Environ. Sci. Technol. 2001, 35, 501-507
Bioavailability of Nonextractable (Bound) Pesticide Residues to Earthworms B O N D I G E V A O , * ,† CATRIONA MORDAUNT,† KIRK T. SEMPLE,† TREVOR G. PIEARCE,‡ AND KEVIN C. JONES† Department of Environmental and Biological Sciences, Institute of Environmental and Natural Sciences, Lancaster University, Lancaster LA1 4YQ, U.K.
There is an ongoing debate regarding whether nonextractable (bound) pesticide residues in soils are occluded or may remain bioavailable in the long term in the environment. This study investigated the release of 14Clabeled residues, which were previously nonextractable after exhaustive extraction with organic solvents in soils, and their uptake by earthworms (Aporrectodea longa). After a 100-day incubation of soils treated with 14C-labeled atrazine, isoproturon, and dicamba and exhaustive Soxhlet extractions with methanol and dichloromethane, nonextracted 14C-labeled residues remaining in the soils were 18, 70, and 67%, respectively. Adding clean soil in the ratio of 7:1 increased the volumes of these extracted soils. After earthworms had lived in these previously extracted soils for 28 days, 0.02-0.2% of previously bound 14C activity was absorbed into the earthworm tissue. Uptake by earthworms was found to be 2-10 times higher in soils containing freshly introduced 14C-labeled pesticides as compared to soils containing nonextractable 14C-labeled residues. The differential bioavailability observed between freshly introduced 14C-labeled pesticides and those previously nonextractable may be related to the ease of transfer of the 14C activity into the solution phase. By the end of the 28day incubation period, 3, 23, and 24% of previously nonextractable 14C-labeled isoproturon, dicamba, and atrazine residues, respectively, were extracted by solvents or mineralized to 14CO2. The amounts of 14C activity released were not significantly different in the presence or in the absence of earthworms in soils containing previously nonextractable residues. However, the formation of bound residues was 2, 2, and 4 times lower for freshly introduced 14C-labeled isoproturon, dicamba, and atrazine, respectively, suggesting that the presence of earthworms retarded bound residue formation.
Introduction Certain organic contaminants may be of concern in terrestrial environments if they cause adverse effects on the indigenous microbial and invertebrate populations or if there is the * Corresponding author e-mail:
[email protected]; fax: (+44) 1524 593985. † Department of Environmental Sciences. ‡ Department of Biological Sciences. 10.1021/es000144d CCC: $20.00 Published on Web 12/21/2000
2001 American Chemical Society
potential for transmission of these chemicals through the food chain. Pesticides are of particular interest in this regard because they are deliberately manufactured, have selective biological activity, and are deliberately applied to terrestrial systems. Two key chemical properties of organic chemicals are important in determining the likelihood of movement through terrestrial food chains. These are (i) persistence, which is the resistance of a compound to chemical and biological degradation, and (ii) polarity, which determines the lipophilicity of a compound and the likelihood of storage in lipids of food chain organisms (1, 2). It is now well-known that most pesticides and their degradation products become nonextractable in soils when bound to organic or mineral constituents of soil (3-6). The formation of nonextractable (bound) pesticide residues in soil is very important in the evaluation of the impact of pesticides and other toxic chemicals on the environment (7, 8). Bound residues “... represent compounds in soils, plants or animals which persist in the matrix in the form of the parent substance or its metabolite(s) after extraction. The extraction method must not substantially change the compounds themselves or the structure of the matrix” (9). Problems pertaining to nonextractable (bound) pesticide residues in soils have been investigated since the late 1960s (10-13). Since then, concerns have been focused on the possible release and delayed environmental impact of these residues. Several concerns have been expressed; one is that changes in cultural practices may liberate previously bound residues, reintroducing them into the soil solution to become bioavailable (3, 14). Likewise, there is concern that continued buildup of bound residues in soils may affect important physical and biochemical processes such as water holding capacity, soil structure, and the processes of nitrification, ammonification, cellulose decomposition, and a host of related processes (14, 15). Microbially mediated release of nonextractable residues has been observed in several biodegradation studies, including some investigations in which no microbial degradation of bound residues were observed. Hsu and Bartha (16) found that small amounts of soil-bound [UL-14C]3,4-dichloroaniline residues were mineralized to 14CO2 upon incubation of soil containing bound residues with fresh soil. This mineralization was decreased with sterilization or anaerobic incubation. Roberts and Standen (17) reported that between 25 and 40% of soil-bound [14C]cypermethrin residues were mineralized to 14CO2 after a 26-week incubation of soil containing bound residues to which fresh soil had been added. Khan and Ivarson (18) studied the potential release of soil-bound [14C]prometryn residues finding no release of bound residues from a soil, which had been sterilized. However, after incubating soil containing bound residues, to which a fresh soil inoculum had been added, 27% of 14C activity that had been initially nonextractable could be extracted. Analysis of the extract revealed the presence of the parent compound as well as several metabolites. Earthworms, which have been added to soil containing bound pesticide residues, have been found to absorb unidentified 14C-labeled residues into their tissues (19-23). The aim of the present study is to assess the long-term fate of nonextractable residues in soils. The strategy employed in this study was to expose previously nonextractable residues of three pesticides (dicamba, isoproturon, and atrazine) with different physicochemical properties to earthworms and microorganisms and to evaluate their potential degradation, release, and uptake into earthworm tissue. VOL. 35, NO. 3, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Structure and Physicochemical Properties of the Studied Pesticides
Materials and Methods [UL-14C-ring]Atrazine
Chemicals. and dicamba were purchased from Sigma-Aldrich, U.K., and isoproturon was obtained from the Radiochemical Centre Amersham International, U.K. The specific activities of the 14C-labeled pesticides were as follows: atrazine, 20.1; dicamba, 4.2; and isoproturon, 72 mCi mmol-1. Table 1 gives information on the molecular structure of the three pesticides as well as some of the more important physicochemical properties. All the compounds were >98% pure. Unlabeled compounds were analytical standards obtained from Sigma-Aldrich, U.K. All solvents used for the extractions and preparation of standards were of pesticide grade. The scintillation and sample oxidizer cocktails were obtained from Canberra Packard. Experimental Organism. Earthworms are the most frequently studied invertebrates with regard to the uptake and effects of organic pollutants (19-23). They can accumulate soil-bound contaminants and are widely used as bioindicators of soil health and in toxicity testing for soilbound chemicals. They also constitute a link in the transport of environmental contaminants from soil to organisms higher up in the terrestrial food chain. The earthworm Aporrectodea longa was used in this study since it selectively ingests the organic-rich fraction of the available soil and lives within the plough layer of agricultural soils (24). Organic matter is the most important fraction of soil in the formation of bound residues (25-29); consequently, this organism was considered to potentially be exposed to elevated concentrations of soil-bound residues. Mature earthworms (A. longa) were purchased from Walker Organics, U.K., and their identity was confirmed. The earthworms were allowed to acclimatize to laboratory conditions for at least 14 days before use. Soil. Surface soil (0-10 cm) was collected from a farm 20 km south of Lancaster, northwest England. The farm had not received any pesticide applications for 25 yr. The soil was air-dried, passed through a 2-mm sieve, and stored at 4 °C for less than 14 days prior to the start of the experiments. Physicochemical properties of the soil were as follows: organic matter, 9.9 ( 0.25%; organic carbon, 5.7 ( 0.5%, clay, 5 ( 0.8%; sand, 23 ( 1.5%; silt, 72 ( 3%, water holding capacity, 45%; and pH, 6.8 ( 0.2. Production of Soil-Bound Residues. Soil (1 kg) was uniformly spiked with 14C-labeled pesticides at ∼10 000 dpm g-1 and the nonradioactive compounds at the U.K. recommended application rates (atrazine, 2; isoproturon, 2.6; and dicamba, 0.2 mg kg-1) and incubated for 100 days. Figure 1 summarizes the experimental setup. At the end of the incubation period, the soil was exhaustively extracted for 24 502
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h with both methanol and then dichloromethane (DCM). During this period, the solvent was replaced with fresh solvent six times. The soil was then extracted with water and airdried. Further extraction of soil with DCM and methanol did not yield any extractable residues. This was considered the extraction end point. The amount of 14C radioactivity left after this period was determined by sample oxidation and is referred to as “initially bound” throughout this paper. Microcosm Setup. Soil (∼250 g) containing the nonextractable 14C-labeled residues was thoroughly mixed with 1750 g of soil that had been spiked with nonlabeled pesticide at the recommended application rate of the chemical in U.K. agricultural soils to increase the bulk volume of the soil and stimulate microbial activity in the system. The moisture content of the mixture was adjusted to 70% of its water holding capacity by the addition of the appropriate volume of distilled water. The soils were incubated in 3-L glass Kilner jars with silicone septa-lined glass caps and a 0.6-cm (i.d.) tubing for connections to chambers and NaOH traps. The headspaces of the chambers were continuously aerated by means of an aquarium pump (Meiko Hi-Tech 3500) at 25 mL min-1. Air was cleaned of atmospheric CO2 prior to introduction to the chambers by passing through a NaOH trap. Treatments. We wanted to compare the accumulation potential of pesticides by earthworms from soils that had been freshly spiked with the pesticide, when no bound residues had formed, with those in soils containing nonextractable residues. For this, the same type of soil was treated with 14C-labeled pesticide and its 12C-labeled analogue at a similar level to the bound radioactivity for that particular compound. For every compound, the following microcosm treatments were designed: microcosms containing nonextractable pesticide residues with earthworms present, microcosms containing nonextractable pesticide residues without earthworms, microcosms containing freshly applied pesticides in the presence of earthworms, and microcosms containing freshly applied pesticides in the absence of earthworms. Control incubations, containing earthworms but no pesticides, were used to determine background 14C activities under the same conditions. All the incubations were carried out in triplicate. Laboratory acclimatized earthworms (n ) 5) were added to the appropriate microcosms and incubated for 28 days. The earthworms were fed once over the incubation period with 10 g of finely ground horse manure. Extraction and Analysis. At the end of the 28-day incubations, triplicate soil samples from each microcosm incubation (5 g wet weight) were shaken for 24 h with 20 mL
FIGURE 1. Schematic diagram of the experimental setup. of 0.01 M CaCl2 solution and methanol in sequence. The mixtures were centrifuged with a Centaur-2 centrifuge (∼3300 rpm) for 20 min, and the supernatant decanted. Triplicate aliquots (3 × 5 mL) of both CaCl2 and methanol supernatants were analyzed by a Tri-carb 2250CA liquid scintillation analyzer. The residue was then Soxhlet extracted with DCM for 6 h. The extract was reduced in volume on the Soxhlet apparatus and made up to 20 mL, and 14C activity of triplicate 5-mL aliquots was determined by liquid scintillation counting (LSC). All the post-extracted soil samples were air-dried in a fume cupboard to allow the solvent to evaporate. When dry, the samples were homogenized, and triplicate aliquots were combusted using a sample oxidizer. The 14CO2 released from the combustion process was trapped in 14C-labeled cocktail scintillation fluid (20 mL), and the 14C radioactivity was measured by LSC. The 14CO2 evolved during the incubation period was trapped in a 1 M NaOH solution and measured at the end of the 28-day incubation period or when the solution became saturated, as indicated by a thymolphthalein indicator. Triplicate aliquots (3 × 3 mL) of the NaOH solution were combined with scintillation fluids (17 mL), and total radioactivity was measured by LSC.
At the end of the incubation period, the earthworms were removed from the microcosms and rinsed three times with distilled water. The earthworms were transferred directly into uncontaminated soil to purge their guts for between 36 and 48 h. The earthworms were lyophilized for 24 h in an Edwards model freeze-dryer at -55 °C and 4-mbar vacuum. To determine the amount of nonextractable 14C activity that the earthworm had incorporated into tissues over the 28-day incubation period, the earthworms were ground into a fine powder, and triplicate subsamples were obtained for sample oxidation.
Results and Discussion General Comments. There was no obvious toxicity of added pesticides to the test organisms during the 28-day exposure period. There were no earthworms on the surface at any point, suggesting that soil avoidance had not occurred and there were no deaths in any of the replicate incubations. However, there was a general decrease in mean wet weight/ individual animal of less than 5%, except for the control were there was a 0.1% weight increase, an indication of suboptimal growth conditions. VOL. 35, NO. 3, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. Percentage of 14C activity of previously nonextractable (bound) residues made extractable, mineralized, or accumulated in earthworm tissue after 28 days of incubation: (a) atrazine, (b) isoproturon, and (c) dicamba. In this study, bioaccumulation has been expressed on a total weight basis only. Tissue to soil ratios (TSR) were determined as TSR ) (14C activity in organism (g of dry weight)-1) ÷ (14C activity in soil (g of dry soil)-1). The activities were not normalized to lipid content of organism or organic carbon (OC) of soil because the experiment was confined to one species and one soil type. As such, differences in uptake by earthworms in this experiment should primarily be related to physicochemical properties of the pesticide and the degree of interaction with the soil and not biological factors such as feeding mechanisms or lipid composition. The bioaccumulation potential of earthworms was assessed without the gut contents. While purging of the gut contents of earthworms acts to reduce the bias from chemicals associated with gut contents, it also has the potential to introduce bias caused by depuration of chemicals from the tissues. Depuration of the compounds from earthworm tissue may occur after transferring the organism to clean soil. However, all organisms in these experiments were allowed to clear their guts with clean soil for the same length of time. Thus, artifacts from depuration and the weight of clean soil in their guts should be similar for all the organisms, and results within this study should be comparable. 14C-Labeled Residues in Soil. After a 100-day incubation of soils containing 10 000 dpm g-1 of 14C-labeled atrazine, 504
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isoproturon, and dicamba, 18 ( 1.1, 70 ( 0.8, and 67 ( 2.3% were present as nonextractable residues, respectively. The bulk volume of the extracted soil was increased by a factor of 7 with uncontaminated soil, and the moisture content was adjusted to 70% of its water holding capacity. After earthworms had lived in these soils for 28 days, 3 ( 0.2, 23 ( 1.2, and 24 ( 1.5% of the previously nonextractable radioactivity originally added as isoproturon, dicamba, and atrazine became available for solvent extraction (mostly as organic soluble [14C]carbon), metabolism to 14CO2, and absorption by earthworms (Figure 2). Although the amounts of 14C activity extracted or mineralized was consistently higher for incubations containing earthworms relative to those without earthworms for all three studied pesticides; the differences were not statistically significant (p > 0.05). This consistency (Figure 2) however suggests that, given time, the difference could be important. From the data in Figure 2, it appears that release of previously bound residues is being facilitated by microbial activity. As the bacteria consume soil organic matter as their primary substrate, the bound pesticide/metabolite molecules are freed from their entrapment within the humic macromolecules. This suggests that binding of contaminants can be reversed through microbial activity. This finding is supported by Khan and Behki (30), who investigated the microbial release of bound atrazine residues by two Pseudomonas strains. At the end of an 83day incubation with soil-bound atrazine residues, 30-35%
FIGURE 3. Percentage of 14C activity of freshly applied pesticides extractable, mineralized, or accumulated in earthworm tissue after 28 days of incubation: (a) atrazine, (b) isoproturon, and (c) dicamba. of the initially bound 14C activity was extractable as compared to only 3% in the sterile control. Gas chromatographic analysis of the released compounds indicated the presence of the parent compound atrazine, hydroxyatrazine, and their associated dealkylated metabolites. Moreover, Wszolek and Alexander (31) conducted similar experiments to determine the effects of microbes on the sorption of n-alkylamines to clays and discovered that microbes facilitated desorption from the clay surfaces. The results for the freshly treated incubations in the presence and absence of earthworms show some very interesting differences (Figure 3). First, the formation of nonextractable 14C radioactivity originally added as isoproturon, dicamba, and atrazine was higher by a factor of 2, 2, and 4, respectively, in incubations without earthworms over a 28-day exposure period (Figure 3). Second, the amount of the pesticide mineralized to 14CO2 was higher for all three compounds in incubations with earthworms. The most dramatic difference was seen for atrazine where 34 ( 3% of the added radioactivity was mineralized in incubations with earthworms as compared with only 0.7 ( 0.1% in for the incubations without earthworms. The enhanced degradation of atrazine in the presence of A. longa has not been previously
observed. It is possible that bacteria capable of mineralizing atrazine are part of the bacterial community in the gut of A. longa and are added to soil during gut passage. The increased microbial activity in earthworm guts, changes in C substrate availability, and changes in soil structure brought about by the presence of earthworms may change the bioavailability and mineralization of organic pollutants in soil. Meharg (32) reported a doubling of atrazine mineralization in the presence of Lumbricus terrestris as compared to the incubations without earthworms. The increased mineralization was explained by an increased availability on exchange sites in the presence of the earthworm. It has been postulated that, during the passage through the gastrointestinal tract of earthworms, soil undergoes a number of physical, chemical, and biological changes that may result in the release of bound residues (33). Earthworms change the biological and physical-chemical properties of soils that come into contact with them. Both burrow linings and excreted casts are altered with respect to bulk soil (34). These changes within casts and burrows lead to a shift in nutrient dynamics, increased microbial activity (34), and changes in fungal and bacteria composition (35). The addition of mucus, urine, and glucose to soil by earthworms leads to VOL. 35, NO. 3, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 2. 14C Activity in Soil, Earthworm Tissues, TSR Values, and Percentage of Initial Activity Accumulated in Earthworm Tissue over a 28-Day Incubation Period soil tissue tissue to soil uptake (% (dpm/g) (dpm/g) ratio (TSR) initial acty) bound atrazine bound dicamba bound isoproturon fresh atrazine fresh dicamba fresh isoproturon
411 988 961 241 1400 1810
136 3123 766 815 9053 11187
0.3 3.2 0.8 3.4 6.5 6.2
0.02 0.21 0.05 0.31 0.65 0.74
a greatly elevated microbial biomass (35, 36). An increase in microbial biomass has been linked to an increase in catabolic activity and hence enhanced microbial activity (18, 32, 37). Increased activity and increased bioavailability will act synergistically to increase pollutant mineralization rate. Bioaccumulation of Soil-Borne Residues. After earthworms had lived in soils containing nonextractable residues and freshly spiked pesticides, the organisms had incorporated some of these residues into their body tissues. TSR values for earthworms exposed to soil containing nonextractable residues ranged from 0.3 to 3.2. Under identical conditions, TSR values for earthworms from freshly spiked soils ranged from 3.4 to 6.5. TSR values were 2-10 times higher in soils containing freshly spiked pesticides as compared to soils containing previously nonextractable residues for the same compounds (Table 2). Mineralization vs Earthworm Uptake. Although laboratory and field studies have shown that aging of chemicals in soil leads to a diminished bioavailability to microorganisms, little evidence exists for declining availability to higher organisms with persistence of organic molecules in soil. It seems likely that bioavailability as a phenomenon is speciesdependent (38, 39) since molecules that are unavailable to certain organisms can be available to other species (40). Observations made in this study support previous observations that, as pollutants age in soil, their availability to microorganisms diminishes (12). Apparently, during prolonged residence or aging in soil, pesticide molecules undergo an entrapment (sequestration) in structural voids and hydrophobic interiors of micelle-like humic aggregates (29, 41, 42) or in micropores of organic matter and clays (41, 43). As a result of these phenomena, chemicals exhibit declining availability to extraction and biodegradation (12, 44, 45). However, it can be inferred from the results of this study that earthworms are able to access the 14C activity within the soil that are no longer accessible to microorganisms. The bioavailable fraction of an organic molecule is generally regarded as the portion that is available for biotic uptake. This simplistic definition masks the difficulty of accurately predicting the exposure and bioavailability of contaminants in soils. A primary reason for the problem of definition is the different ways in which various organisms interact with and influence their local geochemical environments in soils. It is postulated that before a sorbed molecule can be taken up by a bacterium, it has to diffuse within the organic particle to the solid-liquid interface, then it must partition into the water, and finally it has to diffuse through the aqueous phase to the bacterium (46, 47). Whereas microorganisms can only assess pollutants in the aqueous phase, earthworms can access them both in the solution and in the solid phases in the soil and by ingesting and affecting desorption as the soil passes through the gut. This strong interaction between earthworms and their geochemical environment means that they can influence contaminant fate and therefore their own exposure in important ways that are quite different from those of other soil-inhabiting biota. 506
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From a regulatory standpoint, the significance of release is primarily a consideration of the character of the substance released and made bioavailable or chemically extractable by mechanical and/or rigorous solvents. If the chemicals released can be extracted, then the extractable 14C activity should be characterized to determine whether it represents the parent molecule, a closely related metabolite, or merely metabolic fragments derived from the parent compound or its metabolites. At present, we have not identified the chemical identity of the 14C activity in the extract or in the earthworm tissues. This work is ongoing and will be reported at a later stage. However, the present study provides important data about long-term fates of nonextractable soil residues and the potential transfer of pollutants in their “bound state” through the terrestrial food chain. In summary, the present study has shown that soil-bound (operationally defined as nonextractable) pesticides with varying physicochemical properties can be bioavailable to earthworms and, to a lesser extent, microorganisms. Thus, this study supports previous speculations that soil-bound residues of pesticides are not excluded from environmental interactions. It has also been shown that the presence of earthworms in soils suppresses the formation of nonextractable residues, the extent of which varies with chemical properties of the chemical.
Acknowledgments We thank the Pesticide Safety Directorate of the Ministry of Agriculture Fisheries and Food for funding this work.
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Received for review June 26, 2000. Revised manuscript received October 30, 2000. Accepted October 30, 2000. ES000144D
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