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Bioavailability of atrazine to soil microbes in the presence of the earthworm Lumbricus terrestrius (L.)
Soil Biol. Biochem.Vol. 28, No. 415,pp. 555-559,1996 Copyright 0 1996ElsevierScience Ltd Printed in Great Britain. All rights reserved
Pergamon
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0038-0717/96 $15.00 + 0.00
BIOAVAILABILITY OF ATRAZINE TO SOIL MICROBES IN THE PRESENCE OF THE EARTHWORM LUMBRICUS TERRESTRIUS (L.) ANDREW
A. MEHARG
Institute of Terrestrial Ecology, Monks Wood, Abbots Ripton Huntingdon, Cambridgeshire, PE17 2LS, U.K. (Accepted 19 November 1995) Sntnmary-Experiments were conducted to investigate the interactions between an earthworm species (Lumbricus terrestrius) and soil microflora with respect to the bioavailability and mineralisation of ‘C ring-labelled atraxine. Presence of earthworms had no affect on atraxine in soil solution (assayed by soil centrifugation). This soil solution pool was highly time dependent, decreasing considerably as the experiment proceeded. KCI- extractable label was, however, affected by the presence of earthworms, with this pool initially increasing in the presence of the worms. This pool was also highly time-dependent although, the pattern of this dependence did not follow that for label in soil solution. Mineralisation of the atraxine closely followed the KC1 exchangeable pool and not that of the soil solution pool. However, label sorbed to the surface of the worms was closely correlated to the soil solution pool. Minerahsation in the presence of earthworms was double that of the controls. By the end of the experiment 6% of added
radioactivity was present in the earthworm biomass. Copyright 0 1996 Elsevier Science Ltd
INTRODUCTION
Earthworms change the biological (Pedersen and Hendriksen, 1993; Tiwari and Mishra, 1993) and physical-chemical (Trig0 and Lavelle, 1993; Zhang and Schrader, 1993) properties of soils that come into contact with them. Both burrow linings (Edwards et al., 1992; Wolters and Jorgensen, 1992; Stehouwer et al., 1994) and excreted casts (Daniel and Anderson, 1992; Wolters and Jorgensen, 1992; Tiwari and Mishra, 1993) are altered with respect to bulk soil. These changes within casts and burrows are due to shifting of nutrient dynamics (C, N, K) (Daniel and Anderson, 1992; Wolters and Jorgensen, 1992; Basker et al., 1994; Parkin and Berry, 1994), increased microbial activity (Daniel and Anderson, 1992; Wolters and Jorgensen, 1992), changes in fungal or bacterial species composition (Kristufek et al., 1992; Pedersen and Hendriksen, 1993; Tiwari and Mishra, 1993) and changes in soil structure (Zhang and Schrader, 1993). Burrow linings are physically changed, mainly by compaction and mucus excretion (Scheu, 1991; Zhang and Schrader, 1993). Changes in physical structure will also affect the biological activity of soil microbes in the affected zone (the drillosphere). Mucus, as well as affecting soil aggregation, will’ also be a substrate for soi microbes. The daily loss of mucus from the earthworm Octolasion lacteum has been estimated to be 0.2 and 0.5% of total animal C on a daily basis from body surface and casts, respectively (Scheu, 1991).
Given the increased microbial activity in earthworm guts, changes in C substrate availability and changes in soil structure (both in casts and in burrows), the presence of earthworms may change the bioavailability and mineralisation of organic pollutants in soil. Earthworms do change the availability of some inorganic ions in casts and burrow linings (Turner and Steele, 1988; Basker et al., 1994). The presence of earthworms has also been shown to affect soil exchange properties of a range of herbicides (including atrazine), increasing up to 3-fold the pesticide binding affinity of burrow linings compared to bulk soil (Edwards et al., 1992; Stehouwer et al., 1994). These changes in herbicide availability may affect the bioavailabilty of the herbicides to degrading soil organisms. I studied mineralisation rates of “C-atrazine in the presence or absence of the earthworm Lumbricus terrestrius and related atrazine dissipation to changes in soil atrazine pools caused by the presence of earthworms. That is, to investigate if earthworms change the bioavailability of atrazine to soil microbes. MATERIALS AND METHODS
Microcosms were constructed from 125 ml Furan flasks which had recessed silicone Suba Seals (size 51) fitted in their necks. Two, 50 mm long glass tubes (5 mm OD) were inserted through the Suba Seals. One end of one glass tube was connected to a high-volume, variable-speed peristaltic pump 555
556
A. A. Meharg
(Cole-Parmer Instrument Company, model 7553-75) using silicone tubing. The other end was connected to a CO1 trap, again using silicone tubing. The trap was constructed from a 25 ml McCartney bottle with a silicone Suba Seal (size 51) placed in its neck. The outlet tube from the microcosm was connected to a glass diffusion bubbler which was placed through the Suba Seal in the McCartney bottle. An air outlet tube for the McCartney bottle was also created by placing a piece of 50 mm long glass tube though the Suba Seal. The trap was then filled with 5 ml of 4 M NaOH. The microcosms were filled with 40 g (d. wt.) of sieved (2 mm). A horizon from a beech-Scats pine stand (pH 4.4), to which 10 ml of distilled water was added to give a final matrix potential of - 5.8 kPa. The soil was then spiked with uniformly Y-ring labelled atrazine (specific activity, 102.5 MBq mmol - ‘) supplied by Sigma Chemicals to give a final soil activity of 185 kBq kg - ‘. To half the microcosms, one L. terrestrius was introduced, initial starting weight was 1.27 g f. wt. Mature earthworms were collected from a field site and maintained in laboratory culture 1 month prior to experimentation. Microcosms were kept at ambient temperature (1423°C) and aerated using the peristaltic pump, operating at a flow rate of 6 ml min- I per channel and CO* in the aeration train trapped by NaOH. Traps were changed every 7 days and assayed for radioactivity using a liquid scintillation counter (Packard, Tri-Carb, model 2500 TR). One ml of the trap was counted using 2.5 ml of Hi-ionic Fluor scintillation fluid (Packard). Mini counting vials were used. At each harvest, microcosms were destructively sampled. Earthworms were washed in distilled water and then left for 24 h to void their guts. They were then rinsed again in distilled water and then 2 ml of 1 M KC1 was used to desorb surface sorbed radioactivity. This radioactivity was again assayed using Hi-ionic Fluor. Then homogenised worm (100 mg f. wt.) was then digested using 10 ml tissue solubiliser (hydroxide of hyamine 10-X, Packard) and radioactivity assayed using Hi-ionic Fluor scintillation fluid. A sub-sample of soil was dried at 70°C and then extracted with 1 M KC1 and the extract used to assay radioactivity on the soil exchange site. Another sub-sample of fresh soil (i.e. not dried) was centrifuged (2000 rev min - ’for 10 min) to extract soil solution. The soil was centrifuged using 5 ml centrifuge tubes fitted with an insert which had a 220 pm Millipore filter fitted in its base (supplied by Sigma). Soil was placed in this insert to enable soil solution to be separated from the bulk soil. Radioactivity in the soil solution was assayed using Hi-ionic Fluor scintillation fluid. Soil moisture content was determined at each harvest to monitor water loss. By the end of the experiment soil matrix potential was - 7.8 kPa, indicating that there had been only slight water loss
during the experiment (initial matrix potential was - 5.8 kPa). There was no significant difference (statistics not shown) in matrix potential between the earthworm and control treatment. Analysis of variance was used to analyse the results using the Minitab statistical program. RESULTS
Earthworms did not lose or gain weight over the 4 weeks of the experiment (data not shown). Figure 1 shows the 14Caccumulation by the worms over time. Radio-label body burdens increased over 4-fold, comparing harvest at wk 1 to wk 4, with this increasing body burden trend being significant at the 5% level. By wk 4, approximately 6% of the total radioactivity was present in earthworm tissue. This has obvious ecotoxicological implications under field conditions, both for the earthworm and for any predating organisms. Radioactivity sorbed to the earthworms epidermis decreased (P < 0.05) rapidly (Fig. 1). This decrease was over 6-fold between wk 1 and wk 4. Thus total body burdens were not correlated to surface sorbed radioactivity. Results for the soil radioactivity pools (Fig. 2) showed that radioactivity in soil solution also decreased rapidly during this period, with a 4.5-fold total decrease from by the end of the experiment (the decrease with time was significant at the 0.1% level). Thus, the earthworm surface sorbed radioactivity followed closely the trend of decreasing concentrations in soil solution. Soil KCl-extractable I
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1
01 B
Fig. 1. Labelled atrazine residues present in earthworms expressed on a fresh weight basis. A. Whole body burdens, excluding surface sorbed label. B. Surface sorbed label. Bars indicate the standard error of the mean.
Bioavailability of atrazine to soil microbes in the presence of the earthworm f-100
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temporal pattern of mineralisation closely mirrors the KC1 extractable radioactivity (Fig. 2) and bears no relation to radioactivity in soil solution (Fig. 2). It appears that mineral&&on is regulated by substrate availability on exchange sites. Both the plot of mineralisation rate (Fig. 2) and the cumulative plot of mineralised radioactivity (Fig. 3) showed that mineralisation proceeded much more rapidly in the presence of earthworms. By the end of the experiment about double the amount of atrazine had been mineral&d in the presence of earthworms compared to the control (Fig. 3). This increased mineralisation in the presence of earthworms appears to be explained by increased label available on exchange sites in the presence of the worms. Mineralisation rates were slow, less than 1% of the total added atraxine had been mineral&d by wk 4 in the presence of worms. DISCUSSION
Fig. 2. Fate of labelled atrazine within the soil with (squares) or without (circles) earthworms. A. KCl-extractable label. B. Label in soil solution. C. Mineralisation of label. Bars indicate the standard error of the mean.
radioactivity increased over the first 2 wks and then decreased to approximately the original value (Fig. 2). The soil KC1 pool showed no correlation with the surface-sorbed earthworm pool. Analysis of variance (not shown) revealed that there was no difference in radioactivity in soil solution between the treatment with the earthworm present and the no earthworm treatment (at the 5% level). At the start of the experiment only 4% of total activity was present in this pool, decreasing to less than 1% by the end of the experiment. Unlike the soil solution pool, radioactivity on exchange sites was affected by both the presence of earthworms (significant at the 5% level) and time (sign&ant at the 0.1% level), with a non-sign&ant (P = 0.067) time x earthworm interaction (analysis of variance not shown). Thus earthworms did affect binding of atraxine or atraxine metabolites to soil exchange sites. This radioactive pool (KCl-exchangeable) accounted for 4% of the total soil radioactivity at maximum. Mineralisation of atraxine by soil microbes (Fig. 2) showed a pattern that was time dependant (at the 1% level) and was affected by the presence of earthworms (at the 5% level). There was no time x earthworm interaction (analysis of variance not shown). The
In determining the ecotoxicological implications and the environmental fate of chemicals, the bio-availability of the pollutant to target and non-target biota is essential if the ecological effects of that chemical is to be adequately predicted. If an organic pollutant is not very bioavailable its ecological effects will be minim&d, but its residence in the environment will be long if it has a high affinity for soil exchange sites or soil organic matter. The converse is true of highly bioavailable pollutants. Bioavailability will be determined by the physical and chemical properties of the pollutant and by the environment into which it is released. Soil environmental factors, properties, variables affecting bioavailability will include: matrix potential, soil pore size, percent organic matter, number of exchange sites and clay mineral content amongst others. Biotic interactions in the soil will also affect bioavailability as these will alter the nature of the soil physical and chemical environment. Thus, it would be expected
Fig. 3. Cumulative mineralisation of labelled atrazine in the presence (squares) or absence (circles) of earthworms.
558
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that biotic factors should affect pollutant dynamics in soil. This is clearly illustrated by the results of my experiments where the presence of macro-biota (earthworms) shifted the bioavailability of atrazine with respect to the soil microflora. Previous studies have shown that binding of atrazine to soil exchange sites is strongly influenced by soil spatial relationships with respect to earthworm burrows, with binding to exchange sites on the burrow wall being 3-fold greater than in the bulk soil, principally due to mucilage secretion onto the surface of the burrow (Edwards et al., 1992; Stehouwer et al., 1994). Mucilage secretion in the present study may be the obvious reason for increased atrazine and its metabolites binding to soil exchange sites in the presence of earthworms although, changes in soil structure and soil microflora may be at least, partially responsible. This change in binding characteristics is a dynamic process, as can be seen by the data in Fig. 2. Increased bioavailability (assuming that the pattern of atrazine mineralisation reflects the pattern of exchangeable radioactivity) will lead to increased mineralisation, leading to a greater depletion of the soil pools contributing to exchangeable atrazine (and metabolites). This is reflected in the convergence of the plot of KC1 extractable activity in the presence or absence of earthworms (Fig. 2). It is unlikely that exchangeable atrazine is the only soil pool available to mineralisation. Atrazine sorbed to clay minerals and organic matter and atrazine in soil solution may also be mineralised, although the pattern of mineralisation did not follow activity in soil solution. My experiment did not distinguish between bioavailabilty in casts and in burrow linings. The degradation and bioavailability may differ in these two soil compartments. Besides changing the bioavailability of atrazine, earthworms also significantly alter microbial processes (Daniel and Anderson, 1992; Kristufek et al., 1992; Pedersen and Hendriksen, 1993; Wolters and Jorgensen, 1992; Tiwari and Mishra, 1993; Zhang and Schrader, 1993). These principle changes are due to: changes the soil structure, altering the microbes physical environment; earthworm gut passage alters nutrient availability; mucilage excretion into the gut and from the worms surface will be a substrate for microbes; anaerobic conditions prevail on gut passage of soil, changing microbial species composition and changing the metabolic processes for atrazine and; many of these factors combine to give increased microbial activity in the presence of earthworms. So although, earthworms do alter bioavailability they may also increase mineralisation due to increased size of the microbial biomass or to increased activity of that biomass (Daniel and Anderson, 1992; Wolters and Jorgensen, 1992). Increased activity and increased bioavailability will act synergistically to increase the mineralisation rate. The cumulative mineralisation data shows two
distinct phases for both treatments (Fig. 3). An initially high rate when the KCl-extractable label increased (Fig. 2) with degradation occurring much more rapidly in the presence of the worms. As the extractable pool decreased, so did the mineralisation rate. But even when exchangeable label converged for both treatments, the mineralisation rate was still much higher in the presence of earthworms, suggesting that bioavailability and increased microbial fitness are acting synergistically. Bioavailability of the atrazine (and metabolites) also shifted with time with respect to the earthworm, as radioactivity in the soil solution is correlated to radioactivity sorbed onto the surface of the earthworm (Fig. 1). Absorption across the epidermis is only one of the two principle accumulation routes of atrazine to the earthworm, the other being absorption across the gastro-intestinal tract. It is not known what contribution both assimilation pathways contribute to the total body burdens. The decrease in atrazine present in soil solution may have been due to metabolism of atrazine, with the metabolites having stronger affinity for the soil surface (both sorption and by ionic bonds) or incorporation of atrazine metabolites into the soil microbial biomass. The mineralisation rates of atrazine in this soil were extremely slow. These degradation rates are not exceptional for 14C-ring labelled atrazine - Wolf and Martin (1975) reported 18% mineralisation in a top soil after 550 d, Klint et al. (1993) reported 20% mineralisation after 90 d in top soil and 1% for subsoil. These and my results illustrate the potential of atrazine to persist in the environment. Pollutant interactions in the environment must be viewed as a dynamic process, involving many complex processes. By not including such possible interactions when investigating the environmental behaviour of chemicals, gross misunderstandings of the ecological implications (environmental half life, effects on non-target organisms, food chain transfer) of such studies will be made. My study only investigated one earthworm species and excluded the possible influence of the rhizosphere on pollutant degradation (Anderson et al., 1993), and the possible interactions between the rhizosphere and earthworms with respect to pollutant degradation. Given the fact that most soils support a wide range of diverse macrofaunal and floral populations, only field trials (and not laboratory incubations) will adequately assess the environmental behaviour of chemicals. Acknowledgements-I wish to thank Jason Weeks and Claus Svendsen for the earthworm cultures.
REFERENCES
Anderson T. A., Guthrie E. A. and Walton B. T. (1993) Bioremediation in the rhizosphere. Enoironmentaf Science and Technology 27, 2630-3636.
Bioavailability of atrazine to soil mic:robes in the presence of the earthworm Basker A., Kirkman J. H. and Macgregor A. N. (1994) Changes in potassium availability and other soil properties due to soil ingestion by earthworms. Biology and Fertility of Soils 14, 300-303.
Daniel 0. and Anderson J. M. (1992) Microbial biomass and activity in contrasting soil materials after passage through the gut of the earthworm Lumbricus rubellus Hoffmeister. Soil Biology & Biochemistry 24, 465-410. Edwards W. M., Shipitaib M. J., Traina S. J., Edwards C. A. and Owens L. B. (1992) Role of Lumbricus terrestrius (L.) burrows on. the quality of infiltrating water. Soil Biology & Biochemistry 24, 1555-1561. Khnt M., Arvin E. and Jensen B. K. (1993) Degradation of the pesticides mecoprop and atrazine in unpolluted sandy aquifers. Journal of Environmental Quality 22, 262-266. Kristufek V., Ravasz K. and Pizl V. (1992) Changes in densities of bacteria and microfungi during gut transit in Lumbricus rubellus and Aporrectodea caliginosa (Oligochaeta: Lumbricidae). Soil Biology & Biochemistry 24, 1499-1500.
Parkin T. B. and Berry E. C. (1994) Nitrogen transformations associated with earthworm casts. Soil Biology & Biochemistry 26, 1233-1238.
Pedersen J. P. and Hendriksen N. B. (1993) Effect of passage through the intestinal tract of detrivore earthworms (Lumbricus spp. ) on the number of selected Gram-negative and total bacteria. Biology and Fertility of Soils 16, 221-232.
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Scheu S. (1991) Mucus excretion and carbon turnover of endogeic earthworms. Biology and Fertility of Soils 12, 217-220. Stehouwer R. C., Dick W. A. and Traina S. J. (1994) Sorption and retention of herbicides in vertically oriented earthworm and artificial burrows. Journal of Environmental Quality 23, 286292.
Tiwari S. C. and Mishra R. R. (1993) Fungal abundance and diversity in earthworm casts and in uningested soil. Biology and Fertility of Soils 16, 131-134. Trigo D. and Lavelle P. (1993) Changes in respiration rate and some physicochemical properties of soil during gut transit through Allolobophora molleri (Lumbricidae Oligochaeta). Biology and Fertility of Soils 15, 185-188. Turner R. R. and Steele K. F. (1988) Cadmium and manganese sorption by soil macropore linings and fillings. Soil Science 145, 79-86.
Wolf D. C. and Martin J. P. (1975) Microbial decomposition of ring-Y atrazine, cyanuric acid, and 2-chloro-4, 6-diamino-s-triazine. Journal of Environmental Quality 4, 134139.
Walters V. and Jorgensen R. G. (1992) Microbial carbon turnover in beech forest soils worked by Aporrectodea caliginosa (Savigny) (Oligochaeta: Lumbricidae). Soil Biology & Biochemistry 24, 171-177.
Zhang H. and Schrader S. (1993) Earthworm effects on selected physical and chemical properties of soil aggregates. Biology and Fertility of Soils 15, 229-234.
Pergamon
PIT: soo38-0717(95)00165-4
0038-0717/96 $15.00 + 0.00
BIOAVAILABILITY OF ATRAZINE TO SOIL MICROBES IN THE PRESENCE OF THE EARTHWORM LUMBRICUS TERRESTRIUS (L.) ANDREW
A. MEHARG
Institute of Terrestrial Ecology, Monks Wood, Abbots Ripton Huntingdon, Cambridgeshire, PE17 2LS, U.K. (Accepted 19 November 1995) Sntnmary-Experiments were conducted to investigate the interactions between an earthworm species (Lumbricus terrestrius) and soil microflora with respect to the bioavailability and mineralisation of ‘C ring-labelled atraxine. Presence of earthworms had no affect on atraxine in soil solution (assayed by soil centrifugation). This soil solution pool was highly time dependent, decreasing considerably as the experiment proceeded. KCI- extractable label was, however, affected by the presence of earthworms, with this pool initially increasing in the presence of the worms. This pool was also highly time-dependent although, the pattern of this dependence did not follow that for label in soil solution. Mineralisation of the atraxine closely followed the KC1 exchangeable pool and not that of the soil solution pool. However, label sorbed to the surface of the worms was closely correlated to the soil solution pool. Minerahsation in the presence of earthworms was double that of the controls. By the end of the experiment 6% of added
radioactivity was present in the earthworm biomass. Copyright 0 1996 Elsevier Science Ltd
INTRODUCTION
Earthworms change the biological (Pedersen and Hendriksen, 1993; Tiwari and Mishra, 1993) and physical-chemical (Trig0 and Lavelle, 1993; Zhang and Schrader, 1993) properties of soils that come into contact with them. Both burrow linings (Edwards et al., 1992; Wolters and Jorgensen, 1992; Stehouwer et al., 1994) and excreted casts (Daniel and Anderson, 1992; Wolters and Jorgensen, 1992; Tiwari and Mishra, 1993) are altered with respect to bulk soil. These changes within casts and burrows are due to shifting of nutrient dynamics (C, N, K) (Daniel and Anderson, 1992; Wolters and Jorgensen, 1992; Basker et al., 1994; Parkin and Berry, 1994), increased microbial activity (Daniel and Anderson, 1992; Wolters and Jorgensen, 1992), changes in fungal or bacterial species composition (Kristufek et al., 1992; Pedersen and Hendriksen, 1993; Tiwari and Mishra, 1993) and changes in soil structure (Zhang and Schrader, 1993). Burrow linings are physically changed, mainly by compaction and mucus excretion (Scheu, 1991; Zhang and Schrader, 1993). Changes in physical structure will also affect the biological activity of soil microbes in the affected zone (the drillosphere). Mucus, as well as affecting soil aggregation, will’ also be a substrate for soi microbes. The daily loss of mucus from the earthworm Octolasion lacteum has been estimated to be 0.2 and 0.5% of total animal C on a daily basis from body surface and casts, respectively (Scheu, 1991).
Given the increased microbial activity in earthworm guts, changes in C substrate availability and changes in soil structure (both in casts and in burrows), the presence of earthworms may change the bioavailability and mineralisation of organic pollutants in soil. Earthworms do change the availability of some inorganic ions in casts and burrow linings (Turner and Steele, 1988; Basker et al., 1994). The presence of earthworms has also been shown to affect soil exchange properties of a range of herbicides (including atrazine), increasing up to 3-fold the pesticide binding affinity of burrow linings compared to bulk soil (Edwards et al., 1992; Stehouwer et al., 1994). These changes in herbicide availability may affect the bioavailabilty of the herbicides to degrading soil organisms. I studied mineralisation rates of “C-atrazine in the presence or absence of the earthworm Lumbricus terrestrius and related atrazine dissipation to changes in soil atrazine pools caused by the presence of earthworms. That is, to investigate if earthworms change the bioavailability of atrazine to soil microbes. MATERIALS AND METHODS
Microcosms were constructed from 125 ml Furan flasks which had recessed silicone Suba Seals (size 51) fitted in their necks. Two, 50 mm long glass tubes (5 mm OD) were inserted through the Suba Seals. One end of one glass tube was connected to a high-volume, variable-speed peristaltic pump 555
556
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(Cole-Parmer Instrument Company, model 7553-75) using silicone tubing. The other end was connected to a CO1 trap, again using silicone tubing. The trap was constructed from a 25 ml McCartney bottle with a silicone Suba Seal (size 51) placed in its neck. The outlet tube from the microcosm was connected to a glass diffusion bubbler which was placed through the Suba Seal in the McCartney bottle. An air outlet tube for the McCartney bottle was also created by placing a piece of 50 mm long glass tube though the Suba Seal. The trap was then filled with 5 ml of 4 M NaOH. The microcosms were filled with 40 g (d. wt.) of sieved (2 mm). A horizon from a beech-Scats pine stand (pH 4.4), to which 10 ml of distilled water was added to give a final matrix potential of - 5.8 kPa. The soil was then spiked with uniformly Y-ring labelled atrazine (specific activity, 102.5 MBq mmol - ‘) supplied by Sigma Chemicals to give a final soil activity of 185 kBq kg - ‘. To half the microcosms, one L. terrestrius was introduced, initial starting weight was 1.27 g f. wt. Mature earthworms were collected from a field site and maintained in laboratory culture 1 month prior to experimentation. Microcosms were kept at ambient temperature (1423°C) and aerated using the peristaltic pump, operating at a flow rate of 6 ml min- I per channel and CO* in the aeration train trapped by NaOH. Traps were changed every 7 days and assayed for radioactivity using a liquid scintillation counter (Packard, Tri-Carb, model 2500 TR). One ml of the trap was counted using 2.5 ml of Hi-ionic Fluor scintillation fluid (Packard). Mini counting vials were used. At each harvest, microcosms were destructively sampled. Earthworms were washed in distilled water and then left for 24 h to void their guts. They were then rinsed again in distilled water and then 2 ml of 1 M KC1 was used to desorb surface sorbed radioactivity. This radioactivity was again assayed using Hi-ionic Fluor. Then homogenised worm (100 mg f. wt.) was then digested using 10 ml tissue solubiliser (hydroxide of hyamine 10-X, Packard) and radioactivity assayed using Hi-ionic Fluor scintillation fluid. A sub-sample of soil was dried at 70°C and then extracted with 1 M KC1 and the extract used to assay radioactivity on the soil exchange site. Another sub-sample of fresh soil (i.e. not dried) was centrifuged (2000 rev min - ’for 10 min) to extract soil solution. The soil was centrifuged using 5 ml centrifuge tubes fitted with an insert which had a 220 pm Millipore filter fitted in its base (supplied by Sigma). Soil was placed in this insert to enable soil solution to be separated from the bulk soil. Radioactivity in the soil solution was assayed using Hi-ionic Fluor scintillation fluid. Soil moisture content was determined at each harvest to monitor water loss. By the end of the experiment soil matrix potential was - 7.8 kPa, indicating that there had been only slight water loss
during the experiment (initial matrix potential was - 5.8 kPa). There was no significant difference (statistics not shown) in matrix potential between the earthworm and control treatment. Analysis of variance was used to analyse the results using the Minitab statistical program. RESULTS
Earthworms did not lose or gain weight over the 4 weeks of the experiment (data not shown). Figure 1 shows the 14Caccumulation by the worms over time. Radio-label body burdens increased over 4-fold, comparing harvest at wk 1 to wk 4, with this increasing body burden trend being significant at the 5% level. By wk 4, approximately 6% of the total radioactivity was present in earthworm tissue. This has obvious ecotoxicological implications under field conditions, both for the earthworm and for any predating organisms. Radioactivity sorbed to the earthworms epidermis decreased (P < 0.05) rapidly (Fig. 1). This decrease was over 6-fold between wk 1 and wk 4. Thus total body burdens were not correlated to surface sorbed radioactivity. Results for the soil radioactivity pools (Fig. 2) showed that radioactivity in soil solution also decreased rapidly during this period, with a 4.5-fold total decrease from by the end of the experiment (the decrease with time was significant at the 0.1% level). Thus, the earthworm surface sorbed radioactivity followed closely the trend of decreasing concentrations in soil solution. Soil KCl-extractable I
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Fig. 1. Labelled atrazine residues present in earthworms expressed on a fresh weight basis. A. Whole body burdens, excluding surface sorbed label. B. Surface sorbed label. Bars indicate the standard error of the mean.
Bioavailability of atrazine to soil microbes in the presence of the earthworm f-100
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temporal pattern of mineralisation closely mirrors the KC1 extractable radioactivity (Fig. 2) and bears no relation to radioactivity in soil solution (Fig. 2). It appears that mineral&&on is regulated by substrate availability on exchange sites. Both the plot of mineralisation rate (Fig. 2) and the cumulative plot of mineralised radioactivity (Fig. 3) showed that mineralisation proceeded much more rapidly in the presence of earthworms. By the end of the experiment about double the amount of atrazine had been mineral&d in the presence of earthworms compared to the control (Fig. 3). This increased mineralisation in the presence of earthworms appears to be explained by increased label available on exchange sites in the presence of the worms. Mineralisation rates were slow, less than 1% of the total added atraxine had been mineral&d by wk 4 in the presence of worms. DISCUSSION
Fig. 2. Fate of labelled atrazine within the soil with (squares) or without (circles) earthworms. A. KCl-extractable label. B. Label in soil solution. C. Mineralisation of label. Bars indicate the standard error of the mean.
radioactivity increased over the first 2 wks and then decreased to approximately the original value (Fig. 2). The soil KC1 pool showed no correlation with the surface-sorbed earthworm pool. Analysis of variance (not shown) revealed that there was no difference in radioactivity in soil solution between the treatment with the earthworm present and the no earthworm treatment (at the 5% level). At the start of the experiment only 4% of total activity was present in this pool, decreasing to less than 1% by the end of the experiment. Unlike the soil solution pool, radioactivity on exchange sites was affected by both the presence of earthworms (significant at the 5% level) and time (sign&ant at the 0.1% level), with a non-sign&ant (P = 0.067) time x earthworm interaction (analysis of variance not shown). Thus earthworms did affect binding of atraxine or atraxine metabolites to soil exchange sites. This radioactive pool (KCl-exchangeable) accounted for 4% of the total soil radioactivity at maximum. Mineralisation of atraxine by soil microbes (Fig. 2) showed a pattern that was time dependant (at the 1% level) and was affected by the presence of earthworms (at the 5% level). There was no time x earthworm interaction (analysis of variance not shown). The
In determining the ecotoxicological implications and the environmental fate of chemicals, the bio-availability of the pollutant to target and non-target biota is essential if the ecological effects of that chemical is to be adequately predicted. If an organic pollutant is not very bioavailable its ecological effects will be minim&d, but its residence in the environment will be long if it has a high affinity for soil exchange sites or soil organic matter. The converse is true of highly bioavailable pollutants. Bioavailability will be determined by the physical and chemical properties of the pollutant and by the environment into which it is released. Soil environmental factors, properties, variables affecting bioavailability will include: matrix potential, soil pore size, percent organic matter, number of exchange sites and clay mineral content amongst others. Biotic interactions in the soil will also affect bioavailability as these will alter the nature of the soil physical and chemical environment. Thus, it would be expected
Fig. 3. Cumulative mineralisation of labelled atrazine in the presence (squares) or absence (circles) of earthworms.
558
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that biotic factors should affect pollutant dynamics in soil. This is clearly illustrated by the results of my experiments where the presence of macro-biota (earthworms) shifted the bioavailability of atrazine with respect to the soil microflora. Previous studies have shown that binding of atrazine to soil exchange sites is strongly influenced by soil spatial relationships with respect to earthworm burrows, with binding to exchange sites on the burrow wall being 3-fold greater than in the bulk soil, principally due to mucilage secretion onto the surface of the burrow (Edwards et al., 1992; Stehouwer et al., 1994). Mucilage secretion in the present study may be the obvious reason for increased atrazine and its metabolites binding to soil exchange sites in the presence of earthworms although, changes in soil structure and soil microflora may be at least, partially responsible. This change in binding characteristics is a dynamic process, as can be seen by the data in Fig. 2. Increased bioavailability (assuming that the pattern of atrazine mineralisation reflects the pattern of exchangeable radioactivity) will lead to increased mineralisation, leading to a greater depletion of the soil pools contributing to exchangeable atrazine (and metabolites). This is reflected in the convergence of the plot of KC1 extractable activity in the presence or absence of earthworms (Fig. 2). It is unlikely that exchangeable atrazine is the only soil pool available to mineralisation. Atrazine sorbed to clay minerals and organic matter and atrazine in soil solution may also be mineralised, although the pattern of mineralisation did not follow activity in soil solution. My experiment did not distinguish between bioavailabilty in casts and in burrow linings. The degradation and bioavailability may differ in these two soil compartments. Besides changing the bioavailability of atrazine, earthworms also significantly alter microbial processes (Daniel and Anderson, 1992; Kristufek et al., 1992; Pedersen and Hendriksen, 1993; Wolters and Jorgensen, 1992; Tiwari and Mishra, 1993; Zhang and Schrader, 1993). These principle changes are due to: changes the soil structure, altering the microbes physical environment; earthworm gut passage alters nutrient availability; mucilage excretion into the gut and from the worms surface will be a substrate for microbes; anaerobic conditions prevail on gut passage of soil, changing microbial species composition and changing the metabolic processes for atrazine and; many of these factors combine to give increased microbial activity in the presence of earthworms. So although, earthworms do alter bioavailability they may also increase mineralisation due to increased size of the microbial biomass or to increased activity of that biomass (Daniel and Anderson, 1992; Wolters and Jorgensen, 1992). Increased activity and increased bioavailability will act synergistically to increase the mineralisation rate. The cumulative mineralisation data shows two
distinct phases for both treatments (Fig. 3). An initially high rate when the KCl-extractable label increased (Fig. 2) with degradation occurring much more rapidly in the presence of the worms. As the extractable pool decreased, so did the mineralisation rate. But even when exchangeable label converged for both treatments, the mineralisation rate was still much higher in the presence of earthworms, suggesting that bioavailability and increased microbial fitness are acting synergistically. Bioavailability of the atrazine (and metabolites) also shifted with time with respect to the earthworm, as radioactivity in the soil solution is correlated to radioactivity sorbed onto the surface of the earthworm (Fig. 1). Absorption across the epidermis is only one of the two principle accumulation routes of atrazine to the earthworm, the other being absorption across the gastro-intestinal tract. It is not known what contribution both assimilation pathways contribute to the total body burdens. The decrease in atrazine present in soil solution may have been due to metabolism of atrazine, with the metabolites having stronger affinity for the soil surface (both sorption and by ionic bonds) or incorporation of atrazine metabolites into the soil microbial biomass. The mineralisation rates of atrazine in this soil were extremely slow. These degradation rates are not exceptional for 14C-ring labelled atrazine - Wolf and Martin (1975) reported 18% mineralisation in a top soil after 550 d, Klint et al. (1993) reported 20% mineralisation after 90 d in top soil and 1% for subsoil. These and my results illustrate the potential of atrazine to persist in the environment. Pollutant interactions in the environment must be viewed as a dynamic process, involving many complex processes. By not including such possible interactions when investigating the environmental behaviour of chemicals, gross misunderstandings of the ecological implications (environmental half life, effects on non-target organisms, food chain transfer) of such studies will be made. My study only investigated one earthworm species and excluded the possible influence of the rhizosphere on pollutant degradation (Anderson et al., 1993), and the possible interactions between the rhizosphere and earthworms with respect to pollutant degradation. Given the fact that most soils support a wide range of diverse macrofaunal and floral populations, only field trials (and not laboratory incubations) will adequately assess the environmental behaviour of chemicals. Acknowledgements-I wish to thank Jason Weeks and Claus Svendsen for the earthworm cultures.
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