APPWED AND ENVIRONMENTAL MICROBIOLOGY, May 1979, p. 886-8910099-2240/79/05-0886/06$02.00/0

Vol. 37, No. 5

Microbial Cleavage of Various Organophosphorus InsecticidesARTHUR ROSENBERG AND MARTIN ALEXANDER*

Laboratory ofSoil Microbiology, Department ofAgronomy, Cornell University, Ithaca, New York 14853

Received for publication 23 February 1979

Bacteria able to utilize Aspon, Azodrin, Dasanit, diazinon, malathion, Orthene,parathion, Trithion, dimethoate, Dylox, methyl parathion, and Vapona as solephosphorus sources were isolated from soil and sewage. Individual isolates usedfrom 3 to 10 of these insecticides as sole phosphorus sources. The extent of growthof two Pseudomonas strains in media containing diazinon and malathion was inthe range expected from the amount of insecticide supplied, and their proliferationresulted in disappearance of the chemical. Resting cells of the pseudomonadsderived from cultures grown on diazinon or malathion but not orthophosphatecaused extensive destruction of these two organophosphates in the presence orabsence of chloramphenicol. Extracts of the two bacteria derived from organo-phosphate-grown cultures catalyzed the disappearance of Aspon, Azodrin, Das-anit, diazinon, malathion, Orthene, parathion, and Trithion but not dimethoate,Dylox, methyl parathion, and Vapona. Results from gas chromatographic analysissuggested that the extracts formed dimethyl phosphate from azodrin, dimethylphosphorodithioate from malathion, diethyl phosphorodithioate from Trithion,and diethyl phosphorothioate from Dasanit, diazinon, and parathion. Dimethylphosphate, dimethyl phosphorothioate, dimethyl phosphorodithioate, diethylphosphate, and diethyl phosphorothioate were not used by the pseudomonads assole phosphorus sources.

The chlorinated hydrocarbon insecticides arecharacteristically persistent in soils and waters,and their persistence as well as their suscepti-bility to biomagnification and subsequent toxic-ity to higher animals have resulted in the ter-mination of use ofmany of these pesticides. Thereplacement chemicals for many agriculturalpest control operations are the organophos-phates, a group that includes a large number ofcompounds that usually are readily destroyed insoils and waters.The microbial attack on organophosphate in-

secticides is well documented. For example, mi-croorganisms bring about the destruction of Di-syston (2), malathion (3), parathion (6), anddiazinon (10). Some reports of microbial growthon these compounds are questionable, however,because the slight growth obtained may be at-tributable to contaminating orthophosphate inthe medium, and even the demonstration ofpesticide disappearance under such circum-stances is not adequate evidence that the pesti-cide is serving as a phosphorus source for growthbecause many synthetic chemicals are subject tocometabolism. Furthermore, although mostmembers of this class of pesticides have in com-mon the presence of two methoxy or ethoxygroups linked to the phosphorus atom, onlymodest attention has been given to the specific-

ity of microbial enzymes or enzyme preparations(14).The present study was undertaken to deter-

mine the specificity ofenzyme preparations frommicroorganisms able to metabolize organophos-phate insecticides and to establish the productsof enzyme breakdown. Because of the frequentgrowth of bacteria on contaminating orthophos-phate in media receiving synthetic organophos-phates as sole added phosphorus sources, specialcare was taken to minimize the contribution ofthe contaminating nutrients to microbialgrowth.

MATERIALS AND METHODS

Materials. The sources of the chemicals were asfollows: Aspon, methyl parathion, and Trithion werefrom Stauffer Chemical Co., Westpoint, Conn; Azodrinand dimethyl dichlorovinyl phosphate (Vapona) werefrom Shell Chemical Co., San Ramon, Calif.; Dasanitand Dylox were from Chemagro Agricultural Div.,Kansas City, Mo.; diazinon was from Ciba-Geigy,Greensboro, N.C.; dimethoate, malathion, dimethylphosphorothioate, and dimethyl phosphorodithioate(potassium salt) were from American Cynamid Co.,Princeton, N.J.; Orthene was from Chevron ChemicalCorp., San Francisco, Calif.; ethyl parathion was fromMonsanto Co., St. Louis, Mo.; chloramphenicol andsodium barbital were from Calbiochem, San Diego,Calif.; diethyl phosphate was from Eastman Kodak

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ORGANOPHOSPHORUS INSECTICIDE CLEAVAGE 887

Co., Rochester, N.Y.; diethyl phosphorothioate, di-ethyl phosphorodithioate, triethyl phosphorothioate,triethyl phosphate, dimethyl phosphate, trimethylphosphorothioate, trimethyl phosphate, and tri-n-bu-tyl phosphate were from Aldrich Chemical Co., Mil-waukee, Wis.; and crystalline bovine serum albuminwas from Nutritional Biochemicals Corp., ClevelandOhio. All chemicals were of the highest purity availa-ble commercially.

Glassware. Glassware was cleaned by rinsing inwater followed by a 24-h immersion in 20% (vol/vol)HNO3. The nitric acid was removed by thorough wash-ing in tap water followed by washing in distilled water.Medium and cultural conditions. Medium free

of inorganic phosphate was buffered with 10 mMsodium barbital (pH 7.2) and contained (per liter):KCI, 0.20 g; (NH4)2SO4, 0.50 g; NaCl, 0.10 g; MgSO4-7H20, 0.2 g; CaCl2.H20, 50 mg; FeCl36H20, 20 mg;

and 0.2 g each of glucose, glycerol, and sodium succi-nate (1). The basal medium and the insecticides were

sterilized by filtration through sterile Nalgene filterunits (Nalge-Sybron Corp., Rochester, N.Y.) contain-ing 0.2-pm membrane filters. Cultures in screw-cap

tubes (16 by 100 mm) were incubated at 29°C withoutagitation. Growth curves and kinetics of substrateutilization were established with cultures incubated at29°C in 125-ml baffled Erlenmeyer flasks on a gyratoryshaker (New Brunswick Scientific Co., New Bruns-wick, N.J.) at 150 rpm. The cells were harvested bycentrifugation at 15,000 x g and 4°C for 30 min.

Isolation of bacteria. Enrichment cultures were

used to obtain isolates able to utilize a given organo-

phosphate as the sole source of phosphorus. Whenprovided as the sole phosphorus source, the organo-phosphate was present at a concentration of 0.2 mM.The organophosphates were each prepared with 10 mlof 95% ethanol. The enrichment culture (final volume,3.0 ml) received either 0.5 ml of municipal sewagecollected from the primary settling tank and utilizedwithin 30 min of sampling or 0.5 g of soil. When growthgreater than that in organophosphate-free solutionswas observed (usually after 1 to 3 days), the enrich-ments were subcultured into fresh medium. Afterthree successive transfers, enrichments using organo-phosphates as the sole phosphorus sources were

streaked on nutrient agar plates (Difco Laboratories,Detroit, Mich.). Isolates that were able to utilize theorganophosphates were subsequently identified bytheir growth in selective liquid medium.

Preparation of resting cell suspensions. Cellsgrown to the early stationary phase were harvested,washed three times with sodium barbital buffer (10mM, pH 7.2), and suspended in the same buffer to an

optical density of 1.5 at 420 nm. To 5-ml portions ofresting cell suspensions in 25-ml Erlenmeyer flaskswas added 30 to 40,ug of diazinon or malathion per ml.Chloramphenicol (100,g/ml) was used to inhibit pro-tein synthesis. At intervals, 0.5-ml portions were re-

moved for extraction and analysis.Preparation of cell-free extracts. Cells were

grown to the early stationary phase, harvested, andwashed in the barbital buffer. The pellet was sus-

pended to about 5 mg (wet weight) per ml of thebuffer. Extracts were prepared by sonic disruption at4°C by using the microtip of a model W185D cell

disruptor (Heat Systems-Ultrasonic, Inc., Plainview,N.Y.) at 70% power in four 30-s periods, separated by30-s cooling periods in ice. Debris and whole cells wereremoved by centrifugation at 15,000 x g and 4°C for30 min.Enzyme assay. The reaction mixture was assayed

at 29°C. The complete reaction mixture contained, in5.0 ml: 10 mM sodium barbital buffer (pH 7.5); KCI,50,umol; NaCl, 10JUmol; MgSO4. 7H20, 25,umol; CaCl2-2H20, 5.0 umol; FeCl3.6H20, 1.5 Mmol; organophos-phate, 0.1 mM; and 1.0 ml of extract containing 700 to1,000 ug of protein. At intervals, 0.5-ml portions wereremoved for extraction and analysis. Sterile controlsand boiled cell-free extract were used to determineabiotic disappearance of the test compound. Activityis reported as nanomoles of organophosphate de-stroyed per minute per milligram of protein.

Analytical methods. Turbidity was measured at420 nm in 1-cm cuvettes in a Bausch and Lomb Spec-tronic 20 spectrophotometer. Inorganic phosphate wasassayed by the method of Dick and Tabatabai (7), andnone of the organophosphates or organic buffer inter-fered.

Protein was assayed at 660 nm by the method ofLowry et al. (12). Crystalline bovine serum albuminwas used as the standard. The organic buffer did notinterfere in the assay.

Organophosphate disappearance was measuredwith a Perkin-Elmner model 3920B gas-liquid chromat-ograph equipped with a flame ionization detector anda flame photometric detector fitted with phosphorusand sulfur filters. The packing material was 3% OV-17on 100/120-mesh Gas-Chrom Q in a Teflon-lined stain-less steel column (1.83 m by 2-mm ID; Applied ScienceLaboratory, State College, Pa.). The operating tem-peratures were as follows: 140, 200, and 230°C for thecolumn; 190 and 245°C for the injector; and 250 and275°C for the interface (detector). For the flame pho-tometric and flame ionization detectors, the heliumcarrier gas flow rate was 30 ml/min. The organophos-phate concentration was determined from standardcurves of each compound.To extract the organophosphates, portions of the

cultures were mixed with equal volumes of pesticide-grade ethyl acetate (Fisher Scientific Co., Rochester,N.Y.), and the organic layer was removed. The pro-cedure was repeated twice, and the organic phaseswere dried with anhydrous Na2SO4. Based on a stan-dard concentration of each chemical, the two extrac-tions removed more than 95% of the chemical sub-strate from the sample. Portions of the extract wereinjected into the gas chromatograph.To determine water-soluble products of organo-

phosphate breakdown, samples were treated with di-azomethane by the method of Daughton et al. (5). Thederivatized products were extracted twice with ethylacetate and dried with anhydrous Na2SO4 over glasswool before gas chromatography. Derivatives of di-methyl and diethyl phosphate and thiophosphateswere prepared similarly.

RESULTSEnrichment cultures able to use the 12 organ-

ophosphates as sole phosphorus sources were

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888 ROSENBERG AND ALEXANDER

readily obtained from sewage and soil samples.None of the organophosphates could serve as a

sole carbon source; i.e., there was not the ex-

pected increase in turbidity in a medium with10 to 20 mg-atoms of pesticide carbon per liter.To determine the capacity of the isolates to usea variety of organophosphates as phosphorussources, the bacteria were first grown in mediacontaining the organophosphate on which theywere isolated. These cultures were then inocu-lated into tubes containing 0.2 mM organophos-phate in the basal medium. Growth was meas-

ured turbidimetrically and compared with thatin solutions with no added phosphorus. A sum-mary of the results of a study to show the abilityof 12 bacterial isolates to grow on a variety oforganophosphorus insecticides is given in Table1. Organisms isolated from diazinon and mala-thion enrichments were the most versatile, ableto metabolize 10 and 9 compounds, respectively.Azodrin, Dasanit, malathion, and Orthene were

the most widely used, whereas Trithion andVapona were the least frequently used.The two organisms (both from soil) obtained

from diazinon and malathion enrichments were

characterized by the procedures described bySkerman (15). The isolates corresponded to thedescription of species of Pseudomonas (4) inbeing strictly aerobic, gram-negative, motile rodsthat bore polar flagella, produced catalase, wereoxidase positive, had a respiratory metabolism,and had simple growth requirements. The pres-ence of fluorescent pigmentation and arginine

dihydrolase, lack of poly-,8-hydroxybutyrate ac-cumulation, and growth at 4 but not at 410Csuggested that the organism isolated from dia-zinon enrichments was Pseudomonas putida,but because extensive taxonomic tests were notperformed, the bacterium was designated Pseu-domonas 28. The organism isolated from mala-thion enrichments was designated Pseu-domonas 7.To determine the optimum phosphorus con-

centration for growth, Pseudomonas 28 was

grown in basal medium with various concentra-tions of inorganic phosphate, diazinon, or mala-thion. The extent of growth, as measured byturbidity, was linearly related to the concentra-tion of phosphorus, with maximum growth oc-

curring at 0.15 mM (Fig. 1). At this concentra-tion, the molar ratio of carbon to phosphorus inthe medium was 15:1 and approximated the C:Pmolar ratio of bacteria (13). The specific growthrate, which varied with the phosphorus source,was highest for KH2PO4. Similar growth re-

sponses were obtained at temperatures of 20 to450C and pH values of 6.5 to 8.5. The growthresponses and C:P molar ratios in the mediumwere similar with Pseudomonas 7.To confirm that Pseudomonas 28 was utilizing

the insecticides as phosphorus sources and notgrowing on contaminating inorganic phosphate,the organism was grown in basal mediumamended with 0.20 mM diazinon (61 ,ug/ml) or

malathion (66 ,g/ml) as the sole phosphorussource. As Fig. 2 shows, growth ofPseudomonas

TABLE 1. Organophosphorus compounds used as phosphorus sourcesSource of bacterial isolate

Compounds used as sole P source'Common name Chemical structure

Aspon O,O,O,O-tetra-n-propyl dithiopyrophosphate As, Az, Da, Di, Dm, Ma, 0, PAzodrin O,O-dimethyl 0-(2-methylcarbamoyl-1-methyl- As, Az, Da, Di, Dm, Ma, Me, 0

vinyl) phosphateDasanit O,O-diethyl 0-[4-(methylsulfmyl)phenyl] phos- Az, Da, Dm, Dy, Ma, Me, 0, V

phorothioateDiazinon O,O-diethyl 0-(2-isopropyl-4-methyl-6-pyrimidi- As, Az, Da, Di, Dm, Dy, Ma, Me,

nyl) phosphorothioate 0, PDimethoate O,O-dimethyl S-(N-methylcarbamoylmethyl) Az, Da, Dm, Ma

phosphorodithioateDylox Dimethyl(2,2,2-trichloro-1-hydroxyethyl) phos- Az, Da, Di, Dm, Dy, Ma, 0

phonateMalathion O,O-dimethyl S-(1,2-dicarbethoxyethyl) phos- As, Az, Da, Dm, Ma, Me, 0, P, V

phorodithioateMethyl parathion O,O-dimethyl O-p-nitrophenyl phosphorothioate Az, Da, Di, Ma, Me, POrthene O,S-dimethyl acetylphosphoramidothioate As, Az, Da, Di, Ma, 0Parathion 0,0-diethyl O-p-nitrophenyl phosphorothioate Da, Di, Dm, Dy, Ma, 0, PTrithion S-[(p-chlorophenylthio)methyl] O,O-diethyl Da, Di, Dm, Ma, T

phosphorodithioateVapona 2,2-Dichlorovinyl O,O-dimethyl phosphate Da, Dm, Va Abbreviations: As, Aspon; Az, Azodrin; Da, Dasanit; Di, diazinon; Dm, dimethoate; Dy, Dylox; Ma,

malathion; Me, methyl parathion; T, Trithion; V, Vapona; 0, Orthene; and P, parathion. If a compound is notlisted, no turbidity increase compared with P-free solutions was evident.

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ORGANOPHOSPHORUS INSECTICIDE CLEAVAGE 889

1.0

0.5.

006 0.12 0.18CONCENTRATION (mM)

FIG. 1. Growth ofPseudomonas 28 after 48 h withvarious concentrations ofK2HPO4, malathion, or dia-zinon provided as the sole phosphorus source.

28 measured as protein was correlated with in-secticide disappearance. More than 75% of bothdiazinon and malathion disappeared by 84 h.During the growth cycle, the amount of phos-phorus utilized for growth agreed closely withthe amount needed for cell protein, assumingthat approximately 2% of the protein is phos-phorus (13). The growth of Pseudomonas 28 atthe expense of the insecticides and the extent ofgrowth could not be attributed to contaminatinginorganic phosphate from diazinon, malathion,or other medium ingredients. Furthermore, col-orimetric analyses of the organophosphates in-dicated no detectable inorganic phosphate, themethod being sensitive to less than 1.0 fig ofphosphate per ml. Similar results were obtainedwith Pseudomonas 7.Pseudomonas 28 was grown for 36 h in media

containing K2HPO4, diazinon, or malathion asthe sole phosphorus source. Resting cells (opti-cal density, 1.5) were prepared from these cul-tures, and these cells were incubated on a rotaryshaker for 24 h at 29°C with 30 to 40,ug ofdiazinon or malathion per ml in the presence or

absence of chloramphenicol (100 ,ug/ml). Theamount of substrate metabolized was deter-mined by gas chromatography. It is evident fromthe data in Table 2 that only resting cells derived

from cultures grown on the insecticides metab-olized diazinon and malathion to a significantextent. Cells derived from cultures grown on oneof the insecticides metabolized the other. Thevalues reported are corrected for substrate dis-appearance in the presence of cell suspensionsboiled for 10 min, the values for which rangedfrom 2.2 to 4.1 Ag of diazinon or malathion perml in 24 h. Except in one instance, the percent-age of breakdown was unaffected by the pres-ence of chloramphenicol. Similar results wereobtained with Pseudomonas 7. The data thusindicate that diazinon and malathion were me-tabolized by an induced enzyme system.To assess the specificities of the crude enzyme

preparations for various organophosphates, cell-

FIG. 2. Growth ofPseudomonas 28 and disappear-ance oforganophosphorus insecticides. D andM referto protein content of cells grown on diazinon andmalathion, respectively.

TABLE 2. Metabolism of diazinon and malathionby resting-cell suspensions ofPseudomonas 28

Amt of substrate me-

Chloram- tabolized (Ag/ml)P source for growth peioDiazinon Mala-

thion

KH2PO4 + 0.2 0.2- 1.0 1.0

Diazinon + 19 36- 18 22

Malathion + 25 19- 25 18

z

-i

x4

KH2PO40

MALATHION/ ro0

DIAZINON

0~ ~ ~ 0~

7,~~~u

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890 ROSENBERG AND ALEXANDER

free extracts of both organisms were incubatedat 29°C with each of the 12 organophosphoruscompounds at a 0.1 M concentration. The bac-teria were grown on either diazinon or mala-thion, and the extracts contained 700 to 1,000ytg of protein per ml. Extracts from both diazi-non- and malathion-grown cells of both isolatescatalyzed initial phases of the degradation of 8of the 12 organophosphates (Table 3), but ex-tracts of neither organism catalyzed the break-down of dimethoate, Dylox, methyl parathion,or Vapona. The maximum activities reportedare corrected for substrate disappearance in thepresence of extracts boiled for 10 min, the valuesfor which ranged from 2.4 to 8.6 nmol/min permg of protein.To determine whether ionic dialkyl phospho-

rus esters were formed by Pseudomonas 7 andPseudomonas 28, products formed by thesecrude enzyme preparations (after 10 h with 0.10mM substrate) were extracted with ethyl ace-tate, derivatized with diazomethane, and ana-lyzed by gas chromatography. On the basis of acomparison with retention times for the deriva-tized standards, the metabolites of six of theorganophosphorus compounds were tentativelyidentified. Both Pseudomonas 7 and Pseudom-onas 28 liberated the following: (i) dimethylphosphate from Azodrin; (ii) diethyl phospho-rothioate from Dasanit, diazinon, and parathion;(iii) dimethyl phosphorodithioate from mala-thion; and (iv) diethyl phosphorodithioate fromTrithion. The liberation of products tentativelyidentified as ionic dialkyl phosphorus and thio-phosphorus esters suggests that a major path-way for organophosphate degradation in thesebacteria is hydrolytic attack by a phosphatase

TABLE 3. Maximum activity of cell-free extracts ofPseudomonas 7 and Pseudomonas 28 on various

insecticidesMaximum activity (nmol of substrate de-

stroyed per min per mg of protein)

Substrate Pseudomonas 7 Pseudomonas 28

Diazi- Mala- Diazi- Mala-non thion non thion

Aspon 62a 1 loa 44a 88aAzodrin 92a 108a 12la 133aDasanit 238b 212b 205b 210bDiazinon 283c 250c 301C 263CMalathion 222C 275C 257c 288cOrthene 52a 41a 37a 32aParathion 189C 200c 259c 212cTrithion 142b 125b 138b 117ba At 10.0 h.b At 7.0 h.c At 3.5 h.

or phosphotriesterase to liberate the dialkylphosphoester.:Peaks with areas less than 10% ofthe areas of the major dialkyl phosphate peakswere observed, but these were not identified.

Since Pseudomonas 7 and Pseudomonas 28liberated products that appeared to be dialkylphosphate and thiophosphates, an experimentwas conducted to determine whether the orga-nisms could utilize various phosphoesters as solephosphorus sources. The basal medium wasamended with 0.2 mM phosphorus (a nonlimit-ing concentration) as trimethyl phosphate, tri-ethyl phosphate, tri-n-butyl phosphate, di-methyl phosphate, dimethyl phosphorothioate,dimethyl phosphorodithioate, trimethyl phos-phorothioate, diethyl phosphate, diethyl phos-phorothioate, or triethyl phosphorothioate.Growth of Pseudomonas 28 occurred only withtrimethyl, triethyl, and tri-n-butyl phosphates(Fig. 3). The maximum optical densities werebetween 1.0 and 1.2. Similar results were ob-tained with Pseudomonas 7.

0

0

1.0-~~~~~~~~~

Trimethyl phosphatew

0. T

R ~~~Triethyl phosphate -

00.,

HOURS

FIG. 3. Growth ofPseudomonas 28 in media withtrimethyl, triethyl, or tri-n-butylphosphate as the solephosphorus source.

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ORGANOPHOSPHORUS INSECTICIDE CLEAVAGE 891

DISCUSSIONThe results show that the two pseudomonads

used the insecticides as sole sources of phospho-rus and were not utilizing contaminant ortho-phosphate as the phosphorus source. The anom-

alous increase in protein concentration com-

pared with insecticide disappearance between 24and 45 h may be a result of the conversion ofthe substrate to an intermediate phosphorus-containing compound that the organisms can

use for growth. The data also demonstrate thatthe breakdown of such chemicals resulted froman induced enzyme or enzyme system. Thebroad substrate specificity of the enzyme sys-tems in the cell-free extracts is in line with theversatility of the organisms in using various or-

ganophosphates as phosphorus sources forgrowth. Cleavage of the organophosphates bythe enzyme preparations was not prevented byalkyl substituents since methyl, ethyl, and pro-pyl esters were metabolized. Nevertheless, sub-stituents on the phosphorus atom did affect therate of enzymatic action, an observation inagreement with previous reports (8, 9).The inability of the bacteria to utilize the

dialkyl phosphates, phosphorothioates, andphosphorodithioate as phosphorus sources issurprising since they could obtain phosphorusfor growth from the insecticides. A possible ex-

planation is the penetration of the nonionic in-secticides into the cells and their metabolismwithin the cell to the ionic alkyl phosphoruscompounds but the lack of permeability of thebacteria to the exogenously supplied ionic alkylphosphorus compounds. This view is supportedby the finding that the nonionic trialkyl phos-phorus compounds are utilized.

It is not surprising that a single enzyme prep-aration acts on several pesticides. For example,Munnecke (14) found that a crude extract froma mixed bacterial culture growing on parathionhad hydrolase activity not only for parathionbut also for eight other pesticides. Similarly,Kearney and Kaufman (11) reported that a pu-rified enzyme from a pseudomonad grown onisopropyl-N-(3-chlorophenyl)carbamate hydro-lyzed several similar phenyl carbamates, as wellas two acylanilide herbicides. In the presentinstance, the tentative identification of the prod-ucts of cleavage as dimethyl phosphate, di-methyl phosphorodithioate, diethyl phosphoro-thioate, and diethyl phosphorodithioate sug-gests that the enzyme acts by catalyzing hydrol-ysis of the aryl P-O bond, a view supported byother investigators (8, 9).The organophosphorus pesticides examined

were chosen because they contained several al-kyl moieties and because they are among the

most commonly used insecticides in the UnitedStates (16). Thus, the crude enzyme prepara-tions used in this study and in another study(14) can hydrolyze chemicals that represent animportant portion of the marketed insecticidalcompounds. Moreover, the reactions may be akey step in the detoxication ofmany biologicallyactive organophosphorus compounds and thusa major mechanism for destroying significantenvironmental toxicants.

ACKNOWLEDGMENTThis investigation was supported in part by contract

N00014-76C-0019 from the Office of Naval Research.

LITERATURE CITED1. Alexander, M., and B. K. Lustigman. 1966. Effect of

chemical structure on microbial degradation of substi-tuted benzenes. J. Agric. Food Chem. 14:410-413.

2. Bhaskaran, R., D. Kandasamy, G. Oblisami, and T.R. Subramaniam. 1973. Utilization of disyston as car-bon and phosphorus sources by soil microflora. Curr.Sci. 42:835-36.

3. Bourquin, A. W. 1977. Degradation of malathion by salt-marsh microorganisms. Appl. Environ. Microbiol. 33:356-362.

4. Buchanan, R. E., and N. E. Gibbons (ed.). 1974. Ber-gey's manual of determinative bacteriology, 8th ed. TheWilliams & Wilkins Co., Baltimore.

5. Daughton, C. G., D. G. Crosby, R. L Garnas, and D.P. H. Hsieh. 1976. Analysis of phosphorus-containinghydrolytic products of organophosphorus insecticidesin water. J. Agric. Food Chem. 24:236-241.

6. Daughton, C. G., and D. P. H. Hsieh. 1977. Parathionutilization by bacterial symbionts in a chemostat. Appl.Environ. Microbiol. 34:175-184.

7. Dick, W. A., and M. A. Tabatabai. 1977. Determinationof orthophosphate in aqueous solutions containing la-bile organic and inorganic phosphorus compounds. J.Environ. Qual. 6:82-85.

8. Eto, M. 1974. Organophosphorus pesticides: organic andbiological chemistry. CRC Press, Cleveland.

9. Faust, S. D., and H. M. Gomaa. 1972. Chemical hydrol-ysis of some organic phosphorus and carbamate pesti-cides in aquatic environments. Environ. Lett. 3:171-201.

10. Gunner, H. B., and B. M. Zuckerman. 1968. Degrada-tion of "diazinon" by synergistic microbial action. Na-ture (London) 217:1183-1184.

11. Kearney, P. C., and D. D. Kaufman. 1965. Enzymefrom soil bacterium hydrolyzes phenylcarbamate her-bicides. Science 147:740-741.

12. Lowry, 0. H., N. J. Rosebrough, A. L Farr, and R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

13. Luria, S. E. 1960. The bacterial protoplasm: compositionand organization, p. 1-34. In L. C. Gunsalus and R. Y.Stanier (ed.), The bacteria, vol. 1. Academic Press Inc.,New York.

14. Munnecke, D. M. 1976. Enzymatic hydrolysis of organo-phosphate insecticides, a possible pesticide disposalmethod. Appl. Environ. Microbiol. 32:7-13.

15. Skerman, V. B. D. 1967. A guide to the identification ofthe genera of bacteria, 2nd ed. The Williams & WilkinsCo., Baltimore.

16. Task Group on Occupational Exposure to Pesticides.1974. Occupational exposure to pesticides. FederalWorking Group on Pest Management, Washington,D.C.

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