r o f the p y r e t h r o i d e n r i c h m e n t s . M o r p h o l o g i c a l a n d m e t a b o l i c c h a r a c t e r i s t i c s were c o n s i s t e n t with a p r o v i s i o n a l t a x o n o m i c a s s i g n m e n t as Bacillus stearothermophilus. In initial a e r o b i c e n r i c h m e n t s with 50 a n d 100 m g permethrin/1, t r a n s f o r m a t i o n p r o d u c t s were first d e t e c t e d after 14 d a y s i n c u b a t i o n (half-life, 1 4 - 2 1 days). D u r i n g the 2 8 - d a y i n c u b a t i o n p e r i o d , the trans-isomer o f p e r m e t h r i n was p r e f e r e n t i a l l y h y d r o l y s e d . A f t e r t w o s u b c u l t u r e s , i n c r e a s e d rates o f p e r m e t h r i n t r a n s f o r m a t i o n (halfqife = 4 - 5 d a y s ) were o b t a i n e d . T h r e e t r a n s f o r m a t i o n p r o d u c t s were d e t e c t e d a n d identified b y H P L C ; 3 - p h e n o x y b e n z o i c acid, 4 - h y d r o x y - 3 p h e n o x y b e n z o i c a c i d a n d trans-DCVCA after 2, 3 a n d
permethrin
Source of inocula Soil samples from hot springs in Iceland (Hrunihverabakkl, 55 ~C, pH 8.4, and Hveradalir Skalholt, 62 ~ pH 8.6) were used as inocula.
Esterase
Culture conditions Enrichments were incubated at 60 ~ in Erlenmeyer flasks (2 1), containing 700 ml mineral salts medium (pH 7.0), the Icelandic soil sample (5-10 g), the pyrethroid under study (10, 50 or 100 rag/1 as a solution in hexane (0.1 1.0% v/v) and 500-5000 mg Tween 80/1 in an orbital shaker at 200 rpm. Enrichments containing the pyrethroids required a surfactant to maintain them an an emulsified state. Tween 80 (500-5000mg/1) was selected for use in these enrichment cultures on the basis of its low antimicrobial activity (Brink and Tramper 1985), its non-interference with HPLC analysis, and its ability to maintain all the pyrethroids in aqueous suspension for the duration of the experiment. The half-life of a given pyrethrold in microbial culture was estimated according to: 1) The time taken to halve the initial concentration of parent compound, and 2) The time taken to produce half the stoichiometric quantity of a metabolic product, (e.g. trans-DCVCA from permethrm), by hydrolysis of the parent compound. Mixed populations of organisms were purified by plating onto TSA, and mineral salts agar containing permethrin, trans-DCVCA, 3-phenoxybenzoate or Tween 80. Cells from each clearly discernible
\cl
o
3-phenoxybenzylalcohol
trans- & cis- DCVCA
Dehydrogenase
O 3-phenoxybenzoicacid I J Hydroxylase
0 4-hydroxy-3-phenoxybenzoic acid
Fig. 1. Proposed initial transformation steps of permethrin by Bacillus stearothermophilus SM4. Symbol [ ]: presumptive intermediate, not detected in microbial cultures
284 4 days, respectively (see Fig. 1). The identity of these compounds was confirmed using GC-MS. The thermophilic isolate B. stearothermophilus SM4 grew well on both TSA and on mineral salts agar containing 10 g Tween 80/1, but it did not grow on mineral salts agar containing either permethrin, trans-DCVCA or 3-phenoxybenzoate as a sole carbon source. On Tween 80 agar, zones of clearing were produced by the organism after forty-eight hours' incubation at 60 ~ which is typical o f lipolytic thermophiles. Pure cultures of Bacillus stearothermophilus SM4 also transformed the pyrethroids listed in Table 1, within a period of 7-35 days, at 60 ~ Deltamethrin was the most persistent of the five second-generation pyrethroids, whilst flumethrin was the most persistent of both the second and the third-generation pyrethroids. B. stearothermophilus SM4 transformed all eight so-called thirdgeneration pyrethroids, albeit at a slower rate than that of the second-generation pyrethroids. These compounds remained stable in uninoculated flasks at 60 ~ for the duration of the experiment (28 days). B. stearothermophilus SM4 was also able to oxidise 20 and 50 mg 3-phenoxybenzyl alcohol/1 producing 3-phenoxybenzoate within 4 days o f incubation in the absence of Tween 80. However, this isolate was unable to utilize 3-phenoxybenzyl alcohol as a sole source o f carbon and energy, but was apparently fortuitously able to metabo-
Table 1. The half-lives of five second and eight third-generation pyrethroids in the thermophilic culture of SM4 Pyrethroid (50 mg/t)
or (gM) Half-lives (in days)
B. stearothermophilus
4-Hydroxy-3-phenoxybenzoic acid standard 258
100" 80
243
60" 40" 20-
=
r O
g
d
123 ~35149 171 "184 199 21~ 227
sO
1C~)
150
200
250
3-Phenoxybenzoic acid microbial transformation product
t~ 258
rr 100
80 60
243
40-
221
20I
o'
~o
I;o
15o
171 1199211
200
[
25o
Mass/Charge ratio
Fig. 2. Identification by GC-MS of 4-hydroxy-3-phenoxybenzoic acid as the microbial transformation product of 3-phenoxybenzoic acid. Samples were methylated using diazomethane prior to analysis, and were analysed by GC-MS as the dimethylated product, 4-methoxy-3-phenoxybenzoic acid methyl ester. GC-MS analysis was carried out as described previously (Maloney et al. 1988),where approximately 1 gg of sample in hexane was injected. The initial oven temperature was held at 50 ~ for 1 rain, followed by a 4 ~ increase up to 70 ~ and a 20 ~ increase from 70 ~ to 220 ~ m/z 258 dimethylated molecular ion; m/z 243 monomethylated 4-hydroxy-3-phenoxybenzoyl ion; m/z 227 monomethylated 4-hydroxy-3-phenoxybenzonium ion
SM4 at 60 ~ Permethrin
130
4- 5
120
7- 10
120
7-10
100
14-21
100
7-10
115
7-14
120
7-14
115
14-21
85
21-28
75
21-28
110
21- 28
120
21-28
100
28-35
(C21H2oC1203)
Cypermethrin (C~2H19C12NO3) Fenvalerate (C25H22CINO3)
Deltamethrin (C22H19Br2NO3)
Fluvalinate (C26H22N203C1F3)
Cyfluthrin (C22H1sClyNO3) Fastac (C22Hx9C12NO3)
Flucythrinate
lise 3-phenoxybenzyl alcohol whilst consuming cell-carbon energy reserves. The rate of transformation of 3-phenoxybenzyl alcohol was further improved by adding 0.5 g Tween 80/1 or yeast extract (0.1% w/v), when the half-life was reduced from 4 to 2 days. In the presence of 0.5 g Tween 80/1 or yeast extract (0.1% w/v), this isolate also transformed 20 mg 3-phenoxybenzoate/1 to 4-hydroxy-3-phenoxybenzoate, within two days of incubation. The identity of this novel microbial metabolite was confirmed by GC-MS, as shown in Fig. 2. 3-Phenoxybenzyl alcohol and 3-phenoxybenzoate remained unchanged in uninoculated medium for the duration of the experiment (14 days).
(C26H23F2NO4)
Tralocythrin (C22H19ClEBrENO3)
Tralomethrin (CzzH19Br4NO2)
L-Cyhalothrin (C23H2oC1F3NO3) Fenpyrithrin (C21H19ClzN203)
Flumethrin (C28Hz3ClzFNOa)
Data represents the average of three to four replicates with standard deviation 10-20%
Discussion Pyrethroid transformation in this thermophilic aerobic system was shown to be microbially mediated. Bacillus stearothermophilus, strain SM4 was able to promote the hydrolytic cleavage of selected second and third-generation pyrethroids to non-insecticidal transformation products. Mineralization of insecticides, however, was not achieved. The hydrolytic cleavage of the pyrethroids was attributed to a fortuitous cometabolic reaction (Harder
285 1981; Hill and Wright 1978), resulting in production of 3-phenoxybenzoate and the corresponding dihalovinyl or haloacid moiety whilst using Tween 80 as the primary carbon source. After continued subculture increased rates of pyrethroid hydrolysis was achieved. When compared with pure cultures of mesophiles, B. stearothermophilus was consistently more rapid at pyrethroid transformation (Maloney et al. 1988). It is possible that the high temperature may render the pyrethroids more available for transformation by improving their solubility. In the case of permethrin esteratic cleavage generated 3-phenoxybenzoic acid, trans- and cis-DCVCA, as illustrated in Fig. 1. 3-Phenoxybenzyl alcohol was not however detected as an intermediate in these cultures, but was apparently rapidly oxidised to 3-phenoxybenzoate by the dehydrogenase of SM4 (Fig. 1). In racemic mixtures, the trans-isomer of permethrin was preferentially transformed by these microbial isolates. This result agrees with findings in other biological systems, such as mammals, insects and soils (Leahey 1985), in which the trans-isomer was always found to be more readily degraded than the cis-form. 3-Phenoxybenzoic acid was further transformed to 4-hydroxy-3-phenoxybenzoicacid by the hydroxylase of SM4 (Fig. 1). It is believed that this is the first instance in which 4-hydroxy-3-phenoxybenzoic acid has been detected and identified in a thermophilic culture, whereas it has commonly been associated with other biological systems; particularly with mammals (Leahey 1985) and in soil (Kaufman et al. 1977; Roberts and Standen 1977; Roberts 1981). In the present study, permethrin was the most rapidly transformed of the second-generation pyrethroids, and deltamethrin was the most persistent. These results concur with data from the degradation of the insecticides in soil (Chapman et al. 1981) and pure bacterial cultures (Maloney et al. 1988). The eight third-generation pyrethroids were more persistent in microbial culture than the above; a reflection, possibly, of the greater cis-isomer content of some of these compounds. Of these, cyfluthrin and Fastac were the most rapidly transformed, whilst flumethrin was the most persistent. The introduction of various substituent atoms or groups (e.g. fluorine) onto the aromatic moiety, and the addition of chlorophenyl- to the acid moiety of cypermethrin to yield flumethrin, affects the stereochemistry of the molecule, reducing its susceptibility to both hydrolytic and oxidative metabolism (Soderlund and Casida 1977a, b; Casida 1980; Fewson 1988). Selective transformation i.e. preferential hydrolysis of the less insecticidally-active (IR)-transisomer may contribute significantly to the long-term persistence of biologically-active residues of pyrethroids in the environment (Solomon 1986). Neither the pyrethroid parent compounds nor their major transformation products were inhibitory to the growth of B. stearotherrnophilus at concentrations up to 500 mg/1. This is in agreement with the results of Tu (1980) who found that permethrin showed only minor and transient effects on soil microorganisms in situ, but contrasts with the findings of Stratton and Corke (1982 a, b), who showed that permethrin transformation products inhibited the growth of a number of algae, cyanobacteria
and fungi. It is of interest to note that Bacillus stearothermophilus SM4 colonies became more mucoid after prolonged subculture of the organism in media containing Tween 80 and insecticide. This may represent the development of a protection mechanism against the toxic effects of Tween/pesticide. The requirement for a dispersant/surfactant for solubilization and the metabolism of all the pyrethroids was evident early on in these studies. Because of its detergent properties, Tween 80 may be involved in the modification of cell surface properties, promoting enhanced transport or better contact between the pyrethroid and the microbial cells (Brown and Richards 1964; Reese and Maguire 1969). The pyrethroid-transforming isolate was unable to grow in the absence of Tween 80, and appeared to use it, or at least the oleic acid moiety, as the primary carbon source in the transformation of the pyrethroids. As an ester of oleic acid, Tween 80 may induce hydrolase(s) that release oleate (Reese 1972) which, in turn, may induce the enzymes of/~-oxidation. Although cometabolism of the pyrethroid insecticides does not result in complete mineralization of these molecules, it does eliminate their insecticidal activity, as reported with other insecticides, for example dieldrin and DDT (Horvath 1972). There have been few reports of either lipolytic thermophiles (Gowland et al. 1987), or carboxylesterases from thermophilic bacilli (Owusu and Cowan 1991). To date, there have been no previous reports of thermophilic isolates able to transform any of the pyrethroids, either as a sole carbon source or in a co-metabolic reaction. B. stearothermophilus SM4 or its pyrethroid esterase/hydrolase could conceivably be developed for in situ detoxification of pyrethroids where they cause environmental problems. The degradation of commonly used pyrethroid moth-proofing agents found in carpet and wool processing effluent which are often discharged at high temperature may be such a use.
Acknowledgements. We thank the Department of the Environment for funding this work. We acknowledge Mr. Robin Wait of the Division of Pathology, C.A.M.R., for mass spectrophotometric analyses; Shell Research Ltd., ICI, Roussel Uclaf, Zoecon Corp., Mitchell Cotts Chemicals, Wellcome Foundation Ltd., and Rothamsted ExperimentalStation for supplyingsamplesof pyrethroids and metabolites. We also thank Dr. D. White for his help in the identificationofB. stearotherrnophilus, Dr. R. J. Sharp of the Division of Biotechnology,C.A.M.R., for soil samples from Iceland and TonyAtkinsonfor helpfulcommentsin preparing this paper.
References
Balch WE, Fox GE, Magrum LJ, Woese CR, Wolfe RS (1979) Methanogens: reevaluation of a unique biological group. Microbiol Rev 43:260-296 Brink LES, Tramper J (1985) Optimization of organic solvent in multiphasebiocatalysis.BiotechnolBioengXXVII: 1258-1269 Brown MRW, RichardsRME (1964) Effectof Polysorbate (Tween) 80 on the resistance of Pseudornonas aeruginosa to chemical inactivation. J Pharm Pharmacol 16 [Suppl]: 51T-55T Casida JE (1980) Pyrethrum flowers and pyrethroid insecticides. Environ Health Perspect 34:189-202
286 Chapman RA, Tu CM, Harris CR, Cole C (1981) Persistence of five pyrethroid insecticides in sterile and natural, mineral and organic soil. Bull Environ Contain Toxicol 26:513-519 Elliott M, Janes NF, Potter C (1978) The future of pyrethroids in insect control. Ann Rev Entomol 23:443-469 Fewson CA (1988) Biodegradation of xenobiotic and other persistent compounds: the causes of recalcitrance. Trends Biotechnol 6:148-153 Gowland P, Kernick M, Sundaram TK (1987) Thermophilic bacterial isolates producing lipase. FEMS Microbiol Lett 48: 339-343 Harder W, (1981) Enrichment and characterization of degrading organisms. In: Leisinger T, H/itter R, Cook AM, Niiesch J (eds) Microbial degradation of xenobiotics and recalcitrant compounds. FEMS Symposium No. 12. Academic Press, London, pp 77-94 Hill IR, Wright SJL (1978) Pesticide microbiology. Microbiological aspects of pesticide behaviour in the environment. Academic Press, London Hill BD (1983) Persistence of deltamethrin in a lethbridge sandy clay loam. J Environ Sci Health [B] 18:691-703 Horvath RS (1972) Microbial co-metabolism and the degradation of organic compounds in nature. Bacteriol Rev 36:146-155 Jolly AL, Graves JB, Avault JW, Koonce KL (1977/1978) Effects of a new insecticide on aquatic animals. Louisiana Agric 21 : 3-4 Kaufman DD, Haynes SC, Jordan EG, Kayser AJ (1977) Permethrin degradation in soil and microbial cultures. In: Elliott M (ed) Synthetic pyrethroids. American Chemical Society Series No. 42 ACS, San Francisco, Calif., USA, pp 147-161 Leahey JP (1985) The pyrethroid insecticides. Taylor and Francis, London Maloney SM, Maule A, Smith ARW (1988) Microbial transformation of the pyrethroid insecticides: Permethrin, Deltamethrin, Fastac, Fenvalerate and Fluvalinate. Appl Environ Microbiol 54:2874-2876 Miller TA (1988) Mechanisms of resistance to pyrethroid insecticides. Parasitol Today 4:$8-S10 Owusu RK, Cowan DA (1991) Isolation and partial characterization of a novel thermostable carboxylesterase from a thermophilic Bacillus. Enzyme Microbiol Technol 13:158-163
Reese ET, Maguire A (1969) Surfactants as stimulants of enzyme production by microorganisms. Appl Microbiol 17:242-245 Reese ET (1972) Enzyme production from insoluble substrates. Biotechnol Bioeng Symp 3:43-62 Roberts TR, Standen ME (1977) Degradation of cypermethrin and the respective eis- and trans-isomers in soils. Pestic Sci 8: 305-319 Roberts TR (1981) The metabolism of the synthetic pyrethroids in plants and soils. In: Hutson DH, Roberts TR (eds) Progress in pesticide biochemistry, vol 1. John Wiley and Sons, UK, pp 115-146 Soderlund DM, Casida JE (1977a) Substrate specificity of mouseliver microsomal enzymes in pyrethroid metabolism and stereospecificity of pyrethroid metabolism in mammals. In: Elliott M (ed) Synthetic pyrethroids. American Chemical Society Series No. 42, ACS, San Francisco, Calif., USA, pp 162-185 Soderlund DM, Casida JE (1977 b) Effects of pyrethroid structure on rates of hydrolysis and oxidation by mouse liver microsomal enzymes. Pestic Biochem Physiol 7:391-401 Solomon KR (1986) Pyrethroids: their effects on aquatic and terrestrial ecosystems. Associate Committee on Scientific Criteria for Environmental Quality. National Research Council Canada, NRCC, Ontario, Canada. Publication no. 24376 Stratton GW, Corke, CT (1982a) Toxicity of the insecticide permethrin and some degradation products towards algae and cyanobacteria. Environ Pollution [A] 29:71-80 Stratton GW, Corke CT (1982b) Comparative fungitoxicity of the insecticide permethrin and ten degradation products. Pestic Sci 13:679-685 Tu CM (1980) Influence of five pyrethroid insecticides on microbial populations and activities in soil. Microbial Ecol 5:321-327 Tweel WJJ van den, Burg N ter, Kok JB, Bont JAM de (1986) Bioformation of 4-hydroxybenzoate from 4-chlorobenzoate by Alcaligenes denitrificans NTB-1. Appl Microbiol Biotechnol 25: 289-294 Zabel TF, Seager J, Oakley SD (1988) Proposed environmental quality standards for list 11 substances in water. Moth-proofing agents. Water Research Council, Environmental Strategy, Standards and Legislation Unit TR 261, Medmenhem, UK
Archives of
Microbiology
9 Springer-Verlag 1992
Transformation of synthetic pyrethroid insecticides by a thermophilic Bacillus sp. S. E. Maloney ~, A. Maule 1, and A. R. W. Smith 2 1 Divasion of Biotechnology, Public Health Laboratory Service, Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire, SP40JG0 UK z School of Biological and Chemical Sciences, Thames Polytechnic, Wellington Street, London, SE18 6PF, UK Received December 10, 1991/Accepted April 8, 1992
Abstract. Employing a mineral salts medium containing Tween 80 as the primary carbon source, a strain o f Bacillus s t e a r o t h e r m o p h i l u s was isolated which was able to hydrolyse selected second and third-generation pyrethroids to non-insecticidal products. Of a range of pyrethroid insecticides the t r a n s - i s o m e r of permethrin was the most readily transformed by this microbial isolate, whilst flumethrin was the least. 3-Phenoxybenzoic acid and the respective halovinyl or haloacid moieties were detected as the major hydrolytic products of the pyrethroids. It is believed that 3-phenoxybenzoic acid was formed from 3-phenoxybenzyl alcohol which was not however detected as an intermediate in these systems. 3-Phenoxybenzoic acid was further transformed to 4-hydroxy-3-phenoxybenzoic acid. A potential metabolic pathway has been described.
to suggest that the importance and use of these insecticides is likely to decline in the forseeable future. Previous studies (Kaufman et al. 1977; Chapman et al. 1981; Hill 1983), have demonstrated microbially-mediated pyrethroid breakdown in soil and pure culture (Maloney et al. 1988). However, the ability of a thermophilic isolate to hydrolytically cleave pyrethroid insecticides in axenic culture has not been previously demonstrated. We report here the isolation and characterization of a thermophilic Bacillus sp. able to transform a wide-range of pyrethroid insecticides so forming noninsecticidal products.
Key words: Bacillus s t e a r o t h e r m o p h i l u s insecticides - 3-Phenoxybenzoic acid
Chemicals, p y r e t h r o i d s and their m e t a b o l i t e s
-
Pyrethroid
The photostable synthetic pyrethroids are an economically important group of insecticides (Leahey 1985). They are widely used to control agricultural pests, and find increasing usage for the control of arthropods of medical and veterinary importance (Miller 1988). They have been developed over the last decade as replacements for the more toxic and environmentally persistent organochlorine, organophosphorus and methylcarbamate insecticides (Leahey 1985). The pyrethroids exhibit high insecticidal activity combined with low mammalian toxicity and although lipophilic are readily metabolised in biological systems (Elliott et al. 1978). These insecticides are all esters which possess both geometric and optical isomers. These isomers differ in their chemistry, biological activity and persistence (Elliott et al. 1978). Despite some toxic efects on certain non-target organisms (Jolly et al. 1977, 1978; Zabel et al. 1988), there is nothing Correspondence to: S. E. Maloney
Materials and methods
Permethrin (technical grade, 91% pure; analytical grade, 95.5% pure) (cis: trans, 40:60 [mol/mol] proportion by mass), Fastac (technical grade, 96.3% pure) (cis 1 : cis 2, 2.9:97.1 [tool/moll), cypermethrin (technical grade, 94% pure; analytical grade, 97% pure) (cis : trans, 45 : 55) [mol/mol],and fenvalerate (technical grade, 97% pure; analytical grade, 97.8% pure) were supplied by Shell Research Ltd., Sittingbourne, Kent, UK. Deltamethrin (technical grade, > 95 % pure), tralomethrin (technical grade, 96% pure) and tralocythrin (technical grade, 96% pure) was provided by Roussel Uclaf, Romainville, France. Fluvalinate (technical grade, 89% pure) was provided by Zoecon Corp., Surrey, UK. Flueythrinate (analytical grade, > 98% pure), cyfluthrin (technical grade, 96% pure) (cis: trans, 50:50 [mol/mol]), cyhalothrin (technical grade, 92.8% pure) (cis : trans, 50 : 50 [tool/tool]) and flumethrin (technical grade, > 95% pure) were obtained from Promochem Ltd., St. AIbans, Herts., UK. Dow Chemical Co., Midland, Michigan, USA supplied Fenpyrithrin (technical grade, > 98% pure). ICI, Jeallott's Hill, Berkshire, UK, supplied the single isomers of trans-permethrin (analytical grade, 98% pure) and cis-permethrin (analytical grade, 96% pure) and the pyrethroid transformation products 3-(2,2dibromovinyl)-2,2-dimethyleyclopropanecarboxylic acid (analytical grade, 95% pure) and 2- (4-chlorophenyl)butyric acid (analytical grade, 95% pure). Mitchell Cotts Chemicals, Mirfield, West Yorkshire, UK, and Rothamsted Experimental Station, Harpenden. Herts., UK, supplied the trans and cis isomers of 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid (DCVCA; analytical grade, 95% pure). The Wellcome Foundation Ltd., Temple Hill, Dartford, Kent, UK, provided samples of 4-hydroxy3-phenoxybenzoic acid and 2-hydroxy-3-phenoxybenzoic acid. 3-
283 Phenoxybenzyl alcohol, 3-phenoxybenzaldehyde, and 3-phenoxybenolc acid (analytical grade, 99% pure) were obtained from Aldrich Chemical Co. Ltd., Gillingham, Dorset, UK. The surfactant Tween 80 (technical grade) was obtained from BDH Ltd., Poole, Dorset, UK. All solvents used were of pesticide analysis or HPLC grade. Unless otherwise specified, all enrichments were established in the presence of technical grade pyrethroids. All the pyrethroids and their transformation products were routinely identified and quantified by HPLC, and their identities confirmed by gas chromatography-mass spectrometry (GC-MS) (Maloney et al. 1988). Culture growth was assessed by measuring the absorbance at 540 nm.
Media The mineral salts medium used was modified from the formulation of van den Tweel et al. (1986) by replacing 0.1 g yeast extract/1 with 10 ml/1 of the vitamin solution of Balch et al. (1979). The mineral salts medium, pH 7.0, was autoclaved (121 ~ 15 min) prior to the aseptic addition of filter-sterilized vitamin solution. For mineral salts agar, purified agar (1.5% w/v; Difco Laboratories, East Molesey, Surrey, UK) was added to the medium prior to autoclaving. After cooling to 50 ~ one of the following was aseptLcatly added, prior to pouring of plates: (1) 100 and 500 mg permethrin/1, final concentration, as a solution in hexane, (1% v/v); (2) aqueous 50 and 100 mg 3-phenoxybenzoate/1, final concentration (adjusted to pH 7.0 with 5 M KOH); (3) 100 mg trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid (DCVCA), final concentration, as a solution in hexane (1% v/v). For Tween 80 plates, purified agar (1.5% w/v), calcium chloride (0.01% w/v) and 10 g Tween 80/1 were added to the mineral salts medium prior to autoclaving. The above plates were incubated for 5 days a 60 ~ Viable counts, cell purity and separation of the members of the mixed population was achieved using Tryptone Soya Agar (TSA) plates (Oxoid Ltd., Baslngstoke, Hants., UK) after incubation at 60 ~ for 48 h.
colony type were Gram-stained and examined microscopically. Colonies were replated onto both non-selective and selective agar to ensure purity before subculture into Erlenmeyer flasks (250 ml) as described above. When growth became apparent as shown by the development of turbidity, an aliquot of the culture (5 or 10 ml) was used to inoculate mineral salts medium (100 ml) in a fresh flask. The culture was also replated on non-selective and selective agar to confirm purity.
Results
A thermophilic, pyrethroid-hydrolysing isolate designated s t r a i n S M 4 ( D i v i s i o n o f B i o t e c h n o l o g y , P H L S , C A M R , P o r t o n D o w n ) , was i s o l a t e d as the m a j o r c o m p o n e n t m e m b e r o f the p y r e t h r o i d e n r i c h m e n t s . M o r p h o l o g i c a l a n d m e t a b o l i c c h a r a c t e r i s t i c s were c o n s i s t e n t with a p r o v i s i o n a l t a x o n o m i c a s s i g n m e n t as Bacillus stearothermophilus. In initial a e r o b i c e n r i c h m e n t s with 50 a n d 100 m g permethrin/1, t r a n s f o r m a t i o n p r o d u c t s were first d e t e c t e d after 14 d a y s i n c u b a t i o n (half-life, 1 4 - 2 1 days). D u r i n g the 2 8 - d a y i n c u b a t i o n p e r i o d , the trans-isomer o f p e r m e t h r i n was p r e f e r e n t i a l l y h y d r o l y s e d . A f t e r t w o s u b c u l t u r e s , i n c r e a s e d rates o f p e r m e t h r i n t r a n s f o r m a t i o n (halfqife = 4 - 5 d a y s ) were o b t a i n e d . T h r e e t r a n s f o r m a t i o n p r o d u c t s were d e t e c t e d a n d identified b y H P L C ; 3 - p h e n o x y b e n z o i c acid, 4 - h y d r o x y - 3 p h e n o x y b e n z o i c a c i d a n d trans-DCVCA after 2, 3 a n d
permethrin
Source of inocula Soil samples from hot springs in Iceland (Hrunihverabakkl, 55 ~C, pH 8.4, and Hveradalir Skalholt, 62 ~ pH 8.6) were used as inocula.
Esterase
Culture conditions Enrichments were incubated at 60 ~ in Erlenmeyer flasks (2 1), containing 700 ml mineral salts medium (pH 7.0), the Icelandic soil sample (5-10 g), the pyrethroid under study (10, 50 or 100 rag/1 as a solution in hexane (0.1 1.0% v/v) and 500-5000 mg Tween 80/1 in an orbital shaker at 200 rpm. Enrichments containing the pyrethroids required a surfactant to maintain them an an emulsified state. Tween 80 (500-5000mg/1) was selected for use in these enrichment cultures on the basis of its low antimicrobial activity (Brink and Tramper 1985), its non-interference with HPLC analysis, and its ability to maintain all the pyrethroids in aqueous suspension for the duration of the experiment. The half-life of a given pyrethrold in microbial culture was estimated according to: 1) The time taken to halve the initial concentration of parent compound, and 2) The time taken to produce half the stoichiometric quantity of a metabolic product, (e.g. trans-DCVCA from permethrm), by hydrolysis of the parent compound. Mixed populations of organisms were purified by plating onto TSA, and mineral salts agar containing permethrin, trans-DCVCA, 3-phenoxybenzoate or Tween 80. Cells from each clearly discernible
\cl
o
3-phenoxybenzylalcohol
trans- & cis- DCVCA
Dehydrogenase
O 3-phenoxybenzoicacid I J Hydroxylase
0 4-hydroxy-3-phenoxybenzoic acid
Fig. 1. Proposed initial transformation steps of permethrin by Bacillus stearothermophilus SM4. Symbol [ ]: presumptive intermediate, not detected in microbial cultures
284 4 days, respectively (see Fig. 1). The identity of these compounds was confirmed using GC-MS. The thermophilic isolate B. stearothermophilus SM4 grew well on both TSA and on mineral salts agar containing 10 g Tween 80/1, but it did not grow on mineral salts agar containing either permethrin, trans-DCVCA or 3-phenoxybenzoate as a sole carbon source. On Tween 80 agar, zones of clearing were produced by the organism after forty-eight hours' incubation at 60 ~ which is typical o f lipolytic thermophiles. Pure cultures of Bacillus stearothermophilus SM4 also transformed the pyrethroids listed in Table 1, within a period of 7-35 days, at 60 ~ Deltamethrin was the most persistent of the five second-generation pyrethroids, whilst flumethrin was the most persistent of both the second and the third-generation pyrethroids. B. stearothermophilus SM4 transformed all eight so-called thirdgeneration pyrethroids, albeit at a slower rate than that of the second-generation pyrethroids. These compounds remained stable in uninoculated flasks at 60 ~ for the duration of the experiment (28 days). B. stearothermophilus SM4 was also able to oxidise 20 and 50 mg 3-phenoxybenzyl alcohol/1 producing 3-phenoxybenzoate within 4 days o f incubation in the absence of Tween 80. However, this isolate was unable to utilize 3-phenoxybenzyl alcohol as a sole source o f carbon and energy, but was apparently fortuitously able to metabo-
Table 1. The half-lives of five second and eight third-generation pyrethroids in the thermophilic culture of SM4 Pyrethroid (50 mg/t)
or (gM) Half-lives (in days)
B. stearothermophilus
4-Hydroxy-3-phenoxybenzoic acid standard 258
100" 80
243
60" 40" 20-
=
r O
g
d
123 ~35149 171 "184 199 21~ 227
sO
1C~)
150
200
250
3-Phenoxybenzoic acid microbial transformation product
t~ 258
rr 100
80 60
243
40-
221
20I
o'
~o
I;o
15o
171 1199211
200
[
25o
Mass/Charge ratio
Fig. 2. Identification by GC-MS of 4-hydroxy-3-phenoxybenzoic acid as the microbial transformation product of 3-phenoxybenzoic acid. Samples were methylated using diazomethane prior to analysis, and were analysed by GC-MS as the dimethylated product, 4-methoxy-3-phenoxybenzoic acid methyl ester. GC-MS analysis was carried out as described previously (Maloney et al. 1988),where approximately 1 gg of sample in hexane was injected. The initial oven temperature was held at 50 ~ for 1 rain, followed by a 4 ~ increase up to 70 ~ and a 20 ~ increase from 70 ~ to 220 ~ m/z 258 dimethylated molecular ion; m/z 243 monomethylated 4-hydroxy-3-phenoxybenzoyl ion; m/z 227 monomethylated 4-hydroxy-3-phenoxybenzonium ion
SM4 at 60 ~ Permethrin
130
4- 5
120
7- 10
120
7-10
100
14-21
100
7-10
115
7-14
120
7-14
115
14-21
85
21-28
75
21-28
110
21- 28
120
21-28
100
28-35
(C21H2oC1203)
Cypermethrin (C~2H19C12NO3) Fenvalerate (C25H22CINO3)
Deltamethrin (C22H19Br2NO3)
Fluvalinate (C26H22N203C1F3)
Cyfluthrin (C22H1sClyNO3) Fastac (C22Hx9C12NO3)
Flucythrinate
lise 3-phenoxybenzyl alcohol whilst consuming cell-carbon energy reserves. The rate of transformation of 3-phenoxybenzyl alcohol was further improved by adding 0.5 g Tween 80/1 or yeast extract (0.1% w/v), when the half-life was reduced from 4 to 2 days. In the presence of 0.5 g Tween 80/1 or yeast extract (0.1% w/v), this isolate also transformed 20 mg 3-phenoxybenzoate/1 to 4-hydroxy-3-phenoxybenzoate, within two days of incubation. The identity of this novel microbial metabolite was confirmed by GC-MS, as shown in Fig. 2. 3-Phenoxybenzyl alcohol and 3-phenoxybenzoate remained unchanged in uninoculated medium for the duration of the experiment (14 days).
(C26H23F2NO4)
Tralocythrin (C22H19ClEBrENO3)
Tralomethrin (CzzH19Br4NO2)
L-Cyhalothrin (C23H2oC1F3NO3) Fenpyrithrin (C21H19ClzN203)
Flumethrin (C28Hz3ClzFNOa)
Data represents the average of three to four replicates with standard deviation 10-20%
Discussion Pyrethroid transformation in this thermophilic aerobic system was shown to be microbially mediated. Bacillus stearothermophilus, strain SM4 was able to promote the hydrolytic cleavage of selected second and third-generation pyrethroids to non-insecticidal transformation products. Mineralization of insecticides, however, was not achieved. The hydrolytic cleavage of the pyrethroids was attributed to a fortuitous cometabolic reaction (Harder
285 1981; Hill and Wright 1978), resulting in production of 3-phenoxybenzoate and the corresponding dihalovinyl or haloacid moiety whilst using Tween 80 as the primary carbon source. After continued subculture increased rates of pyrethroid hydrolysis was achieved. When compared with pure cultures of mesophiles, B. stearothermophilus was consistently more rapid at pyrethroid transformation (Maloney et al. 1988). It is possible that the high temperature may render the pyrethroids more available for transformation by improving their solubility. In the case of permethrin esteratic cleavage generated 3-phenoxybenzoic acid, trans- and cis-DCVCA, as illustrated in Fig. 1. 3-Phenoxybenzyl alcohol was not however detected as an intermediate in these cultures, but was apparently rapidly oxidised to 3-phenoxybenzoate by the dehydrogenase of SM4 (Fig. 1). In racemic mixtures, the trans-isomer of permethrin was preferentially transformed by these microbial isolates. This result agrees with findings in other biological systems, such as mammals, insects and soils (Leahey 1985), in which the trans-isomer was always found to be more readily degraded than the cis-form. 3-Phenoxybenzoic acid was further transformed to 4-hydroxy-3-phenoxybenzoicacid by the hydroxylase of SM4 (Fig. 1). It is believed that this is the first instance in which 4-hydroxy-3-phenoxybenzoic acid has been detected and identified in a thermophilic culture, whereas it has commonly been associated with other biological systems; particularly with mammals (Leahey 1985) and in soil (Kaufman et al. 1977; Roberts and Standen 1977; Roberts 1981). In the present study, permethrin was the most rapidly transformed of the second-generation pyrethroids, and deltamethrin was the most persistent. These results concur with data from the degradation of the insecticides in soil (Chapman et al. 1981) and pure bacterial cultures (Maloney et al. 1988). The eight third-generation pyrethroids were more persistent in microbial culture than the above; a reflection, possibly, of the greater cis-isomer content of some of these compounds. Of these, cyfluthrin and Fastac were the most rapidly transformed, whilst flumethrin was the most persistent. The introduction of various substituent atoms or groups (e.g. fluorine) onto the aromatic moiety, and the addition of chlorophenyl- to the acid moiety of cypermethrin to yield flumethrin, affects the stereochemistry of the molecule, reducing its susceptibility to both hydrolytic and oxidative metabolism (Soderlund and Casida 1977a, b; Casida 1980; Fewson 1988). Selective transformation i.e. preferential hydrolysis of the less insecticidally-active (IR)-transisomer may contribute significantly to the long-term persistence of biologically-active residues of pyrethroids in the environment (Solomon 1986). Neither the pyrethroid parent compounds nor their major transformation products were inhibitory to the growth of B. stearotherrnophilus at concentrations up to 500 mg/1. This is in agreement with the results of Tu (1980) who found that permethrin showed only minor and transient effects on soil microorganisms in situ, but contrasts with the findings of Stratton and Corke (1982 a, b), who showed that permethrin transformation products inhibited the growth of a number of algae, cyanobacteria
and fungi. It is of interest to note that Bacillus stearothermophilus SM4 colonies became more mucoid after prolonged subculture of the organism in media containing Tween 80 and insecticide. This may represent the development of a protection mechanism against the toxic effects of Tween/pesticide. The requirement for a dispersant/surfactant for solubilization and the metabolism of all the pyrethroids was evident early on in these studies. Because of its detergent properties, Tween 80 may be involved in the modification of cell surface properties, promoting enhanced transport or better contact between the pyrethroid and the microbial cells (Brown and Richards 1964; Reese and Maguire 1969). The pyrethroid-transforming isolate was unable to grow in the absence of Tween 80, and appeared to use it, or at least the oleic acid moiety, as the primary carbon source in the transformation of the pyrethroids. As an ester of oleic acid, Tween 80 may induce hydrolase(s) that release oleate (Reese 1972) which, in turn, may induce the enzymes of/~-oxidation. Although cometabolism of the pyrethroid insecticides does not result in complete mineralization of these molecules, it does eliminate their insecticidal activity, as reported with other insecticides, for example dieldrin and DDT (Horvath 1972). There have been few reports of either lipolytic thermophiles (Gowland et al. 1987), or carboxylesterases from thermophilic bacilli (Owusu and Cowan 1991). To date, there have been no previous reports of thermophilic isolates able to transform any of the pyrethroids, either as a sole carbon source or in a co-metabolic reaction. B. stearothermophilus SM4 or its pyrethroid esterase/hydrolase could conceivably be developed for in situ detoxification of pyrethroids where they cause environmental problems. The degradation of commonly used pyrethroid moth-proofing agents found in carpet and wool processing effluent which are often discharged at high temperature may be such a use.
Acknowledgements. We thank the Department of the Environment for funding this work. We acknowledge Mr. Robin Wait of the Division of Pathology, C.A.M.R., for mass spectrophotometric analyses; Shell Research Ltd., ICI, Roussel Uclaf, Zoecon Corp., Mitchell Cotts Chemicals, Wellcome Foundation Ltd., and Rothamsted ExperimentalStation for supplyingsamplesof pyrethroids and metabolites. We also thank Dr. D. White for his help in the identificationofB. stearotherrnophilus, Dr. R. J. Sharp of the Division of Biotechnology,C.A.M.R., for soil samples from Iceland and TonyAtkinsonfor helpfulcommentsin preparing this paper.
References
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