APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1978, P. 668-672 Vol. 36, No. 5 0099-2240/78/0036-0668$02.00/0 Copyright © 1978 American Society for Microbiology Printed in U.S.A. Phosphorus-Containing Pesticide Breakdown Products: Quantitative Utilization as Phosphorus Sources by Bacteria ALASDAIR M. COOK, CHRISTIAN G. DAUGHTON, AND MARTIN ALEXANDER* Laboratory ofSoil Microbiology, Department ofAgronomy, Cornell University, Ithaca, New York 14853 Received for publication 1 September 1978 Bacteria were isolated that could utilize representatives of the following ionic phosphorus-containing breakdown products of organophosphorus pesticides as sole phosphorus sources: dialkyl phosphates, dialkyl phosphorothioates, dialkyl phosphorodithioates, alkyl arylphosphonates, alkyl arylphosphonothioates, and alkyl alkylphosphonates. Utilization of each organophosphorus compound, which was complete for 7 of 12 compounds studied, was confirmed by determination of protein yield from the amount of phosphorus source consumed. This is the first report of the utilization of an ionic dialkyl thiophosphate or dithiophosphate by microorganisms. The environmental fate of the initial products have been reported to serve as phosphorus of pesticide breakdown is largely unknown be- sources for bacteria (1, 4, 11, 16, 22), whereas the cause research has been devoted principally to failure to obtain enrichments on phosphoryl-thio studying the initial transformations that a pes- analogs has been reported (7; J. M. Tiedje, M. ticide will undergo. The organophosphorus pes- S. thesis, Cornell University, Ithaca, N.Y., 1966). ticides are a major class of biocides that are used The present study was designed to determine in massive quantities worldwide. They generally whether the exceedingly unreactive (12) ionic exhibit very high acute toxicity and sometimes organophosphorus products of pesticide hydrol- delayed neurotoxicity, and they are widely re- ysis are susceptible to degradation by microor- garded as "nonpersistent." These organophos- ganisms. Twelve representative compounds phorus pesticides offer a unique opportunity for were examined. We report the first definitive a simplified study of the potential fate of their evidence for the existence of microorganisms breakdown products since all organophosphorus freshly isolated from natural ecosystems able to pesticides are susceptible to hydrolysis, resulting utilize ionic dialkyl phosphorothioates, dialkyl in the release of an ionic phosphorus-containing phosphorodithioates, and methyl phenylphos- moiety. This moiety is almost invariably an ionic phonate as sole phosphorus sources. dialkyl phosphate or alkyl- or arylphosphonate, or a sulfur analog thereof. Consequently, a few MATERIALS AND METHODS ionic phosphorus compounds can serve as rep- Materials. The organophosphorus compounds resentative models for the entire spectrum of used and their sources are described in Table 1. Ab- phosphorus-containing breakdown products breviations used in this paper are given in Table 1. from these pesticides. These ionic compounds The mole fraction of inorganic orthophosphate (Pi) in are also released to soils or natural waters be- the organophosphorus compounds was less than cause in 0.0005, except for DMP (0.041) and MPn (0.026), and of their extensive use industry, e.g., as the theoretical phosphorus content was observed in ore flotation promoters in mining, additives to all instances (A. M. Cook, C. G. Daughton, and M. lubricants, flame retardants, and wastes from Alexander, Anal. Chem., in press). All other chemicals pesticide manufacture. The limited literature on were of the highest purity available commercially. the metabolism and toxicology of these products Glassware. The glassware was washed in 5 M of pesticide hydrolysis is inconclusive and con- nitric acid to remove contaminative phosphate (4). tradictory and has been reviewed recently by Media. The growth media were buffered at pH 7.4 several authors (4, 5, 7; C. G. Daughton, Ph.D. with either 50 mM tris(hydroxymethyl)methylamine thesis, University of California, Davis, 1976). or 50 mM HEPES [4-(2-hydroxyethyl)-1-piperazine- Definitive evidence for microbial utilization of ethanesulfonic acid] and contained 2.7 mM KCI, 0.8 mM MgSO4, 40 mM NH4Cl, and trace elements (18). an ionic dialkyl phosphorus compound has been This portion of the medium, together with a stable obtained only for dimethyl hydrogen phosphate carbon source, could be autoclaved without formation (9), and indirect evidence for the bacterial deg- of a precipitate. When required, carbon and phospho- radation of diethyl hydrogen phosphate has rus sources were added aseptically. Organophosphorus been presented (2). Several organophosphonates compounds, glucose, and glycerol were sterilized by 668 VOL. 36, 1978 PESTICIDE PRODUCTS AS PHOSPHORUS SOURCES 669 TABLE 1. Ionic organophosphorus compounds Abbreviation Chemical name Cheniical formula DMP 0,0-dimethyl hydrogen phosphatea (CH30)2P(O)OH DEP 0,0-diethyl hydrogen phosphate' (C2H50)2P(O)OH DMTP 0,0-dimethyl potassium phosphorothioatec (CH30)2P(S)OK DETP 0,0-diethyl potassium phosphorothioatec (C2H50)2P(S)OK DMDTP 0,0-dimethyl potassium phosphorodithioatec (CH30)2P(S)SK DEDTP 0,0-diethyl potassium phosphorodithioatec (C2H50)2P(S)SK coPn Dihydrogen phenylphosphonated C6H5P(O)(OH)2 M4Pn 0-methyl hydrogen phenylphosphonated (CH3O)C6H5P(O)OH M4TPn 0-methyl potassium phenylphosphonothioated (CH3O)C6H5P(S)OK MPn Dihydrogen methylphosphonate' CH3P(O)(OH)2 IMPn 0-isopropyl hydrogen methylphosphonatee [(CH3)2CHO]CH3P(O)OH PMPn 0-pinacolyl hydrogen methylphosphonate' [(CH3)3CCH(CH3)0]CH3P(O)OH a Pfaltz & Bauer, Stamford, Conn. b Eastman Organic Chemical Div., Rochester, N.Y. c American Cyanamid, Princeton, N.J. d Velsicol Chemical Corp., Chicago, Ill. 'Chemical Systems Laboratory, Aberdeen Proving Ground, Md. passage through a membrane filter (4). Analytical methods. Protein (5-ml samples) and Enrichment and isolation of bacteria. Sewage turbidity were measured by the methods previously from a primary settling tank was centrifuged (20,000 described (4). A turbidity of 1.0 at 500, 420, and 650 x g for 10 min at 4°C), and the supernatant fluid was nm represented 100, 85, and 170 iLg of protein per ml, discarded to reduce the quantity of extraneous phos- respectively. The assay for Pi and the wet ashing phorus sources. The pellet was suspended in buffered procedure for total phosphorus will be published else- salts solution and used as the inoculum for growth where (Cook et al., Anal. Chem., in press). The con- media containing 0.1 mM phosphorus source as pre- centration of organophosphorus compound in the ex- viously described (4). The resulting enirchment cul- tracellular solution was calculated as the difference tures were streaked on nutrient agar, and each colony between total phosphorus and Pi concentrations. that gave rise to growth in the selective liquid medium (i.e., homologous with the enrichment medium) was RESULTS AND DISCUSSION subcultured again in selective liquid media before re- Isolation of strains. The use of pure phos- streaking on nutrient agar. This procedure was re- phorus compounds is of utmost importance in peated three times to yield colonies of homogeneous studying phosphorus-limited growth. Contami- characteristics. Strain A was stored on nutrient agar nation of the organic phosphorus compounds slants. Most strains lost their activity on the phospho- with small amounts of Pi (2 to 4%) caused no rus compounds when maintained on nutrient agar and when the media thus were maintained by subculturing in selective interference during enrichment liquid media at intervals of 2 to 4 weeks. In addition to were limiting in phosphorus, but 10 of 11 isolates the freshly isolated bacteria, Pseudomonas testoster- enriched and selected after growing in media oni was used; this bacterium was previously designated with commercially available and unpurified as strain 11 (4). phosphorodithioates and hydrolyzed chloro- Quantification of growth and substrate utili- phosphorothioates (DMDTJ?, DEDTP, DMTP, zation. Cultures (30 ml) contained in 50-ml Erlen- and DETP) were unable to grow in media with meyer flasks and closed with cotton plugs were incu- the respective pure compound. Many strains lost bated at 29°C on an orbital shaker (170 rpm, 3-cm the ability to utilize organophosphorus com- eccentricity). Utilization experiments were done in transferred on nutrient media with 0.1 mM of the phosphorus source and 5 pounds when repeatedly mM p-hydroxybenzoate as carbon source, and they agar; as a result, each isolate was purified by were inoculated (3%, vol/vol) with a culture grown alternate growth in selective liquid medium with limiting organophosphorus (0.1 mM). The data (containing the appropriate phosphorus source) for turbidity and protein at zero time were calculated and nutrient agar. Selective solid media could from the corresponding values in the culture used for not be prepared because of the quantity of con- inoculation; Pi and total phosphorus in the extracel- taminative Pi (4). lular fluid were assayed after passage through a mem- Four pure cultures (P. testosteroni and strains brane filter (0.2-um pore diameter). After growth A, D, and K) were able to utilize 11 of the 12 ceased, samples were assayed for turbidity, protein, A control organophosphorus compounds tested (Table 2), and extracellular Pi and total phosphorus. a culture with M0TPn culture with Pi as phosphorus source was grown for and mixed (J) grew slowly each strain. Sterile