Stimulation of Methanogenesis by Aldicarb and Several Other N ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1986, p. 1247-1251

0099-2240/86/061247-05$02.00/0 Copyright © 1986, American Society for Microbiology

Vol. 51, No. 6

Stimulation of Methanogenesis by Aldicarb and Several Other N-Methyl Carbamate Pesticidest RONALD P. KIENE* AND DOUGLAS G. CAPONE Marine Sciences Research Center, State University of New York at Stony Brook, Stony Brook, New York 11794 Received 16 January 1986/Accepted 19 March 1986

Aldicarb and several other N-methyl carbamate pesticides stimulated methane production in anaerobic salt marsh soils and organic-rich aquifer soils. Stimulation was biological and linearly related to the amount of carbamate added. Of the four carbamates studied, methomyl gave the greatest stimulation followed by carbaryl, aldicarb, and baygon. The percent conversions [(moles of CH4 in excess of control/mole of carbamate added) x 100] for methomyl, carbaryl, aldicarb, and baygon were 88, 57, 40, and 11, respectively. Using aldicarb as a model carbamate, we found that monomethylamine (MA) accumulated in sediments as a result of aldicarb addition. MA arises from the N-methyl carbamoyl portion of the carbamates as a result of presumptive biological hydrolysis. MA levels decreased as CH4 production was stimulated, and 2-bromoethane sulfonic acid (a specffic inhibitor of mathanogenesis) partially inhibited the loss of MA. These findings suggest that N-methyl carbamates are readily hydrolyzed to MA in the presence of an active microbial population under anaerobic conditions and that methanogenesis is stimulated as a result of the consumption of MA by methanogenic bacteria. In the early 1960s carbamate pesticides were developed as a biodegradable alternative to highly stable organochlorines such as dichlorodiphenyltrichloroethane (DDT) (2, 12). A variety of esters of methyl carbamic acid have been synthesized which inhibit acetylcholinesterase, an enzyme vital to nervous system function. Carbamate pesticides are used extensively throughout the world for the control of insects and nematodes (13). Recently there has been great interest in the environmental fate of N-methyl carbamates, particularly that of aldicarb (Temik), which has been found to persist in groundwater (1, 21, 25), and carbaryl, which is the most extensively used carbamate pesticide. Although the concentrations which cause acute toxicity in humans are generally higher than those found in the environment (Temik aldicarb pesticide: the Long Island water situation, Union Carbide Technical Report, 1979), the potential for the formation of N-nitrosocarbamates (potent mutagens) under conditions present in the human gut (5, 20) dictates that even low levels of carbamates are of some concern. Relatively little information exists on the fate or distribution of carbamate pesticides in aboveground aquatic environments, even though runoff from agricultural lands and groundwater intrusion are probable sources of these pesticides for lakes, streams, and estuaries. Most studies of carbamate degradation and effects on microorganisms have been carried out in plants, animals, and soils under oxygenated or ill-defined conditions (4, 7, 19, 22). Weber and Rosenberg (23) and Karinen et al. (8) examined the effects of carbaryl on microorganisms and followed the degradation of the pesticide in estuarine waters. Only a few studies have considered that the fate of carbamates may be different under anaerobic conditions compared with aerobic conditions (15, 18). Anaerobic conditions may be present in waterlogged soils, aquatic sediments, and certain groundwaters. Therefore, it is important to understand the effects of * Corresponding author. t This paper is contribution Research Center.

no. 511

carbamate pesticides on anaerobic microflora and potential degradation mechanisms of these compounds under anaerobic conditions. Kiene and Capone (9) observed that aldicarb stimulated CH4 production in salt marsh sediments. In this study we report that a variety of N-methyl carbamate pesticides stimulate methane production in anaerobic salt marsh soil and in freshwater aquifer soil. The most likely mechanism for the observed stimulation of methanogenesis is a relatively rapid hydrolysis of N-methyl carbamates to yield monomethylamine (MA). MA is subsequently utilized by methanogenic bacteria to form methane. (Portions of this work were presented at the annual meeting of the American Society for Microbiology, 1985, Las Vegas Nev. [Abstr. Annu. Meet. Am. Soc. Microbiol. 1985, I14, p. 148].) MATERIALS AND METHODS Compounds and chemicals. The N-methyl carbamates used in this study included the sulfur-containing oximes aldicarb (Temik; Union Carbide) and methomyl (Lannate; Du Pont), and the aromatic compounds carbaryl (Sevin; Union Carbide) and propoxur (Baygon; Bayer). The chemical structures of these compounds are given in Fig. 1. The only functionality common to all of these compounds is the N-methyl carbamoyl (-OCONHCH3) group. Carbamates were obtained from Ultra Scientific Co. (Hope, R.I.) and were >97% pure. Stock solutions were made in acetone and were prepared fresh before each experiment. All other chemicals used in this study were reagent grade. Sediment source and preparation. Soil core samples were obtained from among stands of Spartina alterniflora in the salt marsh at Flax Pond (Old Field, N.Y.) and from an organic-rich portion (3.5-m depth) of the upper aquifer located on the south shore of Long Island (Copaigue, N.Y.). Sediment-soil slurries were prepared as described previously (9). Briefly, soils were homogenized anaerobically and dispensed (25 or 50 ml) into 125-ml Erlenmeyer flasks. Rubber stoppers were used to seal the flasks, which were subsequently flushed for 1 min with N2 to achieve anaerobic

of the Marine Sciences 1247

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KIENE AND CAPONE

~~

0 S-CH3 0II S-CH3 1 fl~ I CH- C-CH=N-u-O-NH-CH3 CH3 C=N-O-C-NH-CH3

CH3

CH3 ALDICARB 0

O-C-N H-CH3

METHOMYL

O-CH (CH3 )2

A

O-C-N H-C H3 0

CARBARYL

BAYGON

FIG. 1. Chemical structure of the carbamate pesticides used in this study. Note that the only common functionality is the N-methyl carbamoyl (-OCONCH3) group. Trade names and manufacturers are given in Materials and Methods.

conditions. Marsh sediment slurries had a salinity of -27 g liter-1 (measured with a refractometer) and a sulfate concentration of -22 mM. Aquifer samples had a salinity of 0 g liter-1. Sulfate was not measured in these samples. Individual treatments were run in duplicate or triplicate. Killed controls were obtained either by autoclaving or by the addition of glutaraldehyde or formaldehyde (0.1% each). Carbamates were added in 0.1 to 0.2 ml of acetone, and controls received an equal amount of acetone. Samples were incubated at 25°C with gentle shaking for 4 to 12 days, depending on the experiment. Methane measurements. Methane was monitored in the headspace of flasks with a Perkin-Elmer Sigma 2B gas chromatograph equipped with a flame ionization detector. The column was stainless steel (2 m by 3 mm) packed with Porapak R (80/100 mesh). Injector, oven, and detector temperatures were 150, 100, and 175°C, respectively. Nitrogen (40 ml/min) was used as the carrier gas. Methane peak heights were measured and compared with those of standards. Standards were prepared by diluting pure CH4 and were cross-checked with commercial standards (Alltech Associates, Inc., Applied Science Div., State College, Pa.). MA measurements. We used aldicarb as a model carbamate to test whether MA was released during our experiments. To monitor MA in sediment slurries we used a modification of the procedure described by King et al. (10). Two milliliters of sediment slurry was removed from each flask with a syringe at selected time intervals. This was then centrifuged, and 1 ml of the supernatant was placed in a 6-ml serum bottle. The pH of the sample was lowered to 90%, and no correction was made for this efficiency. RESULTS AND DISCUSSION The addition of aldicarb to slurries of salt marsh sediments greatly stimulated methanogenesis. Stimulation was directly related to the amount of aldicarb that was added (Fig. 2), which suggests to us that the carbamate is degraded. The production of CH4 from aldicarb was biological because it was blocked by the specific methanogenic inhibitor 2bromoethane sulfonic acid (BES), as well as by autoclaving, by glutaraldehyde, and by formaldehyde (data not shown). The slope of the line in Fig. 2 indicates that approximately 0.5 mol of excess CH4 is produced for each mole of aldicarb that is added. Similar results were obtained in freshwater sediment samples obtained from an anoxic portion of the upper aquifer of western Long Island. The addition of 5.1 ,Lmol of aldicarb to aquifer sediments resulted in fivefold stimulation of methanogenesis and 47% conversion. MA additions to aquifer samples also stimulated methanogenesis, and 54% conversion was observed. These results indicate that the kinds of reactions leading to CH4 stimulation are not restricted to saline sediments. It is unlikely that methanogenic bacteria directly metabolize aldicarb, because they are known only to metabolize a limited number of low-molecular-weight substrates (25). 6 5

[ E I

0

0

4

0

31

0 Co

C) 0

21

X UJ

1

0

0

2

4

ALDICARB ADDED

6

8

10

umol0)

FIG. 2. Excess CH4 above that of controls plotted against the amount of aldicarb added to salt marsh sedment slurries for two separate experiments. Symbols: 0, November 1983; F1, March 1984. The regression line yields a slope of 0.5 mol of CH4 per mole of aldicarb added. Values for excess CH4 were taken after production had leveled off (7 to 8 days).

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METHANOGENESIS FROM N-METHYL CARBAMATE PESTICIDES

Rather, aldicarb is probably degraded to compounds which become substrates for methanogens. There are two portions of the aldicarb molecule which could be sources of immediate CH4 precursors. The S-methyl group (Fig. 1) is similar to methane precursors such as methionine (17), methyl mercaptan (26), and dimethyl sulfide (26; R. P. Kiene, R. S. Orerhiland, A. Catena, L. G. Miller, and D. G. Capone, stbmitted for publication). Experiments with [14C]aldicarb labeled at the S-methyl carbon failed to yield any 14CH4 (measured by gas chromatography-gas proportional counting) in anaerobic marsh sediments over a 2- to 3-week incubation perod (data not shown). In these experiments pretreatment and enrichment with high levels of aldicarb also failed to yield 14CH4, even though stimulation of methanogenesis was observed. The N-methyl group of aldicarb, as well as other carbamates, is knowrf to be released as MA on hydrolysis (11, 14). MAs can serve as substrates for several kinds of methanogenic bacteria (6) and can support methanogenesis in the presence of the high sulfate concentrations that are found in marine surface sediments (10, 16). N-Methyl[14C]aldicarb was not available to us during this study; therefore, we tested whether MA levels in sediments amended with

15

0

10

0

E

5

0

15 0

0

E

w z

10

i -i

2

5 0

1

2

3

4

5

6

7

8

DAYS

FIG. 3. Time course of CH4 production (A) and MA levels (B) in salt marsh sediments (50 ml) treated with aldicarb (42 tmole, in acetone; (D); aldicarb-BES (8 mM; A); BES alone (0); and acetone controls (0.1 ml; 0). Values represent the means from duplicate flasks, and error bars indicate + 1 standard deviation. The detection limit for MA was 5 ,umol per flask, and no methylamnine was detected in acetone controls or in treatments with BES alone.

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20

15

z 10 E

2 0

2

4

8

8

10

DAYS

FIG. 4. Effects of four N-methyl carbamate pesticides and MA on methane production in anoxic slurries of salt marsh soils. Symbols: 0), acetone control; *, carbaryl; F1, baygon; A, aldicarb; A, methomyl; *, MA. Carbamates (8 to 10 ,umol) were added in acetone solution. MA (8 pumol) was added in distilled Water. Plotted data are the mean values for two replicate samples. Standard deviations were less than 10% in all cases and are not shown.

aldicarb were higher than in unamended samples. In Fig. 3 is shown a time course of CH4 production and MA levels in salt marsh slurries which received either no treatment, 840 ,uM aldicarb; aldicarb-8 mM BES (to inhibit methanogenic bacteria), or BES alone. Methane production was clearly stimulated by the addition of aldicarb (Fig. 3A), and this stimulation was blocked by the inhibitor BES. MA levels showed a sharp increase shortly after the addition of aldicarb (Fig. 3B). No MA was detected in acetone controls or after treatment with BES alone. Aftdf the initial increase, MA levels decreased rapidly in uninhibited samjples during the period when CH4 production was stimulated, whereas MA persisted in the aldicarb-BES treatment. There was some decrease of MA levels in the presence of BES, indicating that processes other than methanogenesis are involved in MA consumption. Results similar to those shown in Fig. 3 were obtained in several experiments (data not shown). The observation that MA decreased, despite inhibition of methanogens, is consistent with the findings of King et al. (10), who found that MA could be metabolized to CO2 in sediments, presumably by sulfate-reducing bacteria. Apparent maximum hydrolysis of aldicarb in sediment slurries occurred on the order of 4 to 10 days, based on the progress curves of CH4 production and the results of MA measurements from several experiments. Attempts to determine whether aldicarb hydrolysis (release of MA) in sediment slurries occurs biologically or chemnically were unsuccessful. Autoclaving, formaldehyde, and glutaraldehyde were used in separate experiments; but all proved unacceptable as killed controls for aldicarb hydrolysis because of interferences with MA analysis by gas chromatography. It is likely that the majority of hydrolysis is biological because in sterile aqueous solution at the pH of sediment slurries (7.0 to 7.2), the half-life of aldicarb is 245 days (3). In addition, Miles and Delfino (15) found that the rate of aldicarb hydrolysis slowed as ionic strength increased. Thus, under sterile conditions, aldicarb should persist longer in saline water than in freshwater. The presence of sediment particles and reduced chemical species such as sulfide may affect the

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KIENE AND CAPONE

TABLE 1. Percent conversiona of several carbamate pesticides to CH4 in anaerobic salt marsh sediments Compound

No. of experiments

% conversion (range) to CH4b

Aldicarb (Temik) Carbaryl (Sevin) Baygon (Propoxur) Methomyl (Lannate)

3 2 2 2

57.2 (36.8-71.6) 11.0 (9.3-12.7) 87.5 (81.3-93.8)

39.2 (18.9-59.5)

aPercent conversion was calculated as: (mole excess CH4 above controls/mole carbamate added) x 100. b The range was obtained in several experiments.

degradation of aldicarb (15), and this effect is not know for our system. Other studies have found that aldicarb degradation is much higher in samples with high biological activity (4, 18), which supports our contention that aldicarb hydrolysis is primarily biological. The addition of several other N-methyl carbamates, with various chemical structures, to anaerobic salt marsh sediment slurries stimulated methanogenesis, relative to that in unamended control samples (Fig. 4). The addition of a similar concentration of MA also stimulated methanogenesis, with a time course identical to that of carbamates in salt marsh and aquifer sediments. From results of several experiments, we computed the mean values of the percent conversion to CH4 [(moles of excess CH4 above that of controls/mole of carbamate added) x 100] of the various compounds. The degree of stimulation was dependent on the compound and experiment. Methomyl consistentl' gave the greatest stimulation, followed by carbaryl, aldicarb, and finally baygon (Fig. 4 and Table 1). The differences among compounds were most likely due to differences in the extent of hydrolysis and to potential toxic elfects of hydrolysis products. Commonly observed produicts of aldicarb,hydrolysis are aldicarb oxime and aldicarb nitrile. Aidicarb sulfoxide and aldicarb sulfone are also formned during degradation, although under anaerobic conditions the sulfoxide may be reduced to parent aldicarb (15). Hydrolysis of carbaryl is known to yield 1-napthol (8, 23). The degradation pathways of carbamate residues are complex and appear to be greatly dependent on biological and physiochemical conditions (2, 4, 7, 11, 12, 18). Variability of the percent conversion among experiments could be due to differehces in the characteristics of the slurries prepared on different dates. Unfortunately we could not obtain N-methyl[14Cjcarbamates. These would have allowed us to determine the relative proportion of CH4 and CO2 produced from the various compounds and to verify that excess CH4 was, in fact, coming from the terminal N-methyl group. Several lines of evidence support the conclusion that it is the N-methyl group of carbamnates which is converted to CH4: (i) the N-methyl carbamoyl moiety is the only functionality common to all of the carbamates that stithulated methanogenesis; (ii) MA is a known hydrolysis product of carbamates and appeared in sediments as a result of carbamate addition; (iii) MA, when added directly to sediments, stimulated CH4 production with a time course identical to that of the added carbamates. Metabolism of MA by methanogenic bacteria gives a CH4:C02 ratio of 3:1 (6). Thus, if all of the parent compound was hydrolyzed to MA, and MA was exclusively utilized by methane-producing bacteria, the percent conversion of the carbamates would be 75. The apparent conversions (to CH4) observed (Table 1) for methomyl (87.5%), carbaryl (57.2%), and aldicarb (39.2%) indicate that a large fraction of the

parent conmpounds was hydrolyzed on a time scale of 4 to 10 days. This assumes that no other parts of the carbamates were converted to CH4. Values lower than 75 may inidicate incomplete hydrolysis or possible metabolism of MA to CO2 rather than CH4. The fact that methomyl gave more CH4 than could be derived from the N-methyl group alone indicates that, for this compound at least, degradation beyond hydrolysis occurs. Hydrolysis appears to occur in anaerobic sediment slurries and represents a significant detoxification step for carbamates because cholinergic activity is eliminated and nitrosocarbamate formnation is no longer

possible.

Our results show that carbamates can be readily degraded in the presence of an active microbial population under anaerobic conditions and that methanogenesis can be stimulated as a result of carbamate degradation. We suspect that other N-methyl carbamates will give similar results, with the degree of CH4 stimulation depending on both the extent of hydrolysis and the toxicity of parent molecules and hydrolysis products. While concentrations of carbamates used in most of this study were significantly higher than those found in groundwater (range, 4 to 400 ,ug/l [ppb] for aldicarb) (25; Union Carbide Technical Report, 1979), concentrations of aldicarb as low as 0.8 ,ug/ml (800 ppb) have been observed to stimulate methanogenesis (9; this study), suggesting that the reactions described here occur even at low aldicarb concentrations. The observed interactions of carbamates with anaerobic microflora most likely will be Important where waters contaminated with carbamates become anoxic (sUich as by movetnent of groundwater into regions of high organic content) and, most significantly, in areas where carbamate pesticides are applied to waterlogged soils such as rice paddies and marsh soils. ACKNOWLEDGMENTS We thank Eric Telemaque, Julie McDaniel, and Jennifer Slater for help on various aspects of this study. We also thank Steve Carey of the Suffolk County Department of Health Services and Ken Pearsall of the U.S. Oeological Survey for help in obtaining aquifer cores. Funding for this research was provided by grant 14-83B-12 from the Hudson River Foundation, grant R-809475-01-0 from the Environmental Protection Agency, and grant NA-80-RAD-0057 from the National Oceanic and Atmospheric Administration. LITERATURE CITED 1. Back, R. C., R. R. Romine, and J. L. Hansen. 1984. A rating system for predicting the appearance of Temik aldicarb residues in potable water. Environ. Toxicol. Chem. 3:589-597. 2. Baron, R. L. 1971. Toxicological considerations of metabolism of carbamate insecticides: methomyl and carbaryl, p. 185-197. In A. S. Tahori (ed.), Pesticide terminal residues. Butterworths, London. 3. Chapman, R. A., and C. M. Cole. 1982. Observations of the influence of water and soil pH on the persistence of insecticides. J. Environ. Sci. Health. B17:487-504. 4. Coppedge, J. R., D. A. Lindquist, D. L. Bull, and H. W. Dorough. 1967. Fate of 2-methyl-2-(methylthio)propionaldehyde o-(methylcarbamoyl)oxime (Temik) in cotton plants and soil. J. Agric. Food Chem. 15:902-910. 5. ElespUru, R., W. Lijnsky, and J. K. Setlow. 1974. Nitrosocarbaryl as a potent mutagen of environmental significance. NatUre (London) 247!386-387. 6. Hippe, H., D. Caspari, K. Feibig, and G. Gottschalk. 1979. Utilization of trimethylamine and other N-methyl compounds for growth and methane formation by AMethanosarcina barkeri. Proc. Natl. Acad. Sci. USA 76:494-498. 7. Iwata, Y., W. E. Westlake, J. H. Barkley, G. E. Carman, and

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9.

10. 11. 12.

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F. A. Gunther. 1977. Aldicarb residues in oranges, citrus byproducts, orange leaves and soil after an aldicarb soil application in an orange grove. J. Agric. Food Chem. 25:933-937. Karinen, J. F., J. G. Lamberton, N. E. Stewart, and L. C. Terriere. 1967. Persistence of carbaryl in the marine estuarine environment. Chemical and biological stability in aquarium systems. J. Agric. Food Chem. 15:148-156. Kiene, R. P., and D. G. Capone. 1984. Effects of organic pollutants on methanogenesis, sulfate reduction and carbon dioxide evolution in salt marsh sediments. Mar. Environ. Res. 13:141-160. King, G. M., M. J. Klug, and D. R. Lovley. 1983. Metabolism of acetate, methanol and methylated amines in sediments of Lowes Cove, Maine. Appl. Environ. Microbiol. 45:1848-1850. Knaak, J. B. 1971. Biological and non-biological modifications of carbamates. Bull. W.H.O. 44:121-131. Kuhr, R. J. 1971. The formation and importance of carbamate insecticide metabolites as terminal residues, p. 199-220. In A. S. Tahori (ed.), Pesticide terminal residues. Butterworths, London. Kuhr, R. J., and H. W. Dorough. 1976. Carbmate insecticides: chemistry, biochemistry and toxicology. Chemical Rubber Co., Cleveland. Lemley, A. T., and W. Zhong. 1984. Hydrolysis of aldicarb, aldicarb sulfoxide and aldicarb sulfone at parts per billion levels in aqueous mediums. J. Agpc. Food Chem. 4:714-719. Miles, C. J., and J. J. Delfino. 1985. Fate of aldicarb, aldicarb sulfoxide and aldicarb sulfone in Floridian groundwater. J. Agric. Food Chem. 33:455-460. Oremland, R. S., L. Marsh, and S. Polcin. 1982. Methane production and simultaneous sulphate reduction in anoxic, salt

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marsh sediments. Nature (London) 296:143-145. 17. Oremland, R. S., and S. Polcin. 1982. Methanogenesis and sulfate reduction: competitive and non-competitive substrates. Appl. Environ. Microbiol. 44:1270-1276. 18. Ou, L. T., K. S. V. Edvarsson, and P. S. C. Rao. 1985. Aerobic and anaerobic degradation of aldicarb in soils. J. Agric. Food. Chem. 33:72-78. 19. Richey, E., Jr., W. J. Bartley, and K, P. Sheets. 1977. Laboratory studies on the degradation of (the pesticide) aldicarb in soils. J. Agric. Food Chem. 25:47-51. 20. Rickard, R. W., and H. W. Dorough. 1984. In vivo formation of nitrosocarbamates in the stomach of rats and guinea pigs. J. Toxicol. Environ. Health 14:279-290. 21. Rothschild, E. R., R. J. Manser, and M. P. Anderpon. 1982. Investigation of aldicarb in groundwater in selected areas of the central sand plain of Wisconsin. Ground Water 2Q:437-445. 22. Smelt, J. H., M. Leistra, W. H. Houx, and A. Dekker. 1978. Conversion rates of aldicarb and its oxidation products in soils. III. Aldicarb. Pest. Sci. 9:293-300. 23. Weber, F. H., and F. A. Rosenberg. 1984. Interactions of carbaryl with estuarine bacterial communities. Microbial Ecol. 10:257-269. 24. Winfrey, M. R. 1984. Microbial production of methane, p. 153-219. In R. M. Atlas (ed.), Petroleum microbiology. MacMillan, New York. 25. Zaki, M. H., D. Moran, and D. Harris. 1982. Pesticides in groundwater: the aldicarb story in Suffolk County, New York (USA). Am. J. Public Health 72:1391-1395. 26. Zinder, S. H., and T. D. Brock. 1978. Production of methane and carbon dioxide from methane thiol and dimethyl sulfide by anaerobic lake sediments. Nature (London) 273:226-228.