Literature Cited (1) Tan, K. H., Leonard, R. A., Bertrand, A. R., Wilkinson, S. R., Soil Sei. Soc. Amer. Proc., 35,265-9 (1971). (2) Lassiter, J. W., Mason, J. V., Perkins, H. F., Brown, A. R., Beaty, E. R., Univ. Ga. College Agr., Ga. Agr. Expt. Sta., Res. Bull., 25,23 pp, 1967.
(3) Perkins, H. F., Parker, M. B., Walker, M. L., ibid., Bull. N.S., 123,24pp, 1964. (4) Papanos, S., Brown, B. A., Storrs Agr. Expt. Sta., Univ. Connecticut, INF Series 13, 1-5, 1950. (5) Parker, M. B., Univ. Ga., College Agr., Ga. Agr. Expt. Sta., Bull. N.S., 159,5-15, 1966. (6) Jones, J. B., Jr., Warver, M. H., “Analysis of Plant Ash Solution by Spark Emission Spectroscopy,” in E. L. Grove and A. J. Perkins, Eds., “Development in Applied Spectroscopy,” Vol. 7A, pp 152-60, Plenum Press, New York, N.Y., 1969. (7) Bremner, J. M., “Total Nitrogen,” in C. A. Black (Editor-inChief), “Methods of Soil Analysis,” Part 2, A.S.A. Monograph 9, 1149-78, 1965. (8) Soil Survey Staff, “Soil Series of the United States, Puerto Rico, and the Virgin Islands, Their Taxonomic Classification,” 361 pp, Soil Conservation Service, USDA, 1973. (9) Tan, K . H., McCreery, R. A., Soil Sei. Plant Anal., 1, 75-84 (1970).
(10) Tan, K . H., King, L. D., Morris, H. D., Soil Sei. Soc. Amer.
Proc., 35,718-51 (1971). (11) Weber, J. B . , A m e r . Mineralog., 51,1657 (1966). (12) Greenland, D. J., Oades, J . M., Trans., 9th Intern. Congr. Soil Sci. I, pp 657-78, 1968. (13) Bailey, G. W., White, J. L., Rothberg, T., Soil Sci. SOC. Amer. Proc., 32,222-34 (1968). (14) Inoue, T., Wada, K., Trans., 9th Intern. Congr. Soil Sci. 111, pp 289-98, 1968.
(15) Mortland, M. M . . A d v . Aeronomv. 22.75-117 (19701. (16) Mortenson, J. L., Anderson, D.” M.,’ White, J . L.; “Infrared Spectroscopy,” in C. A. Black (Editor-in-Chief), “Methods of Soil Analysis,”Part 1,A.S.A. Monograph 9,743-70, 1965. (17) Tan, K. H., Clark, F. E., Geoderma, 2,245-55 (1969).
Received for review April 19, 1974. Accepted Oct 15, 1974. Contribution f r o m the University of Georgia, Agricultural Experim‘ent Station, College Station, Athens, Ga., and Soil, Water and Air Sciences, Southern Regional Agricultural Research Service, U.S. Department of Agriculture. Mention of commercial products is for identification only and does not constitute endorsement by any agency of the U.S. Government.
Rates of Degradation of Malathion by Bacteria Isolated from Aquatic System Doris F. Paris,* David L. Lewis, and N. Lee Wolfe
Freshwater Ecosystems Branch, Southeast Environmental Research Laboratory, National Environmental Research Center-Corvallis, U . S . Environmental Protection Agency, Athens, Ga. 30601
A heterogeneous bacterial population that grew in culture solution with 0,O-dimethyl S-(1,2 dicarbethoxy)ethylphosphorodithioate (malathion) as the only extraneous source of carbon has been isolated. Gas-liquid chromatographic analysis of methylated samples from the cultures showed that the major metabolite was @-malathion monoacid. Only 1% of the malathion was transformed to malathion dicarboxylic acid, 0,O-dimethyl phosphorodithioic acid, and diethyl maleate. At low concentrations of malathion (less than the value of K,) and low concentrations of bacteria, the rate of bacterial degradation can be described mathematically by a second order rate expression. System analysts may find this kinetic expression useful in the construction of models.
0,O-dimethyl S -( 1,2 dicarbethoxy)ethylphosphorodithioate (malathion) is a widely used organophosphorus pesticide. Its increased application is evidenced by a steady rise in production, approaching 35 million lb in 1971 ( 1 ) . This nonsystemic insecticide and acaricide has received wide usage primarily because of its low mammalian toxicity and its biological selectivity. I t is used in agriculture for insect control and in mosquito control by direct application to water areas. Malathion can therefore enter the aquatic environment either directly or through agricultural runoff. Because of its wide usage, extensive research has been stimulated concerning the biological degradation of malathion. Most studies to date have revolved around its degradation in soils. Matsumura and Boush ( 2 ) reported rapid degradation of malathion in cultures of the soil fun-
gus, Trichoderma viride, and a bacterium, Pseudomonas sp., isolated from soil, The products of metabolic degradation were mainly carboxylic acid derivatives of malathion, indicating carboxylesterases are probably involved in microbial metabolism. High demethylation activity was also evident in some Trichoderma uiride varieties indicating another degradation pathway. Walker and Stojanovic ( 3 ) found that malathion is readily degraded by an Arthrobacter sp. The four metabolites produced were identified as malathion half-ester, malathion dicarboxylic acid, potassium dimethyl phosphorothioate, and potassium dimethyl phosphorodithioate. The studies of Mostafa et al. ( 4 ) also indicate that malathion is readily degraded. These researchers report that the fungi, Penicillium rota t u m and Aspergillus niger, metabolized 76 and 59% of the malathion in the medium within 10 days. Both fungi degraded the insecticide through carboxylesteratic hydrolysis as well as by a demethylation process. Data concerning the fate of malathion and its degradation products in natural waters are not available. Most information concerning its breakdown by aquatic microorganisms pertains to its toxicity to such organisms. Algal growth was reported to be inhibited by malathion ( 5 ) ; Sanders and Cope (6) found that malathion was toxic to two species of daphnids; and Macek and McAllister (7), in their insecticide susceptibility studies of fish (catfish, bullhead, goldfish, minnow, carp, sunfish, bluegill, bass, rainbow salmon, brown salmon, coho salmon, and perch), reported considerable species differences, the lowest tolerance being coho salmon (96-hrTL50 = 0.101 ppm). An evaluation of the environmental impact of malathion on the aquatic environment requires an understanding of the breakdown processes, biological and otherwise, of the insecticide. An integral part of this underVolume 9, Number 2,February 1975
135
standing is a quantitative description of the rates of degradation. We studied the kinetics of malathion degradation in aqueous medium .by a heterogeneous population of aquatic bacteria and identified the degradation products. The rate of malathion degradation by bacteria reported for our system would not be expected to be the same as that for a natural system. Although the bacterial concentrations used approximate the total bacterial concentrations found in a natural aquatic system, only a fraction of the natural population would be expected to degrade malathion. In the experimental system, malathion was the only carbon source, whereas in a river or lake other sources would be available. Therefore, although our studies do not predict the rate of degradation for natural systems, they do provide a basis for comparison of bacterial rates with chemical and photochemical rates obtained in the laboratory.
Materials and Methods When enrichment culture techniques were used, bacteria capable of degrading malathion were isolated from river water. The basal salts medium had the following "4'21, 0.5 gram (9.35 mmol)/l.; composition: (NH4)2S04, 0.5 gram (3.78 mmol)/l.; MgS04, 0.01 gram (0.83 mmol)/l.; NazHP04, 3.0 grams (21.1 mmol)/l.; and KHzP04, 2.0 grams (14.7 mmol)/l. ( 8 ) .All components of the medium were reagent grade chemicals. (The company determined the purity of these compounds to be 98.799.870.) The pH of the medium was determined with a Beckman Zeromatic pH meter. Media were adjusted to pH 6.8, a pH a t which preliminary studies had shown chemical hydrolysis of malathion to be negligible. Several flasks of the basal salts medium containing 2.5 grams (12.6 mmol) of glucose and 0.5 mg (1.5 pmol) of malathion per liter were inoculated with river water and incubated on a gyratory shaker in an environmental chamber at 28°C. When the bacteria had removed significant amounts of the pesticide, an inoculum containing the mixed population was transferred into fresh medium containing 1.0 gram (5.05 mmol) glucose and 1.01 mg (3.03 pmol) malathion per liter. Transfers into media containing decreasing quantities of glucose and increasing quantities of malathion were carried out until a population was obtained that grew in a medium containing 10.1 mg (30.3 pmol) per liter of the pesticide as the sole carbon source. The final population was lyophilized for use in the current studies. The members of the population have been identified as Flavobacterium meningosepticum, X a n t h o m o n a s species, Comamonas terrigeri, and Pseudomonas cepacia A saturated solution of malathion (American Cyanamid Co., 99.6% purity) was prepared by stirring malathion into the basal salts solution, then sterilized by passing it through a sterile 0.22-pm Millipore filter. Duplicate malathion solutions with varying concentrations were prepared by aseptically diluting the filtrate (concentration approximately 303 pmol/l.) with sterile basal salts medium. Bacteria were grown for 24 hr in a nutrient broth solution diluted by a factor of 10 and spiked with malathion a t a concentration of 5 mg (15 pmol)/l. The bacteria were harvested by centrifugation and washed three times with sterile dilution water. Washed cells were suspended in 100 ml dilution water for 24 hr prior to inoculation in the pesticide medium. Malathion concentration in the experimental flasks was determined a t zero time and at each sampling period by extracting an aliquot of the whole culture (medium and bacteria) with 2,2,4-trimethylpentane (isooctane) and analyzing the extract by gas-liquid chromatography (glc). At a ratio of 1:l isooctane to sample, extraction efficiency was 96-99%. Determinations were performed using a Tra136
Environmental Science & Technology
cor MT200 gas-liquid chromatograph equipped with a high-temperature Nickel-63 electron capture detector. The glc separations were achieved using a 1-meter glass column (4 mm i.d.) packed with Gas Chrom Q 8 O / l O O mesh support coated with 3% silicone SE-30. Typical glc analysis conditions employed for column, detector, and inlet were 170, 240, and 195"C, respectively. Carrier gas (nitrogen) flow rate was 120 ml/min. Under the above conditions, the retention time of malathion was 0.85 min. Peak height data were used to calculate malathion concentration. Viable cell numbers were estimated by plate counts at 0 hr and a t each sampling time (9). Tryptone-glucose-extract agar was used as plating medium, and the cultures were incubated aerobically a t 28°C. When the malathion in the cultures was depleted, the medium was adjusted to pH 2 with 1.OM HC1 and extracted with two 100-ml portions of chloroform. Products in the extract were separated by preparative thin-layer chromatography using plates coated with Camag silica gel, Type D-5. The developing solvent was hexane:acetic acid:ethyl ether ( 7 5 :15:lO) ( 1 0 ) . Products were visualized by spraying a portion of the plate with the reagent of Menn et al. ( 1 1 ) , 0.570 2,6-dibromo-N-chloro-p-quinoneimine (DCQ) in acetone. R; values of the products were the same as the R, values of standards (American Cyanamid Co.). Mass spectra were obtained with a Finnigan 1015-SL quadrupole mass spectrometer (solid probe) and a Systems Industries 150 digital computer. The identifications of three products-diethyl maleate, 0,O-dimethyl phosphorodithioic acid, and 8-malathion monoacid-were confirmed by mass spectra of authentic samples. Mass spectra of the first two compounds are reported in the literature ( 1 2 ) . The major mass spectral fragments for the latter compound (70 eV) m/e (relative intensity) are 256(2), 158(41), 145(43),and 143(42). One product was identified by methylating the organic solvent extracts and determining the methylated product by gas-liquid chromatography (13).
Results and Discussion In the current studies, the heterogeneous bacterial population grew in culture solution with malathion as the only extraneous source of carbon. As the population increased, the decrease in concentration of the insecticide was monitored by glc of isooctane extracts of whole cultures (media plus bacteria). All unmetabolized malathion was therefore detected, including any that may have been adsorbed on the bacterial cell surface. From Monod kinetics (14), removal of malathion from the medium may be described by Equation 1
where p m is the maximum growth rate ( h r - l ) for the bacteria in the basal salts-malathion medium; [SI is malathion concentration (pmol/l.); [ B ]is the number of viable bacteria per liter; Y is the yield factor or number of viable bacteria produced per pmol of pesticide; and K , is a constant numerically equal to the malathion concentration supporting a growth rate of 0.5 p m . To determine p,, a series of media with malathion concentrations ranging from 0.0273-128 pmol/l. were inoculated with suspensions of washed organisms to give viable bacterial concentration of 106 per liter. A rearranged Monod equation was used ( 1 5 ) :
and experimental values [SI/@were plotted as a function of [SI (Figure 1). The slope of the resulting plot, 1/pm, determined by computer program employing least squares analysis, yields a value of 0.37 hr-1 for p m . The value of K , was determined from the intercept ( K s / p m ) to be 2.17 pmol/l. for the experimental system. Equation 1 takes into account the major factors influencing the rate of substrate utilization of batch culture. At high substrate concentrations (greater than five times K,), the equation reduces to
‘Is] df
0.0273 0.0273 0.21 0.21 0.273 0.273 0.33 0.33 a
(4 dt where k is a second-order rate constant (liter organism-1 hr-1) for removal of pesticide by bacteria. Equation 1, the more accurate description of substrate removal kinetics, requires the knowledge of p m , K,, and Y, all of which may be determined from growth kinetics experiments. However, the very low solubility of many pesticides in water often precludes the range of experiments necessary to determine these parameters. The more simplified Equation 4 would be useful if it were found to define accurately the kinetics for bacterial removal of pesticide. To test this, we defined the term p m / { Y ( K s + [SI)} from Equation 1 as the second-order rate coefficient (h’)
x
8.0 8.0 1.8 2.3 3.0 3.0 4.1 2.6 Av. 4.1 2 2.3b
k‘,
I. org-1 hr-1
I. o r g - I h r - 1 x 10-12
2.9 2.5 1.2 2.2 3.5 1.9 3.4 3.3 2.6 0.85
2.1 2.1 8.6 6.8 5.1 5.1 3.6 5.7 4 . 9 % 2.1b
x
10’0
10-12
*
Org = organism. b Standard deviation.
:
- k[S][B] =
k,
Yield, org”/pM
Malathion prnol/l.
$” [ B ]
Substrate removal follows pseudo first-order kinetics and is independent of substrate concentration. At [SI,much less than the value of K s , Equation 1 can be approximated by -d‘S1
Table I. Yield Values and Rate Constants for Removal of Malathion by Bacteria
(CH,O)z-P-SH
5
+ (CH,O)z+-S-CHCOOH
0,O-Oimethylphosphorodithioic acid
HCCOOEt
+
I
CHzCOOEt Malathion a-monoacid
5
(CH,O)z-P-S-CHCOOEt
t
It
CHzCOOH
EtOOCCH Diethyl fumarate
1
Malathion B-monoacld
Hydrolysis, pH 8, 2P
f
ICH,O)z-P-S-FHCOOEt CHrCOOEt Malathion
I I
Bacteria, 280
~ , o - ~ i ~ e t ht ~ICH30)r-P-S-CHCOOH l~h~~+ I phorodithioic acid CHzCOOH Malathion diacid
CHCOOEt
I1
+ Malathion 8-monoacid
CHCOOEt Diethyl maleate
Figure 2. Comparison of chemical and microbial degradation products of malathion
‘I /
pMnx= 0.37 h i ‘ K, =2.17pM/I
60
[SI Figure 1. Lineweaver and Burk (75) plot of specific growth rates and substrate concentration
and calculated k’ using the kinetic data for p m , K,, and Y. The rate constant, k in Equation 4 was determined experimentally and compared with the calculated values of k ’ . These values are in agreement at low bacterial and malathion concentrations. In Table I, h ranges from 1.23.5 x 10-12 liter organism-l hr-1 and k ’ ranges from 2.1-8.6 X liter organism-1 hr-1. Metabolic products (Figure 2 ) were extracted from the medium with chloroform, and the organic layer was analyzed by tlc (16). By comparison of R f values with those of authentic samples (17), the products were identified as malathion mono- and dicarboxylic acids, the 0,O-dimethyl phosphorodithioic acid, and diethyl maleate. The identifications of three of the metabolites were confirmed by mass spectrometry of the isolated products. Identification of the fourth product, the dicarboxylic acid, was confirmed by methylating the organic solvent extract and comparing the retention time of the methylated product by gas-liquid chromatography with that of a methylated standard. To determine the relative amounts of products, the organic extract was methylated with diazomethane and the resulting mixed esters were analyzed quantitatively by glc. Of the malathion degraded, 99% was degraded to the monoacid, shown subsequently by tlc to be the /?-monoacid ( I 7). Under identical conditions the @-monoacid was not degraded to a detectable extent in 41h months. The bacteria were still viable at the end of the experiment. Under similar conditions the diacid was also not degraded. Volume 9, Number 2, February 1975
137
In further studies, filtrates of bacterial cultures did not degrade malathion, suggesting that degradation was not extracellular. Since the @-monoacid could be recovered quantitatively from the bacterial cultures, degradation was not intracellular. We assume, therefore, that malathion was degraded ectocellularly. In a natural system, physical, chemical, and biological removal processes compete and interact and their rates are controlled by environmental conditions that are characteristic of the individual aquatic system. For example, a t pH 6.8-7.0 and 27”C, malathion does not readily hydrolyze; its half-life is about one month ( 1 7 ) .However, a t pH 9.0 and 27”C, the half-life of malathion is about 10 hr. The photolysis rate, on the other hand, is dependent upon the concentration of humic acids. In water containing no humic acids, the photolysis half-life is 990 hr, whereas in water containing humic acids, the photolysis half-life is 15 hr (18).In the absence of humic acids, therefore, photolysis would not be expected to be the dominant degradation pathway. At low concentrations of malathion-degrading bacteria (2 x 106/1.) and low-malathion concentration (3.3 pM/l.), the half-life of the pesticide was calculated to be 41 hr a t 28°C. Therefore, in neutral waters (pH 6.8-7.0) containing little humic acids, bacterial removal may compete successfully. T o date, the individual degradation processes-bacterial, photochemical, and chemical-have been studied independently. Further studies must be done to test the applicability of the kinetic expression to systems in which the three processes are acting simultaneously.
Acknowledgment We wish to thank John M. McGuire and Ann L. Alford of the Analytical Chemistry Branch for confirming the identifications of the metabolites by mass spectrometry, R. C. Blinn of the American Cyanamid Co. for standards of malathion products, and the Center for Disease Control, Atlanta, Ga., for identifying the bacteria. We would also like to express our appreciation to John T. Barnett. Jr., and John Gordon for their technical assistance.
138
Environmental Science & Technology
Literature Cited (1) U.S. Environmental Protection Agency, “The Pollution Potential in Pesticide Manufacturing,” EPA Report TS-00-72-04, 1972. (2) Matsumura, F., Boush, G. M.; “Malathion Degradation by Trichoderma viride and a Pseudomonas Species,” Science, 151, 1278-80 (1966). (3) Walker, W. W., Stojanovic, B. J., “Microbial versus Chemical Degradation of Malathion in Soil,” J . Environ. Qual., 2 (2), 229-32 (1973). (4) Mostafa, I. Y., Bahig, M . R. E., Fakhr, I. M. I., Adam, Y., “Metabolism of Organophosphorous Insecticides. XIV. Malathion Breakdown by Soil Fungi,” 2. AVaturforsch.,27b, 1115-16 (1972). (5) Moore, Richard, Bull. Enuiron. Contam. Toxicol., Vol. 5, 3, 226-30 (1970). (6) Sanders, J. O., Cope, O.B., Trans. A m e r . Fish. Soc., 95, 1659 (1966). (7) Macek, K . J., McAllister, W. A., ibid., 99,ZO-7 (1970). (8) Payne, W. J., Feisal, V. E., Appl. Microbiol., 11, 339-44 (1963). (9) American Public Health Assoc., Inc., “Standard Methods for the Examination of Water and Wastewater 1965,” 1965. (10) Kadoum, A. M., J . Agr. Food C h e m . , 18,542-3 (1970). (11) Menn, J. J., Erwin, W. R., Gordon, H . T., ibid., 5 , 601-2 (1957). (12) Imperial Chemical Industries Ltd., Dyestuff Division, in collaboration with the Mass Spectrometry Data Centre, “Eight PeakIndex of Mass Spectra,” Vol. 1, pp 114 and 133, 1970. (13) U.S. Environmental Protection Agency, “Correct Procedures in GC-MS Analysis of Organics in Water,” EPA Report R2-73277, 1973. (14) Stumm-Zollinger, Elisabeth, Harris, R. H., Chapt. 23 in “Organic Compounds in Aquatic Environments,” S. J. Faust, and J . V. Hunter, Eds.. Marcel Dekker, Inc., New York, N.Y., 1971. (15) Lineweaver, H., Burke, D., J . A m e r . Chem. SOC.,56, 658-66 (1934). (16) , , Welling. W.. Blaakmeer. P. T.. Couier. H.. J . Chromatoe.. ” , 47,281-3 7i97oj. (17) Wolfe, N. L., U.S. Environmental Protection Agency, Southeast Environmental Research Laboratorv. - , Athens:, Ga:. unuublished data, 1974. (18) Zepp, R. G., personal communication, ibid., 1973. ,
I
,
,
1
Received for review April 29, 1974. Accepted September 30, 1974. Mention of commercial products is for identification only and does not constitute endorsement by the Environmental Protection Agency of the C S.Government.
