Microbial degradation of strychnine rodenticide in South Australian agricultural soils: laboratory studies

PII:

Soil Biol. Biochem. Vol. 30, No. 2, pp. 129±134, 1998 # 1997 Published by Elsevier Science Ltd. All rights reserved Printed in Great Britain 0038-0717/97 $19.00 + 0.00 S0038-0717(97)00109-0

MICROBIAL DEGRADATION OF STRYCHNINE RODENTICIDE IN SOUTH AUSTRALIAN AGRICULTURAL SOILS: LABORATORY STUDIES S. L. ROGERS,1,2* R. S. KOOKANA,1,3 D. P. OLIVER1,3 and A. RICHARDS4 Cooperative Research Centre for Soil and Land Management, PMB No. 2, Glen Osmond, South Australia 5064, Australia, 2Department of Soil Science, University of Adelaide, Waite Road, Urrbrae, South Australia 5064, Australia, 3CSIRO Land and Water, PMB No. 2, Glen Osmond, South Australia 5064, Australia and 4Primary Industries South Australia, Farm Chemical Program, 25 Grenfell Street, Adelaide, South Australia 5000, Australia 1

(Accepted 24 March 1997) SummaryÐThe degradation of strychnine a rodenticide used to control a major mouse plague in South Australia during 1993 was studied under laboratory conditions in three agricultural soils (Bute, Booleroo and Mintaro) with contrasting physico-chemical properties (pH, mineralogy, organic matter). Strychnine disappeared rapidly in non-sterile (biologically active) Bute and Booleroo soils, from an initial concentration of 50 mg kgÿ1 soil to less than 1 mg kgÿ1 within 42 d. Strychnine was not degraded in sterilised Booleroo soil, suggesting that microbiological processes were responsible for the degradation of strychnine in these two alkaline soils. Degradation of strychnine in both soils was modelled using a logistic regression model, and was characterised by an 8±14 d ``lag phase'' followed by rapid strychnine disappearance. In contrast strychnine degradation was not observed both in the non-sterile and sterilised Mintaro soil during a 98 d incubation. The di€erent pattern of strychnine degradation between these soils is discussed in terms of the sorption behaviour and bio-availability of strychnine. It is suggested that in the acid pH Mintaro soil (pH 5.5 [10 mM CaCl2]), degradation is unable to proceed most likely due to low strychnine bio-availability, as a result of strong strychnine adsorption on to the soil organic and mineral phases. # 1997 Published by Elsevier Science Ltd. All rights reserved

INTRODUCTION

A major mouse plague in South Australia (SA) and parts of north western Victoria, during Spring 1993 resulted in an estimated loss of A$64 million in agricultural production, damage to buildings, telephone lines, electricity cables, etc. (Caughley et al., 1994). To control mouse numbers strychnine was used as a rodenticide, and strychnine coated grains (baits) were broadcast over 350,000 ha of productive agricultural land (Caughley et al., 1994). Strychnine C21H22N2O2 (Fig. 1) is a heterocyclic aromatic compound (Bollag and Kaiser, 1991), a plant alkaloid isolated from the seeds of the tree Strychnos nux-vomica L. (Windholz, 1983). Strychnine had been used as a rodenticide in Australia during the 1969±1971 growing season (Allen and McAuli€e, 1971). The mammalian toxicity of strychnine is between 0.7±28.0 mg kgÿ1 body wt (USEPA, 1980). As a result of the use of strychnine in areas of productive agriculture, concerns were expressed about the potential persistence of strychnine in soils, and more importantly the potential for plant uptake of strychnine residues from *Author for correspondence. 129

soils and the contamination of high value agricultural exports. Limited data are available regarding the degradation behaviour of strychnine in soils. Starr et al. (1995) observed rapid disappearance of strychnine in sandy loam and clay loam soils, with strychnine half-lives of 24 and 27 d, respectively, and Bucherer (1965) demonstrated degradation of strychnine by pure cultures of three Arthrobacter sp in vitro. Bollag and Kaiser (1991) reviewed the microbial transformation of heterocyclic aromatic compounds, and noted that other heterocyclic plant alkaloids, such as nicotine, anabasin and cavadin that have been used as natural pesticides, were microbially transformed in soils under both aerobic and anaerobic conditions. Miller et al. (1983) investigated the sorption of strychnine by three north western Nevada soils, and plant uptake of strychnine by alfalfa (Medicago sativa L.). They concluded that strychnine was relatively immobile in soils due to sorption on to clays and soil organic matter, and was not expected to leach signi®cantly or enter plant material due to uptake from contaminated soils. Strychnine sorption in soils has been shown to be highly pH dependent. Kookana et al. (1997) studied the sorption and desorption behaviour of strychnine in four

130

S. L. Rogers et al.

H

N H

N

H O

O

Fig. 1. Strychnine molecule.

South Australian agricultural soils (Bute, Booleroo, Freeling and Mintaro). They noted that Mintaro soil (pHCa 5.5) sorbed 97% of the strychnine applied, whereas the Bute soil (pHCa 7.3) sorbed only 54% of applied strychnine. Given the widespread use of strychnine to control mouse plagues in Australia, our aim was to examine the degradation of strychnine in three South Australian agricultural soils representing major soil types in the region. Experiments were done under controlled laboratory conditions to assess the potential for microbial degradation and consequent environmental persistence of strychnine in the soil environment. MATERIALS AND METHODS

Soils Soils were collected from three ®eld sites close to Bute, Booleroo and Mintaro townships within the mid-North and Clare agricultural regions of SA. These three soils represent a range of SA agricultural soil types in terms of pH, chemical and textural characteristics (Table 1). None of these sites at the time of sampling had any history of strychnine bait application. Soils were sampled from the A horizon and returned to the laboratory in looselytied polythene bags. Soils were hand sieved (3 mm) to remove stones, vegetation, soil invertebrates and to facilitate homogenisation of the samples. Samples were then stored in loosely-tied polythene bags at 48C for a maximum of 3 wk. At no time were soils allowed to dry out and handling was kept to a minimum to maintain the microbiological integrity of the ®eld samples. Degradation study It is not known if abiotic (physico-chemical) soil processes (Wolfe et al., 1990) are responsible for strychnine degradation or transformation in soils. To establish the role of abiotic strychnine degra-

dation processes in these soils, sub-samples of two of the soils used in this study (Booleroo and Mintaro) were sterilised by autoclaving (1218C) for 30 min on 3 consecutive days. Soil sterility was con®rmed by plating sterilised soils onto nutrient agar (Oxoid Ltd), and inoculated plates were incubated at 258C for 5 d. No colony growth was evident on plates inoculated with sterilised soil suspensions. Heat sterilisation will not only destroy metabolically-active microbial cells within the soil, but will also de-nature any exocellular microbial enzymes. Soil samples (50 g d.w.) were weighed into screwtop plastic containers (Disposable Products Pty). Containers for use with the autoclaved sterilised soil were initially disinfected with an ethanol rinse. To provide optimum moisture conditions for soil microbial activity, the water content in all soils was adjusted to 70% moisture holding capacity (MHC), at ÿ10 kPa using sterile de-ionised water. Strychnine addition to soil Baited grains for broadcast into mouse-infested areas were coated in a mixture of strychnine, ¯our, icing sugar and sodium bicarbonate of which strychnine comprised only 8% of the ®nal compound on a w/w basis (Kookana et al., 1994). A mixture of strychnine-free base (Sigma Chemical Co.), icing sugar, ¯our and sodium bicarbonate, mixed in the same proportions as that applied to baited grain, was applied to soils in this study. The bait mixture rather than baited grain was used to ensure a homogeneous mix of strychnine throughout the soil. To assess bait homogeneity and complete mixing of strychnine with the other bait components 10 sub-samples of bait were analysed by HPLC. The 10 replicate sub-samples had a SE of the mean of 1.48%, indicating a high degree of reproducibility between samples. Strychnine bait was mixed into the soils with a sterile metal spatula to give a ®nal concentration of 50 mg strychnine kgÿ1 soil (d.w.). The experimental soil concentration was well in excess of strychnine concentrations in bulk soil likely to be experienced in baited areas, where baiting involved broadcasting 1 kg of baited grains haÿ1, with individual grains containing between 50±100 mg strychnine (Kookana et al., 1994). The experimental application rate was selected to replicate localised strychnine concentrations in soil directly surrounding the strychnine-

Table 1. Physical and chemical characteristics of Mintaro, Bute and Booleroo soils Location Mintaro Booleroo centre Bute a

pH (1:5 10 Mm CaCl2)

Total N(%)a

5.51 6.72

0.22 0.09

15.0 6.5

7.32

0.03

4

NH4-N NO3-N (mg kgÿ1)b (mg kgÿ1)b

Total C (%)a

HCO3 P Clay (%) (mg kgÿ1)b

Silt(%)

Sand(%)

CEC (cmol kgÿ1)

46.5 21

2.4 1.2

41 33

25 14

27 26

48 60

20.5 15.1

11

1

35

6

2

92

11.4

Total C and N determined by oxidation and infra-red detection (Leco CNS 2000 analyser). b Inorganic N and bicarbonate P were determined in soil extracts by continuous ¯ow analysis (¯ow solution 3500 autoanalyser).

Microbial degradations of strychnine

coated grains. Pots containing strychnine-laced soil were sealed with para®lm (American National Can2) and a screw-top lid, and kept at 258C in the dark. Sampling involved randomly removing triplicate pots of soil for each treatment every 7 d. At every sampling occasion the moisture content of all remaining soils was adjusted back to 70% MHC with sterile de-ionised water. Moisture loss from the soils between sampling occasions was approximately 1±2%. Analysis of strychnine in soil Strychnine residues in soils from the degradation study were determined in soil extracts by normal phase HPLC. All soil analyses were performed by the State Chemistry Laboratories, South Australia (SCL). The analytical procedures were as follows: 50 g of soil was accurately weighed into a glass bottle (Schott Duran) and 100 ml of saturated NH4Cl was added, the mixture was then ultrasonicated for 30 min (Bransonic model 521, ultrasonic water bath). Following this 150 ml methanol was added to the soil mix, the soil redispersed and ultrasonicated for a further 30 min. Extracts were centrifuged for 10 min at 2500  g (Spintron GT75) and supernatant retained. Strychnine residues in supernatant were analysed by normal phase HPLC (Waters Novapak silica column, 8 mm  100 mm  4 mm; mobile phase: methanol-towater-to-30 mM ammonia in the ratio 850:141:9 ¯ow rate 2 ml minÿ1, UV detector wavelength 245 nm AUFS = 0.2, retention time 4.9 min). The supernatant was analysed against a 2.5 ng strychnine mlÿ1 standard in 1.5:1 methanol-saturated NH4Cl. The strychnine detection limit in soils was 1.0 mg kgÿ1 (P. Harpas, pers. commun.). Strychnine recovery tests were made to test the eciency of the extraction technique over time. Strychnine (50 mg kgÿ1) was added to heat sterilised Bute, Booleroo and Mintaro soils, and samples were stored at 48C for up to 98 d. Triplicate samples were removed at various times throughout the study and strychnine analysis performed. Any decline in the percentage strychnine recovered from the soil was considered a result of declining extractant recovery eciency. Measurement of soil microbial biomass The size of the soil microbial communities in the three soils, soil microbial biomass C was measured by the chloroform fumigation extraction method of Vance et al. (1987). Fumigated and non-fumigated soils were extracted with 0.5 M K2SO4 and organic C in the soil extracts was determined by persulphate oxidation with UV detection (Dohrmann DC 180 total organic C analyser).

131

Statistical analysis All analyses were performed in triplicate and are reported as the mean2SE. The signi®cance of the di€erence between means of treatments was determined using the least signi®cance di€erence (LSD) values at 0.1% probability (PR0.001)

RESULTS

Strychnine recovery The results of the strychnine recovery tests at day 3 and day 98, for the three soils studied are summarised in Table 2. The reduction in the eciency of the extractant to recover strychnine residue from soil between day 3 and day 98 was signi®cant in each soil, most notably in the Mintaro soil where recovery had declined from 60% at day 3 to 33% by day 98. In the degradation experiments described below, the declining recoveries with time were taken into account and data adjusted accordingly. Microbial biomass C There was no signi®cant di€erence in the size of the soil microbial communities in the Bute, Booleroo and Mintaro soils measured as microbial biomass C (Table 3). Strychnine degradation The concentration of strychnine in non-sterile Booleroo soil declined from 50 mg kgÿ1 soil d.w. at day 0 to below the analytical detection limit of 1 mg kgÿ1 within 42 d (Fig. 2). However, strychnine concentrations did not decline in the sterilised soil, none of the values in the sterilised soil were signi®cantly di€erent (LSD P = 0.001), from the initial strychnine concentration at day 0. Strychnine residues in non-sterile Bute soil also declined from 50 mg kgÿ1 at 0 d to below detectable limits at d 42 (Fig. 3). Residues were detected at 49 d (1.18 2 2.04 mg kgÿ1) and at 56 d (6.6924.47 mg kgÿ1), however, a further sample taken at 63 d con®rmed that soil strychnine concentrations were below detectable limits. Non-sterile and sterilised Mintaro soils spiked with strychnine were sampled every 7 d for 98 d (Fig. 4). Di€erences in strychnine concentrations in non-sterile and sterilised soils were not signi®cant (LSD P = 0.001) at all times. At 98 d the concentrations of strychnine in the non-sterile and sterilised soils were not signi®cantly di€erent from the Table 2. Percentage recovery of strychnine from soil using ammonium chloride±methanol extractant after 3 and 98 d Soil Bute Booleroo centre Mintaro

Recovery after 3 d

Recovery after 98 d

84.6 20.7 73.0 21.0 60.2 22.3

66.72 2.7 55.32 5.9 33.72 3.4

All values are the mean of three replicates 2SE.

132

S. L. Rogers et al.

Table 3. Microbial biomass C (fumigation±extraction) in soils Biomass C (mg kgÿ1)

Soil Mintaro Booleroo centre Bute

3157 2132 2931 2137 2637 2102

All values are the mean of three replicates 2SE.

initial concentration (50 mg strychnine kgÿ1 soil). The absence of strychnine degradation in the nonsterile Mintaro soil is in stark contrast to degradation patterns observed in both the Booleroo and Bute soils. Strychnine degradation model To quantify the kinetics of strychnine degradation in non-sterile Booleroo and Bute soils our data were ®tted to a model using a logistic regression of the form: y ˆ a ‡ c=…1 ‡ exp…ÿb…x ÿ m††† Where y = strychnine concentration, x = time (d), a = the lower asymptote (minimal strychnine concentration), c = the upper asymptote (maximum strychnine concentration), b = slope parameter, m = point of in¯ection of the curve for the explanatory variate (d), time at which degradation is at a maximal rate. Figures 5 and 6 show the theoretical model ®tted to the observed data for the Booleroo and the Bute soils, respectively. Model parameters were closely correlated with the observed data, with r2 values of 0.95 for the Bute soil and 0.92 for the Booleroo soil. Degradation in Booleroo soil was not linear with time and was characterised by a lag of 8.5 d; however, once strychnine degradation commenced, disappearance was rapid and log-linear with time. The time taken for 50% loss of strychnine determined from the model was 20.3 d, and the speci®c degradation rate (K) was 1.24 mg strychnine kgÿ1

Fig. 2. Strychnine degradation in sterilised and non-sterile Booleroo soil, incubated at 258C, and sampled every 7 d for 63 d (LSD P = 0.001).

Fig. 3. Strychnine degradation in non-sterile Bute soil, incubated at 258C, and sampled every 7 d for 63 d (LSD P = 0.001).

soil dÿ1. However during the period of active degradation (8.5±40 d), K = 1.58 mg kgÿ1 dÿ1. Strychnine degradation determined from the model in Bute soil (Fig. 6) had a lag of 14.1 d, followed by rapid loglinear degradation; 50% loss of strychnine occurred at 28.1 d and K = 1.06 mg kgÿ1 dÿ1. However, during the main active period of degradation (14.1± 47 d), K = 1.51 mg kgÿ1 dÿ1. DISCUSSION

Recovery tests The results of the strychnine recovery tests clearly indicate that with time, strychnine becomes more dicult to chemically extract from the soil matrix. Soils were maintained under conditions that prevented biological activity (sterilised and stored at 48C), and we have shown in Kookana et al. (1994) that abiotic degradation of strychnine is insigni®cant. Organic chemical ``ageing'' in soils, whereby

Fig. 4. Strychnine degradation in sterilised and non-sterile Mintaro soil, incubated at 258C, and sampled every 7 d for 98 d (LSD P = 0.001).

Microbial degradations of strychnine

Fig. 5. Logistic regression model of strychnine degradation in non-sterile Booleroo soil. Time taken for 50% loss of strychnine 20.3 d, ®t of model to observed data r2 0.95.

with increasing contact time the compound becomes more tightly sorbed on to the soil organic and mineral surfaces and more dicult to chemically extract is discussed in detail by Guerin and Boyd (1993). Strychnine degradation Degradation rates of strychnine in the non-sterile Booleroo and Bute soils are similar to those of Starr et al. (1995), who reported 90% degradation of 10 mg kgÿ1 strychnine in two alkaline soils (pH 7.6±7.9) within 40 d. While the time taken for strychnine to disappear is similar, our experiments had a starting concentration of 50 mg kgÿ1 strychnine, ®ve times that of the Starr et al. study. The pattern of degradation was also similar in our study to that of Starr et al., with a prolonged lag phase followed by rapid strychnine degradation. Saltzman and Yaron (1986) noted that the pattern of lag

Fig. 6. Logistic regression model of strychnine degradation in non-sterile Bute soil. Time taken for 50% loss of strychnine 28.1 d, ®t of model to observed data r2 0.92.

133

phase followed by a rapid degradation of the substrate, as observed in both the Booleroo and Bute soils, is generally speci®c to microbial degradation. Evidence for the predominant role of biological processes in strychnine degradation comes from the absence of degradation in sterilised Booleroo and Mintaro soils in our study. Our results demonstrating the role of microbial degradation of strychnine con®rm those of Bucherer (1965), who showed that pure cultures of the bacterium Arthrobacter strychnovorum and A. belladonnae were able to proliferate in vitro with strychnine as the sole source of C, producing a degradation product described as Hanssen acid (C16 acid). Bucherer also observed that A. strychnophagum had the ability to utilise strychnine as both a C and N source, resulting in the production of unidenti®ed low molecular weight degradation products, indicating that strychnine was being biotransformed as opposed to complete mineralisation. Given the evidence for the formation of strychnine mineralisation products in Bucherer's studies, it is possible that strychnine transformation, rather than degradation had taken place in the soils we studied. Starr et al. (1995) noted the appearance of a secondary HPLC peak during their strychnine degradation study, and assumed that the peak represented a strychnine degradation product; the peak was not identi®ed. Studies of strychnine degradation in eucaryotic mammalian systems have identi®ed strychnine 21,22-epoxide as a stable degradation product (Oguri et al., 1989). However, degradation products resulting from microbial degradation in soil have not been identi®ed. Our studies were limited to investigating the disappearance of the parent compound. Absence of microbial degradation in Mintaro soil The absence of strychnine degradation in the Mintaro soil after 98 d does not correspond to soil conditions considered favourable for microbial activity. In fact, the nutritional and physical characteristics of the Mintaro soil, such as organic C, N and clay content, characteristics that are important indicators of soil microbial activity (Larson and Pierce, 1994) appear more conducive to microbial activity than in the other two soils. As microbial biomass C was no di€erent in the three soils studied, the absence of degradation in the Mintaro soil cannot be ascribed to a smaller microbial community. However, di€erences in the sorption behaviour of strychnine in the Mintaro soil when compared with the other two soils may have had a major e€ect on the degradation of strychnine. Strychnine sorption to the Mintaro soil was much higher than both the Bute and Booleroo soils (Kookana et al., 1997), as was the amount of strychnine retained in the desorption phase (Kookana et al., 1997). The authors

134

S. L. Rogers et al.

explained this behaviour on the basis of protonation of the strychnine molecule. Strychnine has a pK of 8.3 (Kookana et al., 1997), at soil pH < 8.3 strychnine molecules become progressively dominated by the protonated form and sorption to negatively-charged clays and organic matter increases. At the pH of the Mintaro soil (pHCa 5.5) the protonated form of strychnine would be greater, compared with Bute soil (pHCa 7.3) and Booleroo soil (pHCa 6.7). As the Mintaro soil had four times the clay content and 2.5 times the organic matter content of Bute soil, there are more potentially available exchange sites present. Thus, it appears that the relatively stronger anity of sorption in Mintaro soil may have been responsible for lower bio-availability of the chemical and, hence, the absence of degradation. Smith et al. (1992) noted that the biodegradation in soil of quinoline, an Nheterocyclic organic compound, was 30 times more rapid when the compound was present in the solution phase than when it was bound on the soil solid phase. Our results suggest that in neutral to alkaline soils (Bute, Booleroo) the rates of strychnine degradation are rapid due to increased strychnine bioavailability; however, in acid soils with higher clay contents, such as Mintaro, strychnine bioavailability is limited and degradation may be retarded. Our study has highlighted the close relationship between sorption, bioavailability and biodegradation of strychnine in soils, and the dominant role that the physico-chemical behaviour of a compound in soil has on biological degradation. These studies were carried out under controlled laboratory conditions to demonstrate the potential for strychnine degradation in soils. It is highly likely that rates of strychnine degradation and persistence under ®eld conditions will be very di€erent. AcknowledgementsÐThis research was funded by Primary Industries South Australia, Farm Chemical Management and Services Branch. We would like to thank Mrs Ina Dumitrescu for technical assistance, Mr Peter Harpas State Chemistry Laboratory, South Australia for the analysis of strychnine in soils and Ms Angela Reid CSIRO INRE Biometrics Unit for development of the strychnine degradation model. REFERENCES

Allen P. G. and McAuli€e J. D. (1971) 1969/70 distribution of mice in South Australia and broadacre treatment for mice. Agronomy Branch Report No. 22. Department of Agriculture, South Australia. Bollag J. M. and Kaiser J. P. (1991) The transformation of heterocyclic aromatic compounds and their deriva-

tives under anaerobic conditions. Critical Reviews in Environmental Contamination 21, 297±329. Bucherer H. (1965) Uber den mikrobiellen abbau von phenylacetat, strychnin, brucin, vomicin und tubocurarin. Zentralblatt fur Bakteriologie Parasitenkunde Infektionskrenkheiten und Hygiene, Zweite Naturwissenschaftlick Abteilung 119, 232±238. Caughley J., Monamy V. and Heiden K. (1994) Impact of the 1993 Mouse Plague. Grains Research and Development Corporation, Bureau of Resource Sciences Occasional Paper No. 7, Canberra. Guerin W. F. and Boyd S. A. (1993) Bioavailability of sorbed naphthalene to bacteria: in¯uence of contaminant aging and soil organic carbon content. In Sorption and Degradation of Pesticides and Organic Chemicals in Soil (D. M. Linn, T. H. Carski, M. L. Brusseau and F. H. Chang, Eds), pp. 197±208. Soil Science Society of America, Madison. Kookana R. S., Rogers S. L. and Oliver D. P. (1994) Behaviour of Strychnine in Soils. Part 1: Retention, Release and Degradation Studies. Report prepared for Primary Industries South Australia, Cooperative Research Centre for Soil and Land Management, CSIRO Division of Soils, Adelaide, South Australia. Kookana R. S., Rogers S. L. and Oliver D. P. (1997) Sorption and desorption behaviour of strychnine rodenticide in soils. Australian Journal of Soil Research 35 491±502. Larson W. E. and Pierce F. J. (1994) The dynamics of soil quality as a measurement of sustainable management. In De®ning Soil Quality for a Sustainable Environment (J. W. Doran, D. C. Coleman, D. F. Bezdicek and B. A. Stewart, Eds), pp. 37±52. Soil Science Society of America, Madison. Miller G. B., Miller W. A., Hanks L. and Warren J. (1983) Soil sorption and alfalfa uptake of strychnine. Journal of Environmental Quality 12, 526±529. Oguri K., Tanimoto Y., Mishima M. and Yoshimura H. (1989) Metabolic fate of strychnine in rats. Xenobiotica 19, 171±178. Saltzman S. and Yaron B. (1986) Degradation processes: biological versus abiotic transformation. In Pesticides in Soil (S. Saltzman and B. Yaron, Eds), pp. 163±172. Van Nostrand Reinhold, New York. Smith S. C., Ainsworth C. C., Traina S. J. and Hicks R. J. (1992) E€ect of sorption on the biodegradation of quinoline. Soil Science Society of America Journal 56, 737±746. Starr R. I., Timm R. W., Hurlbut D. B. and Volz S. A. (1995) Aerobic biodegradation of strychnine alkaloid rodenticide in soil. International Journal of Biodeterioration and Biodegradation 36, 103±124. USEPA (1980) Strychnine position document no. 2/3. U.S. Environmental Protection Agency, Oce of Pesticides and Toxic Substances, Washington, DC. Vance E. D., Brookes P. C. and Jenkinson D. S. (1987) An extraction method for measuring soil microbial biomass C. Soil Biology & Biochemistry 19, 703±707. Windholz M. (1983) The Merck Index: A Encyclopedia of Chemicals, Drugs and Biologicals. Merck and Co., Rahway. Wolfe N. L., Mingelgrin U. and Miller G. C. (1990) Abiotic transformations in water sediments and soil. In Pesticides in the Soil Environment: Processes Impacts and Modelling (H. H. Cheng, Ed.), pp. 103±168. Soil Science Society of America, Madison.