Environ. Sci. Technol. 2003, 37, 3399-3404
Elucidating the Routes of Exposure for Organic Chemicals in the Earthworm, Eisenia andrei (Oligochaeta) T J A L L I N G J A G E R , * ,† ROEL H. L. J. FLEUREN,‡ ELBERT A. HOGENDOORN,§ AND GERT DE KORTE§ Department of Theoretical Biology, Vrije Universiteit Amsterdam, de Boelelaan 1085, NL-1081 HV Amsterdam, The Netherlands, and Laboratory for Ecotoxicology and Laboratory for Organic-Analytical Chemistry, National Institute of Public Health and the Environment (RIVM), P.O. Box 1, NL-3720 BA Bilthoven, The Netherlands
Earthworms take up organic compounds through their skin as well as from their food, but the quantitative contribution of each route is unclear. In this contribution, we experimentally validate an accumulation model containing a separate compartment for the gut. Uptake from the gut is modeled as passive diffusion from the dissolved phase in the gut contents. For the experiments, we exposed Eisenia andrei in artificial soil spiked with tetrachlorobenzene, hexachlorobenzene, and PCB 153. Apart from the standard accumulation and elimination experiments, we ligatured the worm (using tissue adhesive) to prevent feeding. Model fits were good, thus supporting the validity of the model. The contribution of the gut route increased with increasing hydrophobicity of the chemical, and for PCB 153 the gut route clearly dominated. Despite the importance of the gut route, the final steady-state body residues did not exceed equilibrium partitioning predictions by more than 25%. Rate constants for exchange across the skin and the gut wall could be separately identified. The rate constant across the skin decreases with Kow but was generally higher than data derived from water-only exposure. The relationship with hydrophobicity was less clear for the rate constant across the gut wall.
Introduction Earthworms are able to take up organic chemicals through their skin (1) as well as from their food (2). However, the quantitative contribution of each route remains unclear. As earthworms regularly consume soil, it is difficult to study both routes in isolation in a relevant experimental setup. Earthworms can be exposed in water alone (3), but the relevance of this setup for porewater uptake from soil is not obvious. More work has been done in this area for sediment organisms, showing that ingestion is an important pathway for very hydrophobic chemicals such as pyrene and dioxins (4, 5). For earthworms, Belfroid et al. (6) predicted, on the * Corresponding author: e-mail:
[email protected]; telephone: +31 20 444 7134; fax: +31 20 444 7123. † Vrije Universiteit Amsterdam. ‡ Laboratory for Ecotoxicology, RIVM. § Laboratory for Organic-Analytical Chemistry, RIVM. 10.1021/es0340578 CCC: $25.00 Published on Web 07/02/2003
2003 American Chemical Society
basis of model extrapolations, that food uptake becomes an important exposure route for very hydrophobic chemicals (log Kow > 5). It seems to be a generally held opinion that feeding on soil can lead to the invalidation of equilibrium partitioning (EP), which is why additional safety factors were prompted in European risk assessment guidelines (7). In most case, uptake from food is modeled by simply adding uptake routes (8), but in a previous contribution (9), we proposed a more mechanistic accumulation model. Based on the work of Gobas et al. (10, 11), the model includes a separate compartment for the gut contents and a closed mass balance. The mechanism for uptake from the gut is likely to be the same as for uptake across the skin (i.e., passive diffusion). This assumption is strongly supported by experimental evidence as derived for intact goldfish (10), isolated gut segments of catfish (12), and humans (13). For earthworms, the validity of this assumption was indicated, although a proper validation was impossible because the physiological data regarding the feeding process were lacking (9). Most routine studies with earthworms are carried out with the compost worm (Eisenia andrei/fetida) in an artificial soil medium (14). It is for this system that the essential feeding parameters have recently been identified (15) including gut loading, gut retention time, and digestion efficiency. In this study, we set out to validate the accumulation model with three organic compounds [tetrachlorobenzene (TeCB), hexachlorobenzene (HeCB), and PCB 153] in artificial soil. A series of experiments was performed with these chemicals, starting with a straightforward accumulation and elimination phase. Subsequently, soil from these experiments was reused to see whether the bioavailable phase had been altered, as indications of depletion have been observed (1). Finally, an accumulation experiment was performed with worms sealed with a tissue adhesive, thus preventing feeding. This procedure was pioneered by Vijver et al. (16) to demonstrate uptake routes for heavy metals. Other forms of ligaturing have been applied to demonstrate that pesticides are mainly taken up through the skin in wateronly exposure (1) and that calcium is mainly taken up from the diet (17). However, this study is to our knowledge the first to separate exposure routes for organic chemicals in a soil situation. The data from all these experiments are used together to fit the accumulation model and to identify the rate constants for uptake through the skin and from the gut. Furthermore, the model can shed light on the central questions: which exposure route dominates and does feeding lead to body residues exceeding the predictions made by equilibrium partitioning.
Experimental Section Exposure Media and Spiking Procedure. Artificial soil was used for the experiments (14). The water content was brought to 40% (weight basis, water/dry medium) with a lutetium solution (Lu, hydrated chloride salt, purity 99.9%, Alfa Aesar, Karlsruhe, Germany) to obtain a nominal concentration of 15 mg/kgdwt. The Lu is used as a nonassimilated tracer to compare the feeding activity to earlier experiments (15). After the soil was wetted, it was thoroughly mixed and stored in closed plastic containers at 5 °C for 1 week prior to spiking with organic chemicals. After storage of the containers, the pH (KCl) of the soil was 5.0. 1,2,3,4-Tetrachlorobenzene and hexachlorobenzene were obtained from Riedel de Hae¨n, Seelze, Germany (99% purity, Pestanal); PCB 153 was synthesized at the IRAS, Utrecht, The Netherlands (99% purity). VOL. 37, NO. 15, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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The spiking procedure for these compounds was adapted from Northcott and Jones (18). Because we needed to spike 4 kgdwt of medium, we had to do the procedure in steps (dilution spike). First, the chemicals needed to achieve a nominal concentration of 10 mg/kgdwt for each chemical were dissolved in 100 mL of acetone (pro analysis). Wet soil (1 kg) was placed in a kitchen blender, and the acetone solution was slowly added while mixing. Mixing continued for several minutes (stopping a few times to crush aggregates with a spatula). The spiked soil was left in a fume cabinet overnight after which the acetone and also most of the water had evaporated. Next, one-fifth of the spiked soil was put in the blender with 900 gwwt of uncontaminated medium and the water needed to restore the 40% water content. This was mixed for several minutes, stopping a few times to prevent the medium from overheating and crushing the aggregates. This entire procedure was followed five times until the entire medium was spiked. The moisture content was checked by oven drying at 80 °C and was 41%. The fraction organic matter (Fom) in the soil was 10.5% (loss on ignition). To allow equilibration, the medium was stored at 10 °C in glass jars for 1 week before animals were introduced. One day before animals were introduced, soils were transferred to the test temperature of 20 °C. Test Animals and Experimental Setup. Sub-adult earthworms (E. andrei), were taken from mass cultures at RIVM (Bilthoven, The Netherlands). The animals weighed between 200 and 300 mgwwt. First, the animals were allowed to evacuate their gut contents by keeping them on moist filter paper for 24 h at 20 °C. Next, the animals were transferred to plastic containers with 175 gwwt of uncontaminated medium (four worms per container), and the containers were placed at 20 °C, covered by a black plastic pot to minimize disturbance. The animals were left to acclimatize for 1 week under these conditions. After this, they were exposed to the chemicals in 1-L glass jars using 250 gwwt of spiked medium and four animals per jar. Several jars were used for the determination of the feeding activity (see Supporting Information). For the accumulation experiment, the worms were recaptured following exposure (0, 1, 2, 3, 5, 7, 14, and 21 d) and placed in a Petri dish on moist filter paper for 24 h at 20 °C. After this period, worms were packed in aluminum foil and frozen at -20 °C. Soil samples were taken and frozen in glass jars at -20 °C (four samples at t ) 0 and two samples at days 14 and 21). At t ) 14, three additional jars were emptied, and the worms were recaptured. The worms were transferred to 250 gwwt of uncontaminated medium and allowed to eliminate for 2, 5, and 11 d. The bioavailability of the chemicals may change during the experiment, which is why the spiked soil from the four jars emptied at t ) 14 was reused. Fresh worms were taken from the culture, placed on wet filter paper for 24 h and subsequently on uncontaminated medium for another 24 h, and then introduced in the reused soil. These worms were recaptured after 1, 3, 7, and 11 d. For the ligaturing experiment, worms were taken from the culture and allowed to empty their gut for 24 h on moist filter paper. Their anterior end was ligatured using a tissue adhesive (Indermil, from Loctite Ireland Ltd.), conforming to the procedure by Vijver et al. (16). Gluing turned out to be difficult for this species because the worms were irritated by the procedure and excreted coelomic fluid, which interfered with the setting of the glue. As exposure time, 0, 1, 2, 3, 5, 7, and 14 d were employed using five worms per jar with 250 gwwt of soil. After exposure, the worms were recaptured and placed individually in a Petri dish with moist filter paper overnight. Only the worms that did not evacuate any solids were used for chemical analysis. For t ) 0, five worms were taken; on days 1 and 5, three worms had been successfully exposed; on day 2, only one worm was exposed 3400
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(after longer periods, all worms excreted solid materials and were discarded). Analysis of Organochlorine Compounds. For the sample pretreatment of soil, 10 gwwt was mechanically shaken during 10 min in a glass tube with 25 mL of acetone. Next, 50 mL of light petroleum (a mixture of saturated alkanes with a boiling point of 40-60 °C) were added, and the contents were mechanically shaken for 20 min. After centrifugation (5 min. at 3000 rpm), the liquid phase was transferred into a shaking funnel. The remaining part was extracted again following the same procedure, and the liquid was transferred to the funnel. After the addition of 500 mL of water, the funnel was manually shaken for 1 min. The aqueous phase was discharged, and the upper layer was extracted once more for 1 min with 500 mL of water. The light petroleum phase was passed through a funnel with about 10 g of anhydrous sodium sulfate and concentrated to a volume of 10 mL. For analysis of the worms, a sample of approximately 1 g was placed into a glass tube and weighed. After addition of 100 µL of the internal standard (approximately 100 ng of [13C12]-PCB 153), 9 mL of isopropyl alcohol and 10 mL of cyclohexane were added, and the mixture was macerated with an ultra-speed homogenizer for 2 min. Next, 10 mL of water was added, and the mixture was macerated again for 1 min. The phases were separated by centrifugation for 10 min at 3000 rpm. The upper organic layer was transferred through a funnel with sodium sulfate to a Kuderna-Danish evaporation apparatus by means of a pasteur pipet. The remaining part of the sample was macerated again for 1 min with 10 mL of a mixture of 2-propanol-cyclohexane (13:87, v/v). After centrifugation, the upper layer was added to the first extract, and sodium sulfate was rinsed with 5 mL of cyclohexane; the organic layer was concentrated to 1 mL. For cleanup (fat destruction), the extract was brought onto a chromatography column filled with 0.5 g of silica gel impregnated with sulfuric acid (100 g of silica heated for 4 h at 200°C and 43.5 mL of concentrated sulfuric acid mixed by rotating for 12 h). Next, 5 mL of hexane was passed through the column, and the organic solvent was collected in a calibrated glass tube and brought to a volume of 5 mL. For instrumental analysis with GC/MS operating in the electron impact (EI) mode, 1 mL of extract was transferred into an autosampler vial, and 10 µL of pentachlorobenzene (PeCB, 1 µg) was added as internal standard. A total of 1.5 µL was on-column injected into a fused silica DB-5MS capillary column coated with 5% cross-linked 5% phenyl methyl siloxane with a length of 30 m × 0.25 mm i.d. and a film thickness of 0.25 µm. Helium was applied as carrier gas at a flow of 1 mL/min. Quantification was done using the internal standard PeCB for calibration and, in case of worm samples, the isotope PCB 153 to correct for losses during sample pretreatment. The average recoveries performed at levels between 2 and 10 µg/g ranged between 83 and 103% with SD below 9% (n ) 4 for each analyte-matrix combination). The Model. The model is set up as a three-compartment model with a closed mass balance (Figure 1). Diffusion (the two-way arrows) and advection (one-way arrows) are the basic transport processes. The model has been described earlier (9), and the full model formulation is available in the Supporting Information. Each compartment is assumed to be well-mixed and of constant volume. Although the gut is probably better reflected by a plug-flow reactor (19), the simpler mixed compartment serves as an approximation (11). The chemicals will be taken up into the tissue of the worm from the outside soil as well as from the gut contents; both processes are modeled as passive diffusion from the dissolved phase. The diffusion gradient between soil or gut contents and worm tissue is defined by the concentrations in both
FIGURE 1. Schematic representation of the accumulation model. compartments and the partition coefficient between organic matter (OM) and earthworm tissue (Kws in kgOM/kgwwt). The Kws can be viewed as the ratio of the bioconcentration factor with water (BCF in L/kgwwt) and the organic matter specific sorption coefficient (Kom in L/kgOM). When multiplied by the soil concentration on OM basis, Kws would reflect the equilibrium body residue of a nonfeeding worm. The OMworm partition coefficient is the same for soil to worm as from gut contents to worm. However, the magnitude of the gradient differs between these two uptake routes because the chemical concentration as well as the Fom in the gut contents differs from that in soil (due to selective feeding, compaction, and OM digestion) (10, 11). The net result of the two uptake routes depends on the kinetics of the various transport processes, and the steady-state body residue in a feeding earthworm will thus end up somewhere between equilibrium with the soil and equilibrium with the gut contents. In this study, we chose to ignore degradation in the gut and biotransformation as removal processes. This is acceptable, given the very short gut retention time (Table 1) and lack of indications for biotransformation of chlorobenzenes and PCBs in earthworms (20, 21). However, the analysis results prompted us to include a first-order loss term for the soil (kd). Supported by pilot calculations (9), instantaneous chemical equilibrium between solid and water phases is assumed. Several adaptations to the previous model (9) are made. First, compaction of the gut contents is included, meaning that the gut volume decreases as food is absorbed (15). Also, a slightly different formulation for the Fom in the gut is taken as average of Fom determined in ingesta and egesta instead of egesta only. Model Fitting. The model equations are implemented in matrix form in MatLab Version 6.1 (The Mathworks, Inc.) and solved with the matrix exponential function. For each chemical, we have five data sets (soil concentrations, two accumulation phases, one elimination phase, and accumulation in glued worms) that must be described by the same model and with the same parameter values. Therefore, all data sets must be fitted simultaneously. This is accomplished by defining a likelihood function on the basis of the sums of squares (SSQ) from the model fits on each data set assuming normally distributed data (22): 5
L(θ|data) ∝
∏SSQ(θ; data ) i
-ni/2
(1)
i)1
where θ is the entire set of parameters, and ni is the number of points in data set i. Different likelihood functions may be multiplied, so in this way we end up with one expression for the overall likelihood of the model parameters given all five data sets. For the gluing experiment, the number of worms that were successfully glued was taken as a weight coefficient
in the SSQ. The overall log-likelihood function is maximized by a Nelder-Mead simplex search in MatLab, yielding maximum-likelihood (ML) estimates of the parameters. The likelihood function is also used to construct confidence intervals by calculating the profile likelihood (23), which is more realistic for small data sets than standard asymptotic procedures based on large-sample theory. Even though the gut retention time is only 2.9 h in feeding worms, 24 h depuration on filter paper is insufficient to remove all of the gut contents. However, a longer depuration could lead to bias as also chemicals will be lost from the tissues. We corrected the modeled concentration for the remaining gut contents using an estimate of the remaining fraction (Frem, Table 1, see Supporting Information). The ML estimates are used to estimate the net chemical assimilation efficiency (AE), the biota-soil accumulation factor (BSAF), and the deviation from EP. The AE can be calculated from the uptake flux from the gut contents into the worm tissues and the chemical flux with feeding (see Supporting Information). BSAF (in kgOM/kgwwt) is calculated from the modeled concentration in the worm (Cw in mg/kgwwt) at t ) 21, the concentration in the soil (Cs in mg/kgdwt), and the Fom in soil:
BSAF )
Cw(21)Fom(soil)
(2)
Cs(21)
The magnitude of the BSAF cannot be directly interpreted in relation to EP as we did not measure porewater concentrations and lipid content of the worms. However, the deviation from EP can be assessed in an indirect manner by comparing Cw(21) to equilibrium estimates based on the concentration in soil and the modeled concentration in the gut (Cg):
Cw(EP, soil) )
Cw(EP, gut) )
KwsCs(21)
(3)
Fom(soil) KwsCg(21)
(4)
Fom(gut)
In these equations, we use the parameter estimate for Kws (resulting from the model fit) for the EP estimates. The worm can come to equilibrium with the soil (eq 3) or the gut contents (eq 4) or end up somewhere between (in a nonequilibrium steady state). To obtain a confidence interval on these derived results, we applied a random parameter search. Parameters were randomly drawn from log-uniform distributions. When the likelihood of these parameters (eq 1) was not significantly lower than the ML estimate, the parameter combination was stored. Random parameters were drawn until 200 acceptable parameter combinations were obtained. For each parameter combination in this set, the BSAF, AE, and deviation from EP were calculated. The maximum and minimum values serve as confidence intervals.
Results and Discussion General Observations. We want to apply the parameter values for the feeding activity, as observed previously (Table 1), to our current experiments. For this reason, we first confirmed that these values are indeed representative for this study (see Supporting Information). The worms increased approximately 10% in weight during the 21-d exposure; the potential effects on accumulation kinetics are expected to be small and were ignored. The initial concentration in the soil was 5.8 mg/kgdwt for TeCB, 7.2 for HeCB, and 6.8 for PCB 153. These were lower than the nominal 10 mg/kgdwt, presumably due to losses when evaporating the acetone. However, the homogeneity of the VOL. 37, NO. 15, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Fixed Parameters for the Feeding Process (15)
a
symbol
description
unit
value
SE
Fege Frem Fsel Fdig Fcom Tret Fsolids
egested feces as fraction of body weight fraction of gut contents remaining after 24 h depuration selectivity for OM in diet digestion efficiency of OM factor by which gut contents are compacted gut retention time fraction solids in worm
kgdwt/kgwwt kgdwt/kgdwt (-) (-) (-) h kgdwt/kgwwt
0.12 0.056 2.1 0.35 1.09 2.9 0.14
0.012 0.021 0.053 0.033 0.011 2.5-3.3a 0.0013
90% probability interval.
TABLE 2. Parameter Estimates and Derived Measures, Resulting from the Model Fitsa parameter
unit
TeCB
HeCB
log Kow Kws estimated from QSARs (20, 27)
Chemical Properties and QSAR Estimations 4.64 (25) 5.73 (25) kgOM/kgwwt 0.12 0.20
Kws (first accumulation exp) Kws (second accumulation exp) rate constant skin (ks) rate constant gut wall (kg) degradation and/or volatilization (kd)
kgOM/kgwwt kgOM/kgwwt d-1 d-1 d-1
BSAF at t ) 21
kgOM/kgwwt kgOC/kglipc % % %
assimilation efficiency (maximum) deviation from EP with soil deviation from EP with gut contents
Estimated Model Parameters 0.11 [0.077, 0.13] [-]b 0.74 [0.42, 2.1] 0.27 [0, +∞] 0.0065 [0.0051, 0.0077] Derived Results 0.12 [0.072, 0.13] 7.1 10 [2.6 × 10-4, 50] 7.3 [0,18] -14 [-21, 0]
PCB 153 6.92 (26) 0.33
0.20 [0.19, 0.22] 0.16 [0.15, 0.18] 0.30 [0.22, 0.43] 0.43 [0.18, 0.72] 0.0055 [0.0051, 0.0058]
0.24 [0.23, 0.27] 0.16 [0.13, 0.21] 0.027 [0.020, 0.037] 0.16 [0.13, 0.20] 0.0062 [0.0058, 0.0066]
0.23 [0.21, 0.26] 14 26 [0.19, 42] 13 [1.0, 23] -7.1 [-21, -1.9]
0.30 [0.25, 0.36] 18 16 [12, 22] 24 [18, 27] -3.3 [-6.4, -0.3]
a Maximum-likelihood estimates with 95% likelihood-based confidence intervals. K b Assumed the ws is the OM-worm partition coefficient. same value as in the first accumulation phase. c Calculated, assuming a lipid content of 1% (20) and a factor of 1.7 between organic carbon and organic matter.
FIGURE 2. Model fits for the different accumulation and elimination experiments (top) and the modeled uptake fluxes from soil and gut contents, with 95% confidence intervals (bottom). The EP prediction marks the estimated body residue for a worm in equilibrium with the soil. spiking was acceptable as the standard deviations were 4-5% (n ) 4) of the average value. The soil concentrations appeared to decrease some 10% in the course of the exposure experiment. It is striking that the rate constants for this disappearance (kd, Table 2) are practically identical for all three chemicals. The reasons for this disappearance have not been investigated but could include the formation of 3402
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resistant fractions (24), degradation, or volatilization. The accumulation model can adequately describe the data from the different experiments simultaneously (Figure 2) with only five free parameters (see Table 2), an average of one parameter per data set. Only the data for TeCB are rather scattered (especially in the first accumulation phase). In most cases, the parameters are accurately identified by the data, evidenced from the tight 95% likelihood-based confidence intervals (Table 2). However, it should be noted that these
confidence intervals do not include the uncertainty in the feeding parameters (Table 1). Only for TeCB, the poor fit results in ill-defined estimates. The good fit for the other compounds is consistent with the assumption that gut uptake is mediated through passive diffusion from the dissolved phase in the gut (10, 11). Ligaturing the earthworm allows to isolate exchange across the skin in a soil situation. Even though the worms survive for several days without problems, the treatment is stressful. The stress is indicated by the coelomic fluid, expelled by all the worms in reaction to the glue. Expelling fluid is a normal response of E. andrei to rough handling and has no lasting effects on their health. Other species (e.g., the genus Lumbricus) do not have this response, but E. andrei was the species for which the feeding parameters were available (Table 1). The ligatured worms may show deviating behavior in soil, which can also bias chemical uptake. Nevertheless, the difference between glued and intact worms is remarkably small for both chlorobenzenes, providing some reassurance that the earthworms are sufficiently active to take up the chemicals through the skin. Exposure Routes and Assimilation Efficiency. The estimated uptake fluxes clearly show an increase of the importance of the gut in the total uptake with increasing hydrophobicity (Figure 2). Furthermore, the net fluxes decrease in time as the animal is approaching steady state. Figure 2 shows that for TeCB the skin is probably the most important route, although the large confidence intervals preclude firm conclusions. For HeCB, both routes are approximately equally important, but for PCB 153, the gut route is truly the dominant exposure route. Although we do not agree with the approach taken by Belfroid et al. (6), our conclusion is comparable: the gut begins to become an important route for chemicals with a log Kow above approximately 5 and dominates above 6. This is also reflected in the deviation from EP with soil, which increases with Kow from 7 to 24%. However, the confidence intervals are quite large, and only the deviation for PCB is clearly higher than 0%. Similarly, the deviation from EP with the gut contents decreases with increasing Kow so that for PCB 153 the tissue residues are nearly in equilibrium with the gut contents. The parameter estimates also allow calculation of the net chemical AE from the estimated fluxes in the model. As shown previously (9), the net AE depends on time; therefore, only the maximum is given here. No general trend with Kow is observed, although a trend may be obscured by the large confidence intervals. Partition Coefficients. The OM-worm partition coefficient (Kws) was accurately predicted by the QSARs for wormwater (20) and organic carbon-water partitioning (27), assuming a factor of 1.7 between organic carbon and organic matter (Table 2). Note that Kws in this case is a model parameter; the final body residue in steady state depends on the kinetics and lies between the extremes: equilibrium with the soil or the gut contents (eqs 3 and 4). The actual BSAF is thus a secondary result (eq 2) and is slightly higher than the Kws, indicating that the concentrations in the worms exceed equilibrium with the soil. The BSAFs after lipid and carbon normalization are larger than unity, reflecting that sorption increases less with Kow (27) than does bioconcentration (20). The Kws in the reused soil was generally somewhat lower than the value in the initial accumulation phase. Only for TeCB, a higher Kws was estimated in the reused soil. As we judged this behavior to be an artifact (because of the scatter in the initial accumulation phase), we chose to use only one Kws for both accumulation experiments with this chemical. For PCB 153, we saw a clear decrease in Kws for the reused soil, showing that bioavailability had declined over 2 weeks. This difference could not be modeled as a depletion of the
FIGURE 3. Rate constants for exchange across the skin and across the gut wall vs log Kow. Error bars represent 95% likelihood-based confidence intervals. The broken line indicates elimination rates for earthworms in water only (3). bioavailable phase, as mentioned in the Introduction. Unfortunately, we cannot offer a convincing explanation for this phenomenon, but some form of sequestration (either autonomous or caused by the worms or their feces) remains possible. In the model, we assume the same partition coefficient (Kws) for exchange from soil to worm (via porewater) as from gut contents to worm (via the gut fluid). However, gut fluids differ from porewater in that they include secretions from the worm to aid digestion. Mayer et al. (28) have shown that, in marine deposit feeders, these secretions include surfactants that also act to solubilize organic contaminants above levels expected in seawater. It may appear that the action of gut fluids invalidates the hypothesis of passive diffusion via a water phase. However, we do not believe this to be the case as there is strong support for a diffusion-driven uptake (see Experimental Section), even though surfactants facilitate this transport (13). Furthermore, although secretions with surfaceactive properties will increase the dissolved chemical concentration, they cannot increase the fugacity gradient. Because the gut fluid becomes less polar than water due to these secretions, the chemical has less urge to flee to the earthworm’s tissues. The gut fluid-worm partition coefficient is decreased by the same factor as the solubility is increased, leading to the same net uptake. The same conclusion was reached by Lu et al. (29). We therefore believe that gut secretions act mainly on the gut rate constant (kg) and not on the OM-worm partition coefficient (Kws). Rate Constants. The rate constants for exchange across the skin (ks) and the gut wall (kg) could be separately identified (Table 2). Only for TeCB, a confidence interval for the gut rate constant cannot be made. There is a value that has the highest likelihood, but very high and very low values are not significantly worse (apparently exchange across the skin is so rapid that the gut route cannot compete). The rate constants for chemical exchange are shown in Figure 3, which also gives a fit on data for the elimination from worms in a water-only situation (3). Comparison to literature data for elimination rates in soil is not useful as reported constants will always reflect the total elimination flux through all routes. The skin rate constant (ks) for TeCB is quite comparable to the water-only data, but our value for HeCB is much higher. This is striking given the fact that, in a water-only situation, the contact between worm and water is likely to be more intensive. Furthermore, rate constants for passive diffusive exchange are expected to decrease with Kow with a slope close to 1 (on log scale) in this Kow range because for these compounds the diffusion across a stagnant water layer is rate-limiting (30). However, for the rate constant across the VOL. 37, NO. 15, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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skin, we find a slope that is less steep than for water-only exposure (-0.63 vs -1.3). An explanation may be derived from the earthworm’s physiology. In a soil situation, earthworms lose 10-20% of their body weight in moisture each day due to their respiratory system, which requires the maintenance of a moist outer surface (31). In water-only exposure, the water loss will likely be much less. The water losses need to be replenished, requiring water transport across the skin (also advectively transporting the chemical). This process may explain a higher exchange rate in soil than in water and the different relationship with Kow. The relationship between the rate constant across the gut wall (kg) and Kow cannot be properly assessed because of the large confidence interval for TeCB (Figure 3). However, given the tight confidence intervals for the other chemicals and judging from the maximum likelihood estimate of TeCB, it appears that kg is actually quite constant over the studied Kow range (Figure 3). Again, what we expected was a decrease with a slope around unity. It is possible that surfactants in the gut facilitate the transport across the stagnant water layer, thus influencing the relationship with Kow. This was also proposed for the absorption of lipids in the gut, a process following passive diffusion but with bile salts enhancing the transport (32). Consequences for EP in Risk Assessment. To our knowledge, this is the first time that, in earthworms, the uptake of organic chemicals from soil through the skin has been separated from uptake resulting from feeding on soil particles. The importance of the gut route increases with increasing hydrophobicity, and very hydrophobic chemicals (log Kow > 6) will mainly be absorbed from the gut contents. There is some additional uptake as a result of feeding on soil, but the deviation from EP with the soil is less than a factor of 1.3, which is well within the accuracy of risk assessment applications. The general fear that feeding leads to the invalidation of EP is thus unwarranted. The model presented here can adequately describe the experimental data, and the results are consistent with the diffusion mechanism for gut uptake (10, 11). However, in view of the small deviations, risk assessment can rely on EP, and specific modeling of the gut compartment is usually not necessary. Nevertheless, this model may be useful for specific cases, especially when the worms are not feeding on soil alone but on a diet that is specifically contaminated (e.g., manure from farm animals treated with pharmaceuticals or pesticide residues in leaf litter). In these situations, soil concentrations along with EP are insufficient to predict body residues.
Acknowledgments We thank the Laboratory for Inorganic Chemistry at RIVM for performing the lutetium measurements in soil and worms and the Institute for Risk Assessment Sciences (IRAS, Utrecht) for kindly providing the organic chemicals. Furthermore, we thank Martina Vijver for demonstrating the ligaturing procedure, Rob Baerselman for support in the experiments, and Willie Peijnenburg and Joop Hermens for reviewing drafts of this manuscript.
Supporting Information Available Model equations and data on checking the validity of the feeding parameters of Table 1. This material is available free of charge via the Internet at http://pubs.acs.org.
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Received for review January 20, 2003. Revised manuscript received May 7, 2003. Accepted May 12, 2003. ES0340578
