APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1977, p. 356-362 Copyright © 1977 American Society for Microbiology

Vol. 33, No. 2 Printed in U.S.A.

Degradation of Malathion by Salt-Marsh Microorganisms1 A. W. BOURQUIN U.S. Environmental Protection Agency, Gulf Breeze Environmental Research Laboratory, Sabine Island, Gulf Breeze, Florida 32561 Received for publication 10 June 1976

Numerous bacteria from a salt-marsh environment are capable of degrading malathion, an organophosphate insecticide, when supplied with additional nutrients as energy and carbon sources. Seven isolates exhibited ability (48 to 90%) to degrade malathion as a sole carbon source. Gas and thin-layer chromatography and infrared spectroscopy confirmed malathion to be degraded via malathion-monocarboxylic acid to the dicarboxylic acid and then to various phosphothionates. These techniques also identified desmethyl-malathion, phosphorothionates, and four-carbon dicarboxylic acids as degradation products formed as a result of phosphatase activity.

Malathion, S-(1,2-dicarbethoxyethyl)-O ,Odimethyldithiophosphate, is an organophosphate insecticide used extensively to control adult mosquitoes. It was the most widely used insecticide in the United States in 1972, with an estimated annual production of 1.36 x 107 kg (30 x 106 pounds) (27). Massive programs for mosquito control were established in municipal areas and, in many instances, treatment involved application of malathion on or near saline marshes which serve as nursery grounds for a variety of marine species (24). Like most organophosphates, malathion is considered to be nonpersistent, and it is degraded in soils (11, 19, 21, 23, 28, 29), in aquatic systems (9, 20), and in terrestrial plants and animals (3, 5, 10). Despite the studies on toxicity of malathion to freshwater and marine fishes (7, 8, 14, 15) and marine invertebrates (4), and of fate after application to an estuary (6, 26), little information is available concerning degradation of this chemical in a salt-marsh environment. The objectives of this study were: (i) to isolate from a salt-marsh environment microbes capable of readily degrading malathion, and (ii) to identify the major metabolites formed during biodegradation of this chemical. MATERIALS AND METHODS Organisms. Sediment and water samples were collected from a salt-marsh environment on Santa Rosa Island, Fla., and incubated in the presence of 100 mg of malathion per liter on a rotary shaker at 28°C for 30 days. Samples (10 mg each) of pesticide were added every 7 days and samples were streaked on marine agar (Difco Laboratories) enriched with 100 mg of malathion per liter. Colonies picked from Gulf Breeze Environmental Research Laboratory contribution no. 291.

these plates were used in pesticide utilization tests (Table 1). This technique allowed the isolation of both malathion sole-carbon-degrading bacteria and bacteria capable of degrading malathion only in the presence of an additional substrate. In all cases, degradation is defined as any transformation of the parent molecule including both chemical transformation and microbial mineralization. Extensive information on taxonomy of the bacteria will be presented elsewhere. Information on the morphology and Gram characteristics are given in Table 1. Unless otherwise stated, bacteria used in biochemical studies were pseudomonad-type cultures. Materials. Malathion, malaoxon, malathion monocarboxylic acid (MCA), malathion dicarboxylic acid (DCA), and K-desmethyl-malathion (KDM) were obtained from American Cyanamid Co., Princeton, N.J. Radiolabeled malathion (methoxy-'4C) was obtained from Mallinckrodt Corp., St. Louis, Mo.; analytical-grade standards for chromatography were obtained from U.S. Environmental Protection Agency (EPA), Pesticides Reference Standards Section, Washington, D.C.; and standards for infrared spectroscopy were obtained from EPA, Southeast Environmental Research Laboratory, Athens, Ga. An organic-containing microbial medium was prepared with marine broth (Difco) with or without 2% agar. For some studies, a minimal medium containing malathion as a carbon source was prepared with aged artificial seawater (Rila Marine Mix, Teaneck, N.J.) with or without 0.2% (wt/vol) peptone. Malathion in distilled water was added at concentrations below 50 /ig/ml or in acetone at higher concentrations. All sea-salts media were adjusted to 20%o salinity with distilled water; nitrogen and phosphorous were kept constant by the addition of (per liter): NH4NO3, 2 g; Na2HPO4, 0.8 g, and NaH2PO4, 0.6 g. Final pH before autoclaving was 7.2.

Physicochemical degradation. The physicochemical degradability of malathion was tested in sterile seawater solutions. Ranges of light, temperature, and salinity were similar to those used in the micro356

VOL. 33, 1977

MALATHION DEGRADATION

TABLE 1. Microbial degradation of malathion Culture no.

Morphology and Gram reaction

Degradeda ()

Degradedw with 0.2%

peptone (%)

(-) Short rod 48 91 (-)Coccoid rod 2 81 (-)Medium rod 32 83 (-)Medium rod 66 100 (+)Short rod 36 94 (-) Medium rod 2 82 (-)Medium rod 1 83 (-) Short rod 28 90 Fungus 1 77 Fungus 50 91 (-) Slender rod 24 92 (-) Medium rod 71 100 (+) Slender rod 90 73 (-)Short rod 72 100 87 100 (-) Large ovoid rod a After 10 days of incubation as the sole carbon source, corrected for recoveries in sterile controls. bAfter 5 days of incubation with 0.2% peptone, corrected for recoveries in sterile controls. 1 2 3 4 5 6 7 8 9 10 11 12 44 45 47

bial degradation tests. Sufficient filter-sterilized (0.2-,um filter) malathion was added to obtain a final concentration of 1.0 mg/liter to flasks containing 50 ml of either sterile distilled water or sterile Rila seasalts adjusted with distilled water to salinities of 10, 20 and 30%o. Duplicate flasks of each salinity were incubated at 20 to 22 or 26 to 28°C in continuous darkness (wrapped in aluminum foil) or under 6,000lx illumination from Growlux fluorescent tubes in alternating 12-h periods of light and darkness. Duplicate flasks of each salinity were removed and extracted every 2 days, and residual malathion concentration was determined by gas-liquid chromatography (GLC). Microbial degradation studies. Twenty selected isolates from enrichment cultures were tested for ability to degrade malathion. A 0.2-ml amount of an 18-h culture grown in 10 ml of marine broth was inoculated into 10 ml of sterile sea-salts medium containing 100 jig of malathion per ml and incubated for 5 days at 28°C on a rotary shaker. Cells plus medium were extracted twice with equal volumes of petroleum ether (nanograde) in the incubation tube or flask, and the extract was analyzed for residual insecticide by GLC. The ability of each culture to degrade malathion was compared with that of a sterile uninoculated control. For quantitative determinations of malathion and its metabolites, isolates that exhibited greatest ability to degrade malathion were tested by inoculating approximately 105 cells washed with sterile sea-salts solution from an 18-h agar-slant culture into sea-salts medium containing 46 jig of malathion per ml with or without 0.2% peptone. The test cultures were incubated for 10 days at 28°C on a rotary shaker before extracting malathion for analysis by GLC. Bacteria that were quantitatively most efficient in utilizing malathion as a sole carbon source were tested for ability to incorporate 14C derived from [methoxy-'4Clmalathion. Cultures grown in -marine

357

broth were diluted 1:100 with seawater medium containing 200 ,ug of malathion per ml and 0.1 ,uCi of 14C in Biometer flasks (Bellco Glass, Inc.). Reaction vessels were incubated for 14 days at 28°C, and the evolution of '4CO2 was measured daily. Radioactive CO2 was recovered by adding 1.0 ml of alcoholichyamine solution (1 M; Packard Instrument Co., Inc.) to the side arm of the Biometer flask, incubating for 5 min, and removing the '4CO2]hyamine solution with a syringe. The hyamine solution was added to 10 ml of toluene-scintillation cocktail and analyzed on a Beckman model LS-250 liquid scintillation counter. After 14 days, the culture medium was treated with 0.5 ml of 2% trichloroacetic acid (final pH 2) and immediately extracted twice with equal volumes of petroleum ether. The ether phase was washed with 20 ml of 0.5 M potassium phosphate buffer (pH 7), and all fractions were assayed radiometrically to determine the extent of malathion breakdown. The ether phase contained malathion and some degradation products. As previously established by Matsumura and Boush (21), the aqueous fractions at pH 2 contained the carboxyesterase-metabolized products (confirmed by GLC analysis; see Table 3), and those at pH 7 contained phosphatase and other hydrolysis products. Degradation rates were determined by incubating the cultures in seawater medium containing 46 Ag of malathion per ml and sampling at 2-day intervals for malathion residues. Sterile peptone was added to cultures that showed no greater reduction in malathion than did uninoculated control cultures, and the cultures were analyzed for malathion residues after an additional 4 days of incubation. Several bacterial isolates that were capable of utilizing malathion as the sole carbon source as well as isolates capable of utilizing this chemical in the presence of peptone were inoculated into 10 ml of sea-salts-0.2% peptone medium (20%o salinity) that contained 46 Atg of labeled malathion per ml (0.1 ,uCi of 14C). After 10 days of incubation, cells were removed by centrifugation at 12,000 x g for 10 min, washed with 10 ml of sterile seawater, dried for 24 h at 85°C on membrane filters (0.45 ,um), and analyzed for 14C activity by liquid scintillation counting in a toluene cocktail. The cell-free supernatant was extracted twice with petroleum ether and analyzed for soluble degradation products by assaying for residual 14C activity. To determine malathion metabolites, bacterium no. 45 was incubated in seawater medium and bacterium no. 8 was incubated in seawater medium with 2% peptone, both containing 100 pg of malathion per ml. (In earlier screening studies [Table 1], culture no. 45 degraded malathion as a sole carbon source, whereas culture no. 8 used malathion as a cosubstrate in the presence of peptone.) Cultures were incubated 10 days in the dark at 28°C. Cells and medium were separated by centrifugation, and the cell-free medium was extracted twice with equal volumes of petroleum ether to remove malathion. The aqueous fraction was acidified to pH 1.5 with trichloroacetic acid (0.5%) and extracted with an equal volume of petroleum ether-acetone (1:1) and

358

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then with diethyl ether. The extracts were concentrated and analyzed for malathion and metabolites by thin-layer chromatography (TLC) (1). GLC. Residual malathion and malathion breakdown products extracted from the various physicochemical and microbial degradation studies were quantitated by GLC methods, using Varian-Aerograph model 2100 with a tritium source electron capture detector and a Tracor model MT-220 with a Melpar flame photometric detector. Electron capture GLC determinations employed two columns (0.64 cm by 1.8 m): one contained 2% OV-101 and 100/120-mesh Gas-Chrom Q; the other contained equal parts of of 0.75% OV-17 and 0.85% OV-210 on 100/120 Gas-Chrom Q. Column, detector, and inlet temperatures were 185, 250, and 250°C, respectively. The carrier gas was nitrogen, used at a flow rate of 25 ml/min. Flame photometric analyses employed a detector operated in the phosphorus mode and a column (0.32 cm by 1.8 m) containing 2% OV-101 on 80/100-mesh Gas-Chrom Q. Respective columns, detectors (ignited), and injected temperatures were 180, 160, and 220°C, and gas flow rates for 02, air, H2, and N2 (carrier) were 20, 50, 200, and 60 ml/min, respectively. Amyl derivatives of malathion degradation products were prepared for gas chromatography analyses by methods described previously (25). All samples were quantified by comparing peak heights with standards of known concentration. Percentages of recoveries are given in the respective data tables with corrections for internal standards where appropriate. TLC. Extracts from biodegradation studies were separated on 250-,um-thick Silica Gel H (20 cm by 20 cm) thin-layer glass plates prepared by Quanta/ Gram (A. H. Thomas Co., Philadelphia, Pa.). Plates were spotted with 10 to 20 Al of concentrated extract and developed in the appropriate solvent systems (A) benzene-hexane-acetic acid (40:40:20), and (B) hexane-acetic acid-ether (75:15:10) or (C) hexaneacetic acid-diethyl ether (2:2:1). For qualitative analyses, two-dimensional chromatography was employed, using the solvents (A) and (B) (18). After they were air dried, the plates were sprayed with freshly prepared 0.5% (wt/vol) N-2,6-trichloro-pbenzoquinoneimine (Eastman Kodak Co., Rochester, N.Y.) in nanograde acetone and then developed at 110°C for 10 min (16). Spots representing malathion and its degradation products appeared dark reddish-pink on a light background. Good separation of malathion, malaoxon, KDM, MCA, and DCA was affected with solvent B (Rf values, 0.93, 0.80, 0.63, 0.71, and 0.28) and with solvent C (Rf values, 0.95, 0.77, 0.23, 0.68, and 0.31). IR spectroscopy. To prepare metabolites for infrared (IR) spectral analyses, 1 to 2 ml of the concentrated acetone extract was streaked on a TLC plate and developed with benzene-glacial acetic acid (4:1) or solvent A in one direction. After it was air dried, the plate was covered with another glass plate that allowed about 2 cm of the TLC plate to be exposed, and this was sprayed with N-2,6-trichloro-p-benzoquinoneimine (28). Areas corresponding to metabolite bands were scraped from the plate and extracted with 50 ml of acetone. This extract was concentrated to 1 to 2 ml under vacuum at 35°C, and an appropri-

ate portion was added to dried potassium bromide

(Harshaw Chemical Co., Cleveland, Ohio) for analysis on a Perkin-Elmer model 621 Grating IR spectrophotometer. When appropriate, samples were analyzed by operating the instrument in a 5 x-expanded ordinate scale, using a micro-pellet of KBr. Spectral tracings for malathion degradation products were compared with those for standards supplied by American Cyanamid Co. and EPA Southeast Environmental Research Laboratory. Quality assurance. Recovery data for GLC analyses are given in the respective tables. All calculations for GLC and TLC data are based on known standards (see above). The IR spectrometer was calibrated prior to scanning samples on the polystyrene blank, and appropriate adjustments were made. All assays for radioactivity were corrected for efficiency with a ['4C]toluene internal standard (New England Nuclear Corp., Boston, Mass.).

RESULTS Physicochemical degradation. Malathion degradation in sterile seawater increased with increasing salinity with ranges and averages of residual malathion detected after various lengths of incubation (Fig. 1). Each point represents four samples, one light- and one darkincubated sample from each of two temperatures. Limited malathion degradation was observed in distilled water, the rate of degradation being much slower than in seawater. No differences in degradation due to effect of light or temperatures were observed. Breakdown products from chemical degadation of malathion were identified by flame photometric gas chromatography as malathion monocarboxylic acid (detected after 2 days) and malathion dicarboxylic acid (detected after 7 days). Malaoxon was detected, but quantities did not exceed the trace amounts found in the malathion stock solution. Microbiological degradation. Estuarine bacterial isolates by malathion-enrichment

_

100 so

DW

2 60

~40

IE20

K

%.SW

20L °

2

6 10 14 18 22 26 INCUBATION TIME (DAYS)

30

FIG. 1. Effect of salinity and temperature on malathion stability. A 1 -dg portion of malathion per ml was added to acetone to sterile seawater (SW) at varied salinities. DW, Distilled water. Dashed line represents microbial degradation rates in nonsterile environments, and bars show range of four samples. Malathion was determined by a GLC method.

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techniques were tested for biodegradative ability by incubating washed cells suspended in sea-salts medium containing malathion with and without 2% peptone as a supplementary energy source. Final pH of all cultures after incubation ranged between 6.3 and 6.8. Residues analyzed by GLC after 10 days of incubation are reported in Table 1 as percentage of added malathion degraded. Controls consisted of malathion media without cells. Only 40% of the isolates tested showed 50% or greater utilization of malathion as the sole carbon source, whereas in the presence of peptone, malathion was rapidly degraded by all isolates. When bacterial cultures were grown on I '4C]malathion with peptone, most of the radioactivity became associated with the cell fractions; however, 8 to 28% remained in the supernatant. The supernatant activity (Table 2) probably represented water-soluble metabolites of malathion. To measure radioactive metabo-

lites of malathion, spent medium from tests using ['4C]malathion as a sole carbon source was assayed for carboxyesterase and phosphatase products by differential extraction and liquid scintillation counting, and confirmed by chemical analysis (Table 3). The carboxyesterase products, MCA and DCA, were identified by GLC retention times and quantitated by comparison with peak heights of standards of known concentration. Chemical residue analyses and isotopic analysis for malathion agreed closely. In most cases, MCA was the predominant degradation product of biological as well as chemical reactions, as indicated in the control data. The data indicate a greater portion of phosphatase products due to biological breakdown (cultures 44, 45, and 47), with lesser production of similar products due to chemical breakdown (control). The microbial mixture, indicated in Table 3, represents equal proportions of the aforementioned cultures. Data from the same cultures as in Table 3 indicated that the mixed culture affected the release of "4CO2 TABLE 2. Cell-medium distribution of '4C from from the methoxy groups on malathion within 2 [methoxy- '4C]malathion" days, whereas the single isolates required incuTotal radioactivity" (%) bation for 7 days before "4CO2 was detected. In Culture no. either case, the total "4CO2 released was insigWet cells Supernatant nificant compared to the radioactivity associ24 1 76 ated with the cells and medium in the sterile 92 8 2 control, 0.02% of the total malathion label 87 3 13 (methoxy- 4C) was detected as '4CO2, and the 72 28 4 mixed culture was only slightly higher at 11 89 5 0.08%. 90 6 10 Figure 2 shows the degradation of malathion 81 7 19 in seawater medium by three different bacteria 93 7 8 and one fungus. Two cultures (no. 1 and 12) 80 9 20 readily utilized malathion, whereas two other 83 17 10 cultures (no. 6 and 9), incubated for 10 days, 87 11 13 12 91 9 showed little degradation of the molecule be"Incubation medium was seawater medium con- yond that of chemical degradation. However, taining 46 ,ug of ['4C]malathion per ml plus 0.2% the latter cultures readily transformed the insecticide when 0.2% peptone was added. peptone in all cases. Isolation and identification of microbial ', Total radioactivity recovered represented 70 to metabolites. Spent seawater malathion me80% of the added activity. TABLE 3. Radiometric and chemical analyses of microbial growth on [methoxy- 4CImalathion Radiometric analysis: total radioactivity remaining in expended medium (%) Culture no.

Residue analyses: flame photometric GLC analysis

Products of:

Recovery (mg/liter)

Recovery

44 45 47 Mixture

Malathion

Carboxyes-

Phospha-

Malathion

MCA

DCA

Total

25.7 8.3 6.5 7.4 10.5

36.8 48.2 61.3 87.2 82.4

37.5 43.5 32.2 5.4 7.1

19 5 75 75 9

85 159 80 11 168

40

144

72

8 23 55 7

172 178 141 184

86 89 71 92

terase

tase

Control" 'l Sterile medium plus 1 '4C]malathion; products are due to chemical breakdown.

(

360

APPL. ENVIRON. MICROBIOL.

BOURQUIN 10

a ____a

gPEPTONE

I

5 zI 2'

TLC SEPARATION: MALATHION & METABLITES fl!.JS!!= minW-90 ala SIFANSARM

x|1 CONTROL

8S 10

st

_______

O

1100)

( (n)AL70 \15INA%)

(100)

(92)~

;I

41

21

SECOND DIMINSION (75St1510) (Nox..ns Acetic AsddiEthr) u

2

4

6 a 12 10 INCUBATION (DAYS)

14

FIG. 2. Bacterial degradation of 46 pg of malathion per ml in seawater medium. Sterile peptone (0.2%, final) added after 10 days of incubation; extent of chemical degradation shown by percentage of original malathion concentration in sterile controls at various incubation times. Values of malathion remaining in cultures are given as percentages of malathion in the sterile controls. Symbols: O, Culture no. 6; *, fungus; O, culture no. 1; and *, culture no. 12.

dium from cultures 8 and 45, with and without added nutrient, was extracted, concentrated, and assayed by TLC to separate and tentatively identify degradation products. Good separation of malathion and its metabolites was achieved on a two-dimensional TLC. Figure 3 shows typical chromatograms from microbial degradation and control media (malathion but no cells). Four compounds the two carboxylic acids, malathion, and KDM were identified by comparison with Rf values of known compounds (see above). Other metabolites, indicated by numbers 5, 6, 7, and 8, were probably phosphothionates, judging from published Rf values in the same solvent system (23), but no standards were available to us for comparison. With the exception of spot number 8, most of the latter compounds were not present on the control plate and, therefore were considered microbial metabolites. All compounds isolated from culture 8 extracts were also isolated from culture 45. Explanations of infrared spectral analysis of malathion and metabolites are taken from Bellamy (2), Jones (17), and Walker and Stojanovic (30). Adsorption peaks at 2,960 and 1,450 cm-' represent asymmetrical C-H stretches, whereas those at 2,940 and 1,375 cm-' represent asymmetrical C-H stretches. Bands at 2,250 cm-' represent S-H bonds. A strong band at 1,730 cm-' represents a C=O, whereas a band 1,000 to 1,200 cm-' indicates a C-O (1,170 cm-'). Methyl and C=C groups adsorb at 1,380 and 1,640 cm-', respectively. The band at 1,010 cm-' indicated P-0-C bonding and C-C stretch bonds are indicated at 1,100 cm-'. The band at 655 cm-' represents P S bond and the weak peaks at 515 and 490 cm-' possibly represent P-S. -

-

-

FIG. 3. Thin-layer chromatogram of malathionseawater medium with (culture extract) and without

(control) an inoculum of bacterium no. 45. Reference standards are included for comparison of spots.

The IR spectral tracing of the compound from TLC spot 1 is identical to that of malathion with major adsorption bands at 655, 1,100, 1,170, and 1,380 cm-'. The spectral tracing of malaoxon differs in several ways from those of all other metabolites and malathion. Malaoxon was not present as a metabolite in this culture fluid. The spectral tracings of MCA and spot 2 were identical with major bands at 655, 1,380, and 1,730 cm-' and reduced adsorption compared to malathion at 1,100 and 1,170 cm-'. DCA and spot 3 were shown to be identical with major bands at 655, 1,100 cm-' and a sharp band at 1,730 cm-' indicating the C=O bands. All peaks were identical, indicating that the spot represents that compound. The IR spectral tracing of TLC spot 4 was identical to KDM for most peaks except the P=S adsorption at 655 cm-' and the minor band at 2,200 to 2,300 cm-' indicating an S-H bond. Other metabolites eluted from spots on the thin-layer plates were not in sufficient quantity to obtain quality IR spectra. Comparisons with available standards showed little similarity to these metabolites; therefore, the compounds cannot be conclusively identified. The IR spectra of the chemical from TLC spot 5 showed major adsorption bands at 670 cm-', P-S, at 655 cm-'. P=S, and at 1,030 cm-', P-O-C, indicating a possible methylated thiophosphate group. However, the P-H stretch at 2,350 cm-' indicates that at least one methyl group has been cleaved from the malathion molecule. The bands at 1,380, 1,450, and 1,550 cm-' indicate an ethyl group, and the band at 1,740 cm-' is a weak acid adsorption band. This probably indicates a MCA derivative of the parent molecule. No conclusive evidence could be obtained from these samples for positive identification of the metabolites found in spots 5 through 9 because of low quantities of sample. However, from TLC data and earlier published studies a number of phosphorothionate derivatives would be expected from phosphatase activity on this chemical (13, 18, 19, 22).

VOL. 33, 1977

DISCUSSION The half-life of malathion in seawater was reported to be approximately 92 to 96 h, and the chemical degradation was influenced by both temperature (27°C) and salinity (12). The pH was adjusted in our studies with Na2HPO,NaH2PO4 in seawater to pH 7.2; the ionic strength of the seawater (increasing with salinity) was probably partially responsible for the degradation exhibited. Previous reports indicated that malathion was quite stable under neutral or acid pH conditions (similar to our degradation studies), and that its susceptibility to hydrolysis increased with increasing alkalinity (12, 28). Likewise, malathion is thermostable at temperatures of 21°C and below, but rapidly dissipates at temperatures of 27 to 32°C (12). Eleven of 15 bacterial cultures isolated from salt-marsh environments after malathion enrichment degraded malathion as a sole carbon source, whereas all 15 isolates degraded the compound within 5 days when an additional carbon source was added. In radiolabeled studies, little malathion carbon contributed to the cell mass of the cultures tested primarily because the "4C tracer was associated only with the methoxy groups. Other cell-mass carbon contributions could have been from the ethyl groups, due to carboxyesterase cleavage to form the malathion-carboxylic acids; however, the ethyl radical was not radiolabeled, and the resulting cell mass would not have been radioactive. Most biological activity seems to be associated with an effective carboxyesterase system that causes early breakdown to the acids. The extent of degradation of malathion by a mixed culture system was greater than that by individual cultures. The microbial systems all had an effective carboxyesterase system that caused rapid breakdown of malathion to the acids, with a delayed demethylation reaction to produce desmethyl-malathion. Some, microbial systems apparently catalyze demethylation earlier, resulting in a more rapid release of CO2 from the malathion methoxy group. Both type cultures, sole-carbon-degrading and co-metabolizing bacteria, yielded 10 metabolites on TLC which were identified by TLC, IR as monocarboxylic acid malathion; DCA (both chemical and biological degradation products); KDM (small amount produced by chemical degradation); and a number of compounds believed to be phosphodithionates (found only as metabolites), for which no standards were available. The low concentration of the latter metabolites is believed to be a consequence of the nature of these compounds, i.e., easily degraded by most

MALATHION DEGRADATION

361

bacteria. It should be noted, however, that toxicity, although reduced, is still sufficiently high in the phosphorylated derivatives to cause reduction in fish brain acetylcholinesterase (D. Coppage, personal communication, Gulf Breeze Environmental Research Laboratory, Gulf Breeze, Fla.). It is unknown if the thiophosphates remain stable in the environment; however, they apparently do remain cholinesterase inhibitors. Malathion is degraded, in vitro, by saltmarsh bacteria using metabolic pathways similar to those found in soil bacteria and fungi and in animals. Application of malathion to a saltmarsh area would result in a rapid disappearance of parent compound by carboxyesterase activities with delayed demethylation and phosphatase activities. The major metabolites are, however, not detoxified for nontarget animals tested, and acetylcholinesterase inhibition persists for some time after the parent compound is no longer detectable. ACKNOWLEDGMENTS I thank L. Kiefer and S. Cassidy for technical assistance in this work, and J. Kakareka for interpretation of IR spectra.

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life management. National Mosquito Control-Fish and Wildlife Management Coordinating Committee, Washington, D.C. Culley, D. D., and H. G. Applegate. 1967. Residues in fish, wildlife and estuaries. Pestic. Monit. J. 1:21-28. Eaton, J. G. 1970. Chronic malathion toxicity to the bluegill. Water Res. 4:673-684. Gardner, A. M., J. N. Damico, E. A. Hansen, E. Lustig, and R. W. Storherr. 1969. Previously unreported homolog of malathion found as residues on crops. J. Agric. Food Chem. 17:1181-1185. Getzin, L. W. 1971. Partial purification and properties

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of soil enzyme that degrades the insecticide malathion. Biochim. Biophys. Acta 235:442-453. Guerrant, G. O., L. E. Fetzer, Jr., and J. W. Miles. 1970. Pesticide residues in Hale County, Texas before and after ultra-low volume aerial application of malathion. Pestic. Monit. J. 4:14-20. Gunther, F. A., and R. C. Blinn. 1956. Persisting insecticide residues in plant materials. Annu. Rev. Entomol. 1:167-180. Hansen, D. J., E. Matthews, S. L. Nall, and D. P. Dumas. 1972. Avoidance of pesticides by untrained mosquitofish, Bambus affinis. Bull. Environ. Contam. Toxicol. 8:46-51. Hansen, D. J., S. C. Schimmel, and J. M. Keltner, Jr. 1973. Avoidance of pesticides by grass shrimp (Palaemonetes pugio). Bull. Environ. Contam. Toxicol. 9:129-133. Jaglan, P. S., and F. A. Gunther. 1970. A thin-layer chromatographic procedure for separating desmethyl methyl parathion (O-methyl-O-p-nitrophenyl phosphorothionate) and its S-isomer (S-methyl-O-p-nitrophenyl phosphorothionate). Bull. Environ. Contam. Toxicol. 5:47-49. Jones, R. N. 1959. Infrared spectra of organic compounds: summary charts of principal group frequencies. National Research Council, Ottawa, Canada, NRS Bulletin no. 6. Kadoum, A. M. 1970. Thin-layer chromatographic separation and colorimetric detection of malathion and some of its metabolites from stored grains. J. Agric. Food Chem. 18:542-543. Konrad, J. G., G. Chesters, and D. E. Armstrong. 1969. Soil degradation of malathion, a phosphorodithioate insecticide. Soil Sci. Soc. Am. Proc. 33:259-262. Lewis, D. L., D. F. Paris, and G. L. Baughman. 1975.

21. 22.

23.

24.

25. 26.

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