Toxicity, bioaccumulation and metabolism of pesticides in the earthworm

Abstract

The toxic effects of pesticides on earthworms, one of the most important bioindicators in the terrestrial environment, are closely related to their body burden determined by uptake, metabolism and excretion processes. Not only the passive diffusion via the outer skin from a dissolved fraction of pesticide but also the ingestion of contaminated soil and food governs the uptake process, with each contribution controlled by either the hydrophobicity of the pesticide or the soil organic matter. Although the available information is limited, earthworms are likely to metabolize pesticides via hydrolysis and oxidation (Phase I) followed by conjugation (Phase II), and low bioaccumulation is observed as a result for most pesticides. The acute toxicity in the soil exposure can be partly explained by the dissolved fraction of pesticide in pore water, but the contribution of dietary uptake and metabolism should be further studied to correctly evaluate pesticide toxicity.

Introduction

Soil animals represented by earthworms, springtails and isopods, are key species in breaking down of various dead plants and animal matter into organic and inorganic constituents; they markedly contribute to soil fertility.^1,2) These animals inhabiting farmland, pastures, and meadows, are possibly exposed to pesticides during various agricultural practices. Their limited ability to escape may result in the bioaccumulation of pesticides that greatly influence soil fertility due to their toxic effects on these animals.^2–4) As they are typical prey for terrestrial animals such as shrews and birds in higher trophic levels, biomagnification of pesticides via the food web is a concern. Based upon their wide distribution and abundance (30–110 g/m²), earthworms are one of the most important soil animals.⁴⁾ The burrowing of many earthworm species in soil causes changes in the soil structure, resulting in the modification of water flow and microbial activity therein.^1,5,6) In addition to the ecological importance of the earthworms whose physiology is well understood, pesticide exposure via either the epidermal surface or soil ingestion is suitable for examining the terrestrial eco-toxicity of pesticides;⁷⁾ standardized toxicity test protocols are available.^8,9) From these viewpoints, the sublethal effects of pesticides on the growth, reproduction and behavior of the earthworm by soil exposure should be examined in its EU registration, together with risk assessment of bioaccumulation in birds and mammals when the log K_ow (n-octanol/water partition coefficient) value of pesticide is >3.¹⁰⁾ The uptake routes of pesticides in earthworms greatly depend on their location in the soil, whether dissolved in pore water or adsorbed to soil particles, as shown in Fig. 1. The physico-chemical properties of pesticides, controlling either adsorption/desorption and sequestration in soil particles or its association with dissolved organic matter, should be taken into account.^7,11,12) To better understand the toxic potential and possible bioaccumulation of pesticides, its body burden, with tissue distribution and information on metabolites should be clarified by studying the metabolism.³⁾ Furthermore, the ecology of earthworms is to be noted for the practical risk assessment of pesticides in the soil environment. As summarized in Table 1, earthworms are conveniently classified according to habitat, food preference and burrowing ability. Since pesticides are generally applied by the spraying of a diluted formulation or as granules, earthworms living near the soil surface and fed on plant leaves tend to be more exposed to pesticides, while those making vertically deep burrows may be less exposed to them.⁴⁾

Fig. 1. Conceptual flow of pesticide bioavailability and toxicokinetics in the earthworm.

Table 1. Ecological classification of the earthworm

Classification	Species	Ecology
Compost	Eisenia fetida, Eisenia andrei, Eisenia venta	Most likely to live in manure, and prefer warm and moist environments
	Pheretima posthuma, Dendrobaena veneta
Epigeic	Lumbricus rubellus, Dendrobaena octaedra	Live on soil surface, dwell in leaf litter and tend not to make burrows
	Dendrodrilus rubidus
Endogeic	Allolobophora caliginosa, Allolobophora rosea	Live in and feed on soil, and make horizontal burrows through soil
	Allolobophora chlorotica, Aporrectodea caliginosa
Anecic	Lumbricus terrestris, Metaphire guillelmi	Make vertically deep burrows in soil and feed on leaves on soil surface
	Aporrectodea longa

References: http://www.earthwormsoc.org.uk/ and 4).

With this in mind, this review first deals with acute toxicity and bioconcentration/bioaccumulation of pesticides in earthworms. Their relationship with pesticide hydrophobicity (log K_ow) is discussed by keeping bioavailability and the role of metabolism in mind. Second, the metabolic profiles of pesticides are summarized with the review of relevant enzymes (oxidases, esterases and glutathione-S-transferases) involved in their metabolism. Unless specified, the earthworms taxonomically identified are adults with well-developed clitellum. Through these reviews, issues to be further investigated are proposed in order to gain a better understanding of the toxic effects of pesticides on earthworms.

1. Methods of Exposure

Either the toxic effects of pesticides with their longevity or their bioaccumulation in the environment should be assessed by field studies in accordance with practical usage manners. However, not only the considerable variability in soil composition, meteorological conditions and earthworm ecology but also the complicated environmental behavior of pesticides makes it difficult to interpret the field results. The topical application of a pesticide solution is the simplest method for examining its toxicity, but the presence of mucus causes its poor contact with the earthworm and therefore, various types of laboratory studies have been developed instead under controlled conditions.³⁾ The injection of a pesticide solution into the coelomic cavity of earthworms was conducted in some toxicity and metabolism studies,^13–17) but it is a cumbersome way that brings concern of a solvent effect, even for larger earthworms such as anecic Lumbricus terrestris. The direct gavage technique was tried for L. terrestris in the metabolism study¹⁸⁾ but was considered impractical for a toxicity study using many individuals. Immersing earthworms into an aqueous solution of pesticide is another convenient approach for examining its potential effect, but its application is limited by the water solubility of the pesticide.^{13,14,17,19–21)} Exposing the earthworm to the natural soil treated with pesticide is the most realistic scenario. However, many environmental factors greatly influence the effects of a pesticide on earthworms; soil properties such as pH and organic matter content,^11,22) dissolved organic carbon in pore water,²³⁾ adsorption-desorption hysteresis with sequestration in the soil,^12,24–26) and microbial degradation.^20,27,28) Detailed kinetic analysis that assumes compartments of pore water, soil particles and biota has been applied to understand the behavior of a chemical in such a complex system based on the equilibrium partitioning (EP) theory.^28–30) Recently, a cylindrical soil monolith treated with organophosphorus and pyrethroid insecticides has been utilized to examine their effects on enzymatic activities by considering earthworm ecology.³¹⁾ However, these approaches are too complicated to concisely examine the effects of many pesticides. To effectively collect reproducible results by taking into account a realistic exposure situation as far as possible, paper contact and artificial soil toxicity tests are very useful, and they are standardized as OECD guidelines.^8,9) Compost earthworms Eisenia fetida and Eisenia andrei are the recommended species due to their commercial availability and ease of breeding. Paper contact tests can specifically examine the dermal exposure from pesticide dissolved in pore water, while artificial soil toxicity tests additionally evaluate the dietary uptake route under conditions where the complicated interactions of pesticide with the natural soil are minimized.

2. Toxicity

2.1. Acute toxicity

The toxicological effect of a pesticide depends on its application rate and mode of action. Both coiling of the body and longitudinal muscle contraction are most frequently observed as acute signs, followed by rigidness of the body and swellings on the body surface.²⁾ Chronic effects on growth and reproduction are also known.³⁾ This review focuses on acute toxicity since significantly more information on a wide variety of pesticides is available. Insecticides and fungicides, as represented by carbamates and organophosphorus compounds that inhibit acetylcholine esterases, tend to cause more toxic effects on earthworms than do herbicides.^3,32) Paper contact tests on E. fetida showed very high acute toxicities of the chemical classes of organochlorines, organophosphorus compounds, carbamates, pyrethroids, and neonicotinoids.^33–35) For several earthworm species, the acute LC₅₀ values obtained by this test are poorly correlated with those from the 14-day artificial soil test.^36,37) The relatively decreased toxicity in the latter test was observed for pyrethroids and some other pesticides,^34,35) possibly due to lower bioavailability by tight adsorption to the substrate, faster degradation therein or more metabolic detoxification in the earthworm via dietary uptake. Metabolic transformation generally decreases a pesticide’s toxicity, but the formation of more toxic metabolites should be noted, for example the oxon of an organophosphorus pesticide and the phenol of a phenoxy herbicide.^17,33) Species sensitivity has also been studied for many combinations of pesticides and earthworms. The comparative acute toxicity in the artificial soil was reported for E. fetida and L. terrestris,³⁸⁾ while E. fetida was found to be less sensitive to pesticides than were other species.^37,39,40) Meta-analysis showed that Eisenia sp. was not comparatively sensitive to pesticide exposure.⁴¹⁾ Recent species-sensitivity distribution analysis has clearly shown the least toxicity in E. fetida among twelve species mostly against eleven pesticides.⁴²⁾ In addition, enantioselective toxicity has been studied for some chiral pesticides. In the paper contact test, the (R)-isomer of fipronil was twice as toxic, due to the preferential metabolic degradation of the (S)-isomer.⁴³⁾ The structurally similar acylanilide fungicides metalaxyl and benalaxyl exhibited the opposite enantioselective toxicities, which was accounted for by the difference in elimination rates between the enantiomers.^44,45) In the case of alpha-cypermethrin, the more insecticidally active (1R)-cis-αS isomer exhibited more toxicity in E. fetida.⁴⁶⁾

Many commercial pesticide formulations contain several active ingredients, so earthworms are likely to be exposed to pesticide mixtures. The toxic effects of a new pesticide should be thoroughly investigated before its registration, but those of a mixture have not been well understood. The concentration addition model is suggested for pesticides having similar modes of action (MOAs), while the independent action model is preferable for those having dissimilar MOAs.⁴⁷⁾ Synergism or antagonism appears with deviation from the above models and is important for assessing the practical risk of a pesticide’s formulation. In paper contact tests, binary mixtures of chlorpyrifos and atrazine or cyanazine, likely narcotics for earthworms, showed higher acute toxicity in E. fetida than did chlorpyrifos alone by a factor of 2–8.⁴⁸⁾ Both the insignificant change in the uptake rate of chlorpyrifos and greater formation of phenol and polar metabolites strongly suggest that triazines accelerate the oxidative formation of the more neurotoxic chlorpyrifos-oxon. The acute toxicity of temephos in E. andrei was enhanced by metolachlor which was likely to activate oxidative desulfurization.⁴⁹⁾ The binary mixture of cypermethrin and chlorpyrifos in the OECD artificial soil showed higher acute and chronic toxicities in E. andrei than did the application of each singly.⁵⁰⁾ The inhibition of esterases by chlorpyrifos might decrease the detoxification of cypermethrin. In contrast, the herbicide formulation including mecoprop, 2,4-D and dicamba greatly reduced the acute toxicity of malathion for E. fetida in soil, which was accounted for by the deactivation of a mixed-function oxidase system by these herbicides.⁵¹⁾ The DNA damage of E. fetida was evaluated by comet assay of the coelomocytes taken after artificial soil exposure, and the damage from the coexistence of Cd²⁺ and atrazine was found to be less than the additive effect.⁵²⁾ Less bioavailability due to the complex formation between atrazine and Cd²⁺ or more activation of detoxifying enzymes might result in less toxicity.

In contrast to traditional toxicological studies using live earthworms, the accumulation of sequencing information and the availability of custom cDNA microarrays enable toxicogenomic approaches to be taken, especially for E. fetida, E. andrei and Lumbricus rubellus.^53,54) Among them, metabolomics using NMR and/or GC-MS have become popular for quantifying endogenous metabolites in response to pesticide exposure, which is useful for understanding toxicological MOAs.⁵⁵⁾ Exposure of E. fetida to DDT or endosulfan in paper contact tests increased the level of alanine, a universal stress indicator, as evidenced by the PCA multivariate analysis.⁵⁶⁾ A similar increase of alanine together with decreased amounts of sugar was observed by the exposure of Metaphire posthuma to carbofuran-treated soil.⁵⁷⁾ In addition to these changes, the various NMR analyses of coelomic fluids taken from E. fetida exposed to endosulfan, a GABA-gated chloride channel antagonist, in the OECD artificial soil clearly indicated significant fluctuation in the content of each endogeneous metabolite participating in the glutamine/GABA-glutamate cycle.⁵⁸⁾

2.2. Relationship with pesticide hydrophobicity

According to the EP theory, only pesticide dissolved in the aqueous phase of soil is bio-available.³⁰⁾ This fraction is primarily controlled by its hydrophobicity and is supposed to be taken up via either the outer skin of the earthworm or its gut wall after soil ingestion, as shown in Fig. 1. Detailed kinetic analysis of hydrophobic polychlorinated aromatics suggested that their absorption via the gut wall was more important than through the outer skin in E. andrei.⁵⁹⁾ By estimating the concentration of a chemical in pore water, Gestel and Ma⁶⁰⁾ have found a satisfactory relationship (r²=0.87–0.98) between the 14-day LC₅₀ value (µmol/L) in E. andrei and L. rubellus and log K_ow for chlorinated benzenes, phenols and anilines in natural soils. A similar QSAR approach succeeded in describing the acute toxicity of chloroanilines to the soil-dwelling springtail Folsomia candida.⁶¹⁾ Therefore, log K_ow is a useful descriptor for analyzing the acute toxicity of pesticides. The 48-hr LC₅₀ values (µg/cm²) from the paper contact tests using E. fetida for 86 pesticides and seven phenol derivatives^33–37,40) were first analyzed against log K_ow (U.S. EPA EPI-Suite; http://www.epa.gov/opptintr/exposure/pubs/episuite.htm). The correlation was found to be insignificant in total (data not shown) but it was slightly improved if limited to organophosphorus pesticides (n=13, r²=0.48). The poor correlation may originate from the large variability in LC₅₀ as the screening character of this method.³⁾ Second, the 14-day LC₅₀ (µmol/L) values in the OECD artificial soil were analyzed against log K_ow for 152 pesticides, as shown in Fig. 2. Assuming the linear adsorption and the organic carbon fraction of 0.0466,⁶²⁾ the toxic unit was converted from ‘mg/kg soil’ to ‘µmol/L’ by using the mean organic carbon normalized adsorption coefficient (K_oc) and the molecular weight (mol. wt.) of each pesticide. Most chemical classes were clustered due to narrow log K_ow ranges, and the overall correlation was found to be poor (r²=0.46). Both the good correlation in each chemical class of chlorinated aromatics^60,61) and the significant correlation in the present analysis for carbamates (r²=0.73, n=9) and organophosphorus pesticides (r²=0.85, n=10 except glyphosate and glufosinate) suggest that the difference in a toxicological MOA at least accounts for the poor correlation. Furthermore, the neglect of metabolic transformation in the earthworm is likely to contribute to the present poor result because the elimination of polycyclic aromatic hydrocarbons (PAHs) from E. fetida and L. rubellus was found to correlate highly with the metabolic rate.⁶³⁾

Fig. 2. Relationship between log K_ow and acute LC₅₀ of pesticides in the artificial soil exposure.

The 14-day-LC₅₀ (mg/kg soil; ‘>x’ assumes ‘x’) and mean K_oc values of each pesticide are cited from “Conclusion on the peer review of the pesticide risk assessment” in EFSA Journal (http://www.efsa.europa.eu/en/publications/efsajournal.htm). ●, neonicotinoid; ▲, urea; ◇, carbamate; △, anilide and amide; *, sulfonylurea; □, strobilurin; ■, macrolide; ○, pyrethroid; ◆, organophosphorus; —, aryloxyacetate; , triazine and azole; +, miscellaneous pesticide.

3. Bioconcentration and bioaccumulation

Bioconcentration means the increased concentration of pesticide in the earthworm, resulting from its uptake via the body surface from pore water in the soil, while bioaccumulation includes both bioconcentration and the dietary uptake of pesticide from soil and foods. The study protocol of bioaccumulation in the earthworm is standardized in OECD 317, and the few factors defined below are utilized to describe these processes.⁶⁴⁾   

C_e, C_w and C_s are the steady-state concentrations of pesticide in the earthworm, the water phase and the soil, respectively. f_oc and f_L are the fractions of organic carbon in the soil and lipid in the earthworm, respectively. The lipid and protein contents of several earthworm species are listed in Table 2. Protein is the main component (60–70% on a dry weight basis),^68,69) and the lipid contents are in a range similar to that observed for aquatic oligochaetes.³⁰⁾ The main components of the lipids are phospholipids (40–60%), followed by triglycerides, glycolipids and cholesterol, with the fatty acid moieties being polyunsaturated (C₁₆–C₂₀).^70,71,74) The BCF, BAF and BSAF factors of pesticides are listed in Table 3, together with log K_ow and log K_oa (n-octanol/air partition coefficient). The mechanistic bioaccumulation model recently developed by Armitage and Gobas⁷⁸⁾ suggests that chemicals with log K_ow of 1.75–12 and log K_oa of >5.25 have a biomagnification potential via the terrestrial food web. Since most pesticides fall under these criteria, the metabolic contribution becomes important when these factors are discussed.

Table 2. Lipid and protein contents of the earthworms

Species	% Protein1)	% Lipid1)	Ref.
E. fetida	—	2.3–5.2	65, 66
E. andrei	—	2.3	67
L. terrestris	9–12	1.0–1.5	68–72
Ap. caliginosa	5–6	1–2	67, 73
Al. caliginosa2)	—	1.2–2.5	71, 74
L. rubellus	—	0.6–1.2	75, 76
M. postuma	ca. 12	ca. 3	77

1) On the fresh weight basis. 2) Mixed with L. rubellus.

Table 3. Bioconcentration and bioaccumulation of pesticides in the earthworm

Chemical	log Koa/Kow1)	Species	Med.2)	Per.3)	BCF4)	BAF4)	BSAF4)	Ref.
DDT	9.82/6.91	L. terrestris	c	28		0.7		22
DDE	9.68/6.51	E. fetida	ns	8		8.2		25
		Ap. caliginosa	ns	8		1.6		126
Aldrin	8.08/6.50	P. posthuma	ns	28		0.3		79
Dieldrin	8.13/5.40	Ap. caliginosa	ns	28			0.8–2	29
		L. terrestris	ns	28		1.4		80
Endosulfan	8.64/3.83	P. posthuma	ns	28		0.6		79
Lindane	8.84/4.14	P. posthuma	ns	28		0.2		79
Carbofuran	9.22/2.32	E. fetida	w	8#	1.6			19
Aldicarb	8.36/1.13	L. terrestris	w	6#	0.5–1.0*			20
Chlorpyrifos	8.88/4.96	Al. caliginosa	ns	7		0.6–0.7		81
		L. rubellus	as	7		0.2		82
Chlorfenvinphos	9.74/3.81	E. fetida	ns	28			0.6–2.3*	83
Malathion	9.06/2.36	L. terrestris	ns	3		0.3		84
Butachlor	10.2/4.50	Al. caliginosa	ns	7		0.1–0.2		81
Metalaxyl	8.63/1.65	E. fetida	ns	31		0.1–0.3		45
Benalaxyl	8.74/3.40	E. fetida	ns	32		0.46–0.53		44
Cypermethrin	11.7/6.60	Al. caliginosa	ns	56		30*		85
		L. terrestris	ns	56		10*		85
Alpha-cypermethrin	11.7/6.60	E. fetida	ns	28			0.1–0.4*	83
		E. fetida	ns	10			0.04–0.05	46
Pentachlorophenol	11.1/5.12	Al. caliginosa	w, as	1, 14	2.2–2.9*	8–13*		21
		E. fetida	ns	15		0.5–0.8		86
Atrazine	9.63/2.61	E. fetida	as	21		6.7		52
Simazine	9.59/2.18	E. fetida	ns	90		0.9–6.9*		87
Diniconazole	9.09/4.30	E. fetida	as	21		1.8–8.5+		88
Tebuconazole	11.8/3.70	E. fetida	ns	36			0.3–2.6	89
Myclobutanil	9.70/2.94	Al. caliginosa	ns	7		0.3		81
Fipronil	11.5/4.0	E. fetida	as, ns	14–18		1.2–1.3		43
Avermectin B1a	29.7/4.48	E. fetida	as	18		0.05–0.08		90

1) Collected from the U.S. EPA EPI-Suite [http://www.epa.gov/opptintr/exposure/pubs/episuite.htm]. 2) Exposure medium: w, aqueous solution; as, artificial soil; ns, natural soil; c, compost. 3) Period of exposure in day or hr (#). 4) BCF, bioconcentration factor in L/kg; BAF, bioaccumulation factor in kg soil/kg worm; BSAF, biota-soil accumulation factor in kg organic carbon/kg lipid; *, based on radioactivity; +, calculated using kinetic parameters.

The ¹⁴C-based BCF values of carbofuran,¹⁹⁾ aldicarb²⁰⁾ and pentachlorophenol²¹⁾ were simplistically estimated to be less than 3 by dipping the earthworm into aqueous solution of each pesticide, similarly to the bioconcentration study in fish. The lower log K_ow or partial dissociation together with the metabolic transformation results in lower BCF values. The transfer of pesticide into the earthworm can be thermodynamically described as a partition process, and its distribution from water to the macerated L. terrestris or its whole body was found mostly proportional to log K_ow with slopes of 0.48–0.52.⁸⁰⁾ The similar relationship with log K_ow was reported in E. andrei for polychlorinated benzenes but with a larger slope of 1.1.⁹¹⁾ The pore-water-based BCF of polychlorinated phenols gave lower responses to log K_ow (slope, 0.4–0.5) when L. rubellus and E. andrei were exposed in natural soils.⁹²⁾ These slopes seem to be smaller than those reported for fish (0.8–1.1),⁹³⁾ which may partly originate from the composition of an earthworm’s body. About 80% of the cuticle of the earthworm’s body surface is protein with the remaining being mostly polysaccharides such as galactose; most of the protein is collagen consisting of glycine (one-third of the total residues), hydroxyproline, alanine and serine.^94,95) Endo et al.⁹⁶⁾ have recently reported that the partition of neutral organic molecules from water to a structured protein such as collagen (K_pw) is much smaller than that to a lipid phase, and hydrogen-bonding plays an important role in the partitioning. These authors showed a slope of 0.42 in the log K_pw vs. log K_ow plot, much smaller than 1, although the chemical classes analyzed were limited. As far as the dermal uptake is concerned, not only hydrophobicity but also the hydrogen-bonding ability may control BCF in the earthworm.

Most of the bioaccumulation reported for the earthworm is expressed as BAF in the soil exposure, and about half of these BAFs were less than unity (Table 3). Since the uptake amount of a pesticide correlates with its hydrophobicity (log K_ow),⁸¹⁾ these low values partly originate from the metabolism of the earthworm. Among the pesticides with BAF>1, the total radioactive residues of C_e and C_s without consideration of metabolism accounted for the apparently high BAF values of cypermethrin, pentachlorophenol and the triazines. In the BAF calculation, the extractable pesticide fraction in soil by a water-miscible organic solvent is used as C_s, which is not always equal to and may be larger than the bioavailable fraction for the earthworm. Therefore, BAF may underestimate the bioaccumulation potential of a pesticide. Furthermore, the release into pore water of a soil-sorbed hydrophobic chemical by dissolved organic carbon may increase the bioavailable fraction in natural soils.^23,30) To more precisely estimate the exposure level from pore water, a solid-phase microextraction (SPME) method using a poly(dimethylsiloxane)-coated fiber has recently been utilized for the bioaccumulation of dieldrin to E. fetida and Aporrectodea caliginosa.⁶⁷⁾ In the dietary uptake route, gut secretions may affect the release of a chemical from the gut contents if they have a surfactant-like property. Surfactants such as Triton X-100 and Tween-80 greatly increased the amount of DDE taken up by E. fetida and L. terrestris.⁹⁷⁾ Although the aging of soil generally decreased the BAFs of pesticides due to their reduced bioavailablity by the formation of unextractable bound residues,^98,99) the uptake of pesticides even from the bound fraction of exhaustively extracted soils showed their partial release during digestion.^24,100,101)

The EP theory has been successfully introduced to describe the bioaccumulation (BSAF) of pesticides in aquatic species,³⁰⁾ while its application to the earthworm is still limited for polychlorinated aromatics and PAHs.¹¹⁾ The BSAF values of a few pesticides are estimated to be low (Table 3) and comparable to those in aquatic species.³⁰⁾ The accumulation of PAHs from soil to earthworm lipids (BSAF) vs. an exposure period generally exhibits a convex shape.^28,102) The combination of the slow mass transfer of PAHs from the soil-sorbed phase to pore water with its microbial biodegradation therein is likely to result in these profiles. The partition of a chemical between water and the earthworm is proportional to log K_ow similarly to that between water and soil, and so no dependency of BSAF on log K_ow is predicted by the EP theory.^30,72,75) However, the contribution of a dietary uptake route causes deviation from this theory.⁷⁵⁾ For the bioaccumulation of a chemical with log K_ow>5–6, the contribution of a water exposure route decreases but a dietary contribution predominates especially in soil with a higher organic matter content.^11,103) Using detailed kinetic analysis of the bioaccumulation of polychlorinated aromatics in E. andrei, Jager et al.^59,104) have indicated that their uptake rate constants through skin decrease in proportion to log K_ow, while those via the gut wall are kept almost constant, and the latter route becomes dominant at log K_ow>5–6. The importance of dietary uptake for hydrophobic chemicals was also supported by the immunofluorescence detection method. Sforzini et al.¹⁰⁵⁾ reported a much greater localization of benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin taken up from the artificial soil for 10–28 days in the gut region (intestine, chloragen and coelomocytes) of E. andrei than in the body wall. Incidentally, the biotransformation in the earthworm was of minor importance in the dissipation of PAHs from the kinetic analyses,^76,102) but no systemic information regarding its importance in pesticides is available.

The enantioselective bioaccumulation has been examined by chiral HPLC analyses of tissue and soil extracts. The (R)-isomer of benalaxyl was slightly more bioaccumulated in E. fetida due to its slower elimination than was the (S)-isomer.⁴⁴⁾ The opposite and higher enantioselectivity in bioaccumulation was observed for the structurally analogous metalaxyl.⁴⁵⁾ E. fetida bioaccumulated 3–9-fold more (R)-isomer of tebuconazole in soil, which was accounted for by its faster uptake and slower elimination than that of the (S)-isomer.⁸⁹⁾ Another azole fungicide diniconazole showed slight enantioselectivity dependent on the exposure concentration.⁸⁷⁾ In the case of fipronil⁴³⁾ and α-HCH,¹⁰⁶⁾ no significant selectivity in bioaccumulation from soil was observed in E. fetida. More residue of the (1S)-cis-(αR) isomer in E. fetida with more rapid degradation of the (1R)-cis-(αS) isomer in soil resulted in the higher BSAF of the former isomer for alpha-cypermethrin.⁴⁶⁾ The differences in uptake, elimination, and metabolic rates in earthworms as well as those in microbial degradation in soil are likely to control the enantioselectivity in bioaccumulation.

4. Metabolism

4.1. Relevant enzymes

Metabolic transformation of a pesticide is one of the major factors for controlling its bioaccumulation and toxicity, but the information on earthworms is limited, as compared with that for other species such as fish. Enzymatic oxidation and hydrolysis are the typical primary Phase I reactions also in earthworms, and the resultant metabolites are conjugated with endogeneous molecules (Phase II).^107,108) The relevant enzymes generally involved in earthworm metabolism are mixed-function oxidases including cytochrome P450 (CYP), carboxylesterases (CaE) and glutathione-S-transferases (GST), and their participation can be indirectly confirmed by the metabolite profiles. The activity of these enzymes, as well as that of acetylcholine esterases, catalases and superoxide dismutases, has been investigated as useful biomarkers of exposure to contaminants such as pesticides, PAHs and heavy metals.^109,110) Biochemical approaches including fractionation of tissue homogenates, followed by chromatographic separation and spectrophotometric analyses are generally taken to investigate these enzymes; however, direct evidence as to the contribution of characterized enzyme(s) to pesticide metabolism has scarcely been reported. Recently, gene analysis using expressed sequence tags (EST; LumbriBase, http://www.earthworms.org/) has been applied to earthworms.^53,54) The existence of various CYPs, GSTs and CaEs is predicted in earthworms by the significant sequence similarity to other species.^53,111) Transcriptomic profiling has shown that atrazine and PAHs upregulate genes related to Phase I and II enzymes in L. rubellus.^112,113)

4.1.1. Oxidases

The oxidase activity in various earthworm species has been examined by using either post-mitochondrial or microsomal fractions, as listed in Table 4. The requirement of O₂ and NADPH as well as the inhibition of enzyme activity by CO or SKF-525A with an optimal pH of 7–8 strongly suggested the presence of CYPs in earthworms.^{117,122,124,125)} However, the difference spectrum of the reduced CO-treated microsomal fraction showed the peak at 420 nm instead of the 450 nm peak typical for CYPs, most likely due to the interference by giant hemoglobin characteristic to the earthworms.^{114,122,123,129)} The presence of CYP in the microsomal fraction was spectrophotometrically supported after removal of the hemoglobin through successive chromatographic purifications.^114,123,124) The estimated CYP content in E. fetida,¹¹⁴⁾ L. terrestris^123,124) and Dendrobaena veneta¹³⁰⁾ was 0.008–0.16 nmol/mg protein, depending on the purification level and the experimental conditions in the enzyme kinetic assays. Many studies show that both the CYP content and its activity are lower than those in rats and fish.^117,130)

Table 4. Oxidases in the earthworms

Species	Enzyme activity1) of oxidases2)
	BROD	MROD	EROD	PROD	ECOD	BPH	AE	ODM	TH	Ref.
E. fetida	Y		N	Y			Y	Y	N	114–116
E. andrei	Y/N	Y	N	Y/N						117–121
L. terrestris		N	N		Y	N	N			122–124
L. rubellus	N		N	N	Y					117, 125, 126
Ap. caliginosa	Y/N		Y/N	Y/N	Y					117, 125, 127, 131
Ap. tuberculanta			Y							128

1) Presence (Y) and absence (N) of the corresponding oxidase activity. 2) Substrate in the parentheses: BROD, 7-benzyloxyresorufin O-debenzylase (7-benzyloxyresorufin); MROD, 7-methoxyresorufin-O-demethylase (7-methoxyresorufin) ; EROD, 7-ethoxyresorufin-O-deethylase (7-ethoxyresorufin); PROD, 7-pentoxyresorufin O-depentylase (7-pentoxyresorufin); ECOD, 7-ethoxycoumarin O-deethylase (7-ethoxycoumarin); BPH, benzo[a]pyrene hydroxylase (benzo[a]pyrene); AE, aldrin epoxidase (aldrin); ODM, p-nitroanisole O-demethylase (p-nitroanisole); TH, testosterone 6β-hydroxylase (testosterone).

Although not systematically investigated, each earthworm species is likely to have an oxidase activity relating to CYP1 and CYP2 families, judging from the substrate specificity. In contrast to the presence of CYP1 predicted by the ESTs,⁵³⁾ EROD activity was not observed in L. rubellus. The TH activity shows the existence of a CYP3 family in E. fetida.¹¹⁶⁾ In the microsomal fraction of E. fetida, three CYP isoforms with mol. wts. of 54, 56 and 58 kDa were identified by SDS-PAGE; the smallest one weakly reacted with the polyclonal antibody to perch hepatic CYP1A in western blotting but with trace EROD activity observed.¹¹⁴⁾ The largest isoform was separately reported in the same species together with AE and ODM activities.¹¹⁵⁾ L. terrestris was found to possess at least three CYP isoforms (48, 51 and 53 kDa) only with BROD activity, none of which reacted with the polyclonal antibodies against intact rat liver CYPs or those induced by 3-methylcholanthrene (3MC) or phenobarbital (PB).¹²⁴⁾

The induction level of CYP by 3MC, PB and other chemicals such as PAHs is very different among aquatic species.³⁰⁾ CYP induction was not observed in D. veneta by 3MC and PB¹³⁰⁾ and was also absent in Ap. caliginosa and L. rubellus with exposure to BaP.¹¹⁷⁾ No or insignificant induction may be accounted for by the high pressure of exposure to many kinds of organic food sources in the environment, which has already increased the levels of some CYP isoforms. The soil exposure of E. andrei to carbaryl caused a gradual decrease in MROD activity by about 20% for 14 days.¹²⁰⁾ In contrast, acetochlor significantly induced both AE and ODM activities in E. fetida,¹¹⁵⁾ as Cu²⁺/Zn²⁺ induced EROD activity in Ap. tuberculata.¹²⁸⁾ The concentration-dependent convex profiles (induction at lower concentrations but insignificant change or inhibition at higher ones) were observed for CYP content or EROD activity when the earthworm was exposed to BaP (E. fetida),¹²⁹⁾ heavy metals (E. fetida)¹¹⁶⁾ and chlorpyrifos (Ap. caliginosa).¹³¹⁾

4.1.2. Esterases

Among the B-type esterases typically inhibited by organophosphorus compounds, CaEs are known to play a role in detoxifying pesticides that have an ester linkage.¹³²⁾ Their activity in the post-mitochondrial fraction is generally examined by spectrophotometrically monitoring hydrolysis product of a phenyl (thio)acetate derivative. Fenitrothion (E. fetida),¹³³⁾ pirimiphos-methyl and deltamethrin (E. andrei),¹³⁴⁾ and dimethoate (E. andrei)¹³⁵⁾ significantly decreased the CaE activity in the paper contact test. Dichlorvos, paraoxon and chlorpyrifos were also potent inhibitors of CaEs through the soil exposure of L. terrestris.^136,137) The species-dependent inhibition of CaE activity by pirimiphos-methyl was examined, using a cylindrical soil microcosm.³¹⁾ This pesticide markedly decreased the CaE activity of E. andrei and moderately did so for L. rubellus, while a concentration-dependent response was observed for L. terrestris.

The presence of seven CaEs (mol. wts. of 60, 70 and 72 kDa) and one arylesterase (62 kDa) was shown in L. terrestris by PAGE; they were differently localized among the body wall, the pharynx, the intestine, and the reproductive organs.¹³⁸⁾ As detected by PAGE of the microsomal fraction from E. fetida, paraoxon-insensitive bands with esterase activity not inhibited by Hg²⁺ were likely to be acetylesterases (57 kDa), while paraoxon-sensitive bands not inhibited by eserine might be CaEs (41 kDa).¹³⁹⁾ Sanchez-Hernandez et al.^{136,140–142)} examined the tissue distribution of CaE activity in L. terrestris by using three different substrates. The activity was maximal in the crop/gizzard and foregut with tissue-specific inhibition by chlorpyrifos-oxon. Six of twelve protein bands by native PAGE were common to the post-mitochondrial fraction of each tissue, indicating multiple CaE isoforms.¹⁴⁰⁾ Kinetic analysis showed one-order of magnitude higher hydrolytic activity in the gut content of L. terrestris than in the gut wall.¹³⁶⁾ The luminal microenvironment of a gastrointestinal tract had higher CaE activity than did feces or bulk soil in Ap. caliginosa.¹⁴²⁾ These are likely to indicate an intestinal secretion of CaEs; this may play a role in protecting earthworms from exposure to xenobiotics.

Various esterases that detoxify organophosphorus compounds have also been reported. Through soil exposure of L. terrestris to bis(p-nitrophenyl)phosphate, both phosphomono- and phosphodiesterases were detected in its enteric tissues.⁴⁰⁾ From the product distribution in E. fetida after the injection of parathion, the presence of phosphotriesterase (PTE) was deduced.¹⁷⁾ Lee et al.¹⁴⁴⁾ chromatographically purified the enzyme that hydrolyzes paraoxon mainly from the gut tissues of E. andrei. The authors characterized it as an alkaline PTE (260 kDa, pH_opt=9) that was sensitive to chelating agents whose inhibition was recovered by Ca²⁺. The PTE-hydrolyzing chlorpyrifos-oxon was also detected in L. terrestris with different localizations in the anterior intestine and the wall muscle of CaEs.¹⁴⁰⁾

4.1.3. Glutathione-S-transferases (GST)

GSTs, mainly distributed in a cytosolic fraction, catalyze the transfer of glutathione to an electrophilic substrate; they have been extensively studied regarding their substrate-specific activity and enzymology. Stenersen et al.^145,146) reported that their activity against the most common substrate 1-chloro-2,4-dinitrobenzene (CDNB) is present in various earthworm species but is much lower than that of rats, and is distributed in any organ of Allolobophora longa and L. terrestris. The GST activity against ethacrynic acid (ETHA) was one-fourth to one-tenth lower as compared with that of CDNB and very low or absent for several other substrates including 1,2-epoxy-3-(4-nitrophenoxy)propane (ENPP) and 1,2-dichloro-4-nitrobenzene (DCNB). These results suggest a greater abundance of π-class GSTs than of θ and µ GSTs.³⁰⁾ Although the GST activity on these potential substrates has been frequently reported, relevant information on GSTs detoxifying pesticides is very limited. The cytosolic fraction from each of eight earthworm species degraded quintozene to hydrophilic products under optimal conditions (30°C, pH 8) but did not show any reactivity on parathion-methyl and atrazine.¹⁴⁶⁾ GST activity was found to be greatly lower against fenoxaprop and metolachlor, as compared with CDNB and DCNB.¹⁴⁷⁾ No induction of GST activity against CDNB, DCNB or ETHA was seen with exposure to PB, 3MC or trans-stilbene in E. andrei and Eisenia venta.^148,149) GST activity against CDNB was not markedly affected by exposure to chlorpyrifos in Ap. caliginosa¹⁵⁰⁾ or acetochlor in E. fetida,¹⁵¹⁾ while it was slightly induced by three organochlorine pesticides in P. posthuma,⁷⁹⁾ fenoxaprop,¹⁴⁷⁾ metolachlor¹⁴⁷⁾ or pyrene¹⁵²⁾ in E. fetida.

The presence of various GST isoforms was demonstrated through isoelectric focusing of the cytosolic fractions from six species by using CDNB as a substrate.¹⁴⁵⁾ Among them, Lumbricus sp. exhibited the main GST activity at pH around 4, while many peaks were observed in Allolobophora and Eisenia sp. at all pH regions and a neutral pH, respectively. Four CDNB-active regions were chromatographically separated from the cytosolic fraction of E. fetida¹⁴⁶⁾ and E. andrei.¹⁴⁸⁾ The major CDNB-active fractions in E. fetida were further separated into three by ion exchange and hydroxyapatite chromatography, one of which showed GST activity against ETHA. GST is a homo- or heterodimer consisting of subunits with mol. wts. of 23–28 kDa.³⁰⁾ Gel filtration indicated the mol. wt. of the CDNB-active GST to be 46.2 kDa¹⁴⁶⁾ with an optimal pH of 7.5 for activity¹⁵³⁾ in Eisenia sp. GST isoforms in the post-mitochondrial fraction of E. fetida, partially purified by GSH-affinity chromatography, were subjected to SDS-PAGE, and four subunits with mol. wts. of 25.5–29.5 kDa were detected.¹⁴⁷⁾ Borgeraas et al.¹⁴⁹⁾ collected GSTs (<1% of the total protein) from the cytosolic fraction of E. venta and E. andrei by GSH-affinity chromatography and showed that they consist of four and six (EaGST I–VI) CDNB-active fractions in the ion-exchange chromatography, respectively. The authors have clarified through reverse-phase HPLC, SDS-PAGE and enzyme activity measurements against several potential substrates that EaGST IV is a homodimer of 26.5 kDa subunits and is most likely to be a π-class due to the high similarity in the N-terminal amino acid sequence to other animals; furthermore, the heterodimer (EaGST I) of 26.3-kDa subunits showed high activity on cumene hydroperoxide (CHP). Recently, one ETHA-active π-GST and CHP-active σ-GST were identified in L. rubellus by a sophisticated approach combining chromatography, ESI-MS, tandem-MS/MS and prediction of a gene sequence using ESTs.¹⁵⁴⁾

4.1.4. Other enzymes

Conjugation with glucose, glucuronic acid or sulfate is one of the most popular Phase II reactions in many species. Although the glucose and sulfate conjugates of some PAHs have been reported for springtail and terrestrial isopods together with the basic reaction profiles of UDP-glucosyltransferase and aryl sulfotransferase,^155,156) corresponding information is not available for earthworms. Cytosolic methyl transferase that catalyzed the methylation of sodium arsenite into monomethyl and dimethyl acids was reported in L. terrestris, and it required S-adenosyl-L-methionine as a methyl donor.¹⁵⁷⁾ In vitro metabolism of 2,4,6-trinitrotoluene using homogenates of E. andrei gave 2- and 4-amino derivatives as the main products with greater formation at a higher temperature,¹⁵⁸⁾ strongly suggesting the involvement of nitroreductases. In relation to the toxic mechanism of paraquat, reduction by NAD(P)H-cytochrome c reductase was shown in the post-mitochondrial fraction from Allolobophora chlorotica.¹⁵⁹⁾ Similar enzymes may operate in the NADPH-dependent reduction of aliphatic keto esters reported in L. rubellus.¹⁶⁰⁾

4.2. Metabolism of pesticides

Most information on the metabolic profiles of pesticides in earthworms has been obtained through studies of their toxicity and bioaccumulation, but it is very limited, as seen in Table 5. Many studies have been conducted by soil exposure simulating an environmental situation, but the possible metabolic contribution by various microbes in either soil or the earthworm gut¹⁶⁶⁾ cannot be fully excluded. Residue monitoring of polychlorinated phenols used for wood preservation in Finnish sawmills showed the formation of O-methylated phenols in only a few earthworm species, but their formation in soil followed by uptake to the earthworms could not be excluded.¹⁶⁷⁾ Sulfoxide phenol from fenamiphos was detected in E. fetida by soil exposure, but the absence of this metabolite’s formation when using earthworm homogenates suggests that its presence was due to uptake from the soil.²⁷⁾ Microbial degradation in soil can be minimized by using sterile natural or artificial soil in the exposure, applying pesticide via water exposure or direct injection, or conducting in vitro metabolism. The contribution of gut microbes has rarely been examined, but no contribution of gut microbes was found when using their inocula on the metabolism of fenamiphos in E. fetida.²⁷⁾ In the metabolism of aniline derivatives in E. venta, pretreatment of the earthworm in agar plates that included several antibiotics was conducted to exclude microbial degradation.¹⁶⁸⁾ Incidentally, the material balance is part of the basic information used to evaluate the validity of a study. The usage of a radiolabel is most convenient with the radio-analysis of all compartments including the exposure medium, but closed systems equipped with volatile traps have only been applied in a few cases.^18,169)

Table 5. In vivo metabolism of pesticides in the earthworm

Chemical	Species1)	Appl.2)	Per.3)	Metabolic profiles4)	Ref.
Aldrin	E. fetida	f, as	4#, 56	O2	161
Chlordane	L. terrestris	inj	2	HD, O1, U	15
DDT	L. terrestris*	c	28	DH, DC	22
	Al. caliginosa	c	28	DH
	P. posthuma	ns	63	DH, DC	162, 163
	L. terrestris	g	7	DH, DC, O1	18
Dicamba	L. terrestris	inj	2	O3, U (1)	16
Chloramben	L. terrestris	inj	2	DE, U (4)	16
Carbofuran	L. terrestris	w, inj	1–2	O1, H1, U (1–2)	13,14
	E. fetida	w	2	O1, H1	14
Aldicarb	L. terrestris	w, ns	6#	O5, H1	20
Oxamyl	Al. caliginosa, L. terrestris	w	1	H1, U (2)	19
	Al. rosea, L. rubellus, E. fetida
Isoproturon	Ap. caliginosa	ns	15	NDA, H1	164
Terbufos	L. terrestris	as	2–32	O5	165
Parathion	E. fetida	inj	1.25	ODA, H2, O4	17
Paraoxon	E. fetida	w	0.5#	H2	17
Chlorpyrifos	E. fetida	f	4	H2, O4	48
Chlorfenvinphos	E. fetida	ns	28	U (1)	83
Fenamiphos	E. fetida	ns	28	O5	27
Cypermethrin	L. terrestris, Al. caliginosa	ns	56	H3, C (spermine)	82
Alpha-cypermethrin	E. fetida	ns	28	H3, U (1)	83
Atrazine	L. terrestris	inj	2	NDA, HD, U (3)	16
Diniconazole	E. fetida	as	21	O1	88
Pentachlorophenol	Al. caliginosa	w, as	1–7	U	21

1) Mature with well-developed clitellum: *, immature. 2) Application method: w, aqueous solution; f, filter paper; as, artificial soil; ns, natural soil; c, compost; inj, injection to body; g, gavage. 3) Period of metabolism in day or hr (#). 4) C, conjugation with its type in the parentheses; DC, dechlorination; DE, decarboxylation; DH, dehydrochlorination; HD, hydrolytic dechlorination; NDA, N-dealkylation; H1, cleavage of carbamate (or urea) linkage; H2, cleavage of P-Oaryl linkage; H3, cleavage of carboxylate ester linkage; ODA, O-dealkylation; O1, alkyl hydroxylation; O2, epoxidation; O3, ring hydroxylation; O4, oxidative desulfuration; O5, S-oxidation; U, unknown metabolites with their number in the parentheses.

The metabolic reactions reported for earthworms are mainly hydrolysis, oxidation and dechlorination (Table 5), which are common to other species. Direct evidence of reductive pathways in the earthworm is not available for pesticides, but the corresponding metabolic ability can be confirmed from reduction of the nitro group for industrial chemicals in E. andrei.¹⁵⁸⁾ A bioaccumulation study without any information on metabolism is reported for fipronil,⁴³⁾ but the sulfide formation in springtail F. candida¹⁷⁰⁾ might suggest a similar reductive pathway in the earthworm. One unique reaction is the decarboxylation reported for chloramben injected to L. terrestris¹⁶⁾; it is also observed in the stepwise degradation of methyl paraben in E. fetida.¹⁷¹⁾ A further novel reaction is the nitration at the 2-position of the phenyl ring through the metabolism of a 4-nonylphenol isomer in Metaphire guillelmi,¹⁶⁹⁾ but a similar reaction has never been reported for pesticides.

Unknown polar metabolites including conjugates have been reported for many pesticides, but their chemical identities have rarely been confirmed. By exposure to soil treated with ¹⁴C-cypermethrin,⁸²⁾ the acid hydrolysis of the polar extracts from earthworms released the corresponding chrysanthemic acid from the [acid-¹⁴C] label. A great deal of harsh acid hydrolysis was necessary to effectively release the 3-phenoxybenzoic acid (PBacid) from the [alc-¹⁴C] extracts. The [alc-¹⁴C] fraction, isolated by HPLC and then derivatized, was subjected to MS and NMR analyses, demonstrating that the main metabolite is the PBacid conjugate with spermine, the main endogeneous polyamine in earthworms.¹⁷²⁾ Incidentally, few conjugation reactions were reported for some chemicals other than pesticides. The γ-glutamyl conjugate at the amino group was the main metabolite from 5-hydroxytryptamine injected to L. terrestris¹⁷³⁾ and 4-fluoroaniline derivatives by paper contact exposure in E. venta.^168,174) In the latter species, glucose conjugates of aniline derivatives were also identified by ¹H-/¹⁹F-NMR and LC-MS.^168,174,175) Pyrene was first hydroxylated at the 1 position, followed by conjugation with glucose or sulfate in wood lice Porcellio scaber or with glucose or malonylglucose in springtail F. candida, while no corresponding conjugate was detected in E. venta.¹⁵⁶⁾ The enzymatic hydrolysis with β-glucuronidase released 4-nonylphenol and other metabolites from the extracts of M. guillelmi,¹⁷⁶⁾ indicating the possible formation of glucuronic acid conjugates.

Conclusion

The application method and formulation type control the initial residue of pesticide on the soil surface, and the distribution of pesticide in the soil is depth dependent due to its physico-chemical properties and microbial activity. Therefore, different earthworm ecologies concerning the location of habitat, feeding and burrowing result in species-dependent exposure to pesticide and its metabolites in the real terrestrial environment. However, the earthworm species used for investigating the effect of pesticide are mostly limited to E. fetida, E. andrei and L. terrestris, due to the ability to obtain earthworms of a uniform quality. For a more appropriate assessment of pesticide exposure, species’ differences in toxicity, bioaccumulation and metabolism should be better examined.

The potential acute and chronic toxicity of various pesticides and industrial chemicals has been studied by paper contact and soil exposure methods. The toxic level is usually expressed by the nominal application rate or the concentration of pesticide in the exposure medium, but its body burden is essential for correctly evaluating the toxic effects. Bioaccumulation and metabolism studies should provide useful information for determining the practical body burden of pesticides in earthworms. Furthermore, pesticide toxicity has not been investigated extensively from the viewpoint of MOA, which is very important for evaluating the combined toxicity of a pesticide mixture. The toxicogenomic approach, especially metabolomics using MS and NMR, should be applied more frequently.

The bioaccumulation potential of a pesticide should be examined at least on the basis of the equilibrium partition theory, successfully introduced in the assessment of aquatic sediment dwellers. The dissolved fraction of a pesticide in the pore water of soil has been estimated conveniently by using its soil adsorption coefficient, but the hysteresis in desorption during aging would cause an overestimated concentration. The SPME technique would be better applied to evaluate the bioavailable fraction of pesticide in the soil. Furthermore, the behavior of a pesticide including metabolism should be examined using radiolabeled forms not only in the earthworm but also in the exposure medium such as soil. Kinetic analysis based on this information plays a role in improving pesticides’ poor correlation between the acute LC₅₀ value and log K_ow or more precisely predicting the bioaccumulation, since the contribution of metabolism is often neglected due to simplification. To know the intrinsic metabolic activity of the earthworm, the contribution of gut microbes on the metabolism of pesticides should be confirmed by in vitro metabolism using a post-mitochondrial fraction or in vivo in the presence of antibiotics. Incidentally, many chiral pesticides have been developed from the viewpoint of higher activity with less environmental burden or toxicity. More attention should be paid to enantioselectivity not only in toxicity but also in bioaccumulation and metabolism for more precise risk assessment of each stereoisomer.

The chemical structure of metabolite(s) is useful information, not only for identifying relevant enzymes that participate in the detoxification of a pesticide but also for assessing the effect of pesticide via the food web on many species feeding on earthworms as prey. Since available metabolic profiles of pesticides mostly originate from old-fashioned studies, up-to-date analyses, such as various LC-MS techniques should be applied to confirm the chemical identity of a polar metabolite (conjugate). This approach should shed light on inconsistencies such as the abundance of GSTs without detection of the corresponding metabolites and the absence of information on the γ-glutamyl conjugate from pesticides and their metabolites. Since the biochemical investigation of CYPs, GSTs and CaEs relevant to pesticide metabolism has been conducted together with their transcriptomic analyses, this approach should be extended to various enzymes such as glucosyl, sulfo and acyltransferases in parallel to metabolite identification.

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