Bioaccumulation and bound-residue formation of 14C-decabromodiphenyl ether in an earthworm-soil system

Journal of Hazardous Materials 321 (2017) 591–599

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Bioaccumulation and bound-residue formation of 14 C-decabromodiphenyl ether in an earthworm-soil system Lei Huang, Wei Wang ∗ , Sufen Zhang, Shenghua Tang, Pengfei Zhao, Qingfu Ye ∗ Institute of Nuclear-Agricultural Sciences, Zhejiang University, Hangzhou 310029, China

h i g h l i g h t s

g r a p h i c a l

a b s t r a c t

• The paper introduces the first infor-

• • • •

mation of bound residue of decabromodiphenyl ether(DecaBDE) in soil and earthworm. DecaBDE could be absorbed but not bio-accumulated in earthworm body. The distribution of DecaBDE in Earthworm (Pheretima guillemi) was evaluated. The accumulation of DecaBDE in earthworm was mainly through ingestion. No significant affections of DecaBDE-degrading onto the fate/transformation of DBDE in system.

a r t i c l e

i n f o

Article history: Received 9 April 2016 Received in revised form 15 September 2016 Accepted 19 September 2016 Available online 19 September 2016 Keywords: Decabromodiphenyl ether Bound residue Geophagous earthworm Soil Risk assessment

a b s t r a c t Decabromodiphenyl ether (DecaBDE) is one of the most frequently detected flame retardants in terrestrial environments. However, the fate of DecaBDE and its transport in an earthworm-soil system with and without a DecaBDE-degrading strain have rarely been evaluated. In this study, 14 C-DecaBDE was self-synthesized, and a DBDE-degrading strain, Rhodococcus erythropolis, was used in an earthworm-soil system. DecaBDE showed limited degradation and mineralization after 35 days of all treatments. The bound-residue (BR) formation in soil was <2.5% in the system containing earthworms, which was significantly higher (p < 0.05) than that observed in the absence of earthworms (<0.45%). DecaBDE could be adsorbed by the earthworms with a BSAF of ≤0.31. The distribution of 14 C-DecaBDE concentrations in the earthworm roughly followed the pattern of crop gizzard > digestive system > head > tail > body wall, suggesting that DecaBDE was mainly uptaken through ingestion. Up to 31% of the 14 C-DecaBDE in the earthworms was not extractable, revealing that the total concentration of accumulated 14 C-DecaBDE was underestimated. The results also showed that the presence of DecaBDE-degrading bacteria did not significantly affect the fate of DecaBDE and its accumulation in earthworms. The study indicates that the conventional assessment of the bioaccumulation and ecological effects of DecaBDE, which is based only on extractable concentrations, may underestimate the risks. © 2016 Elsevier B.V. All rights reserved.

∗ Corresponding authors. E-mail addresses: wei [email protected] (W. Wang), [email protected] (Q. Ye). http://dx.doi.org/10.1016/j.jhazmat.2016.09.041 0304-3894/© 2016 Elsevier B.V. All rights reserved.

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L. Huang et al. / Journal of Hazardous Materials 321 (2017) 591–599

1. Introduction Polybrominated diphenyl ethers (PBDEs) are an important class of brominated flame retardants (BFR) that are used extensively in both commercial and household products [1,2], but these compounds are not chemically bound to the products and tend to be released into the environment [3]. During the past few decades, PBDEs have been frequently detected in soil [2], sediments [4], sewage sludge [5], water, and air as well as animal/human tissues [6–8]. Because of their toxicity and adverse effects on wildlife and humans, the congeners of penta- and octa-BDEs have been classified as persistent organic pollutants and phased out of use in the European Union (EU) and the United States (US) [9]. Out of all PBDEs, decabromodiphenyl ether (DecaBDE) is the most important, and its production constitutes approximately 80% of the total world production of PBDEs [10,11]. With its extreme lipophilicity (with a Kow value of approximately 10), DecaBDE is generally considered less toxic and remains in use in parts of the US and many other countries [10,12]. However, the risks posed by DecaBDE are still a subject of ongoing discussion. Numerous studies on DecaBDE have been conducted in recent years to assess its environmental risks, such as bioavailability, persistence and toxicity [9,13,14]. Researchers have found that DecaBDE could be adsorbed by vegetables (such as alfalfa, radishes and pumpkins), and that the accumulated DecaBDE could be metabolized to hydroxylated PBDEs, which are more toxic than DecaBDE itself [15,16]. Wan et al. [12] observed the accumulation of DecaBDE in the liver, gills, and intestines of Chinese sturgeon and found a maximum concentration of >50 ng/g, and Riu et al. found that DecaBDE could be efficiently adsorbed and distributed in pregnant rats and further transformed to lower brominated metabolites as well as hydroxylated octa-BDE [9]. However, most of the existing studies on DecaBDEs are based on extractable residues (ER) that can be recovered by solvent extraction. When entering the environmental matrix or biota, organic contaminants might also be sequestrated in various manner, such as through strong ionic binding or through hydrogen, and as a result, these contaminants cannot be recovered through conventional exhaustive solvent extraction methods and are thus generally overlooked or just considered a detoxification approach [15,17,18]. Based on the potentially hazardous impact of the release and formation of bound residues (BR) from many xenobiotics (e.g., atrazine [19], nonylphenol isomers [20], TBBPAs, cycloxaprid [18], and carbamazepine [21]) and the limited number of studies on the bound-residue formation of PBDEs, we believe that it is necessary to gather more information regarding the formation of bound-residues from PBDEs in both the soil and the biota. Earthworms are important deposit-feeders in terrestrial ecosystems; they can accumulate many organic contaminants and are thus commonly regarded as important indicator organisms for risk assessment [13,22]. In fact, the residue and accumulation of PBDEs in earthworms were recently observed in soils from electronic waste-dismantling areas and soils amended with biosolids [23]. In addition, several laboratory studies have investigated the accumulation of DecaBDE in the earthworm Eisenia fetida (E. fetida) [13,22,24]. As suggested by a previously published manuscript, bioaccumulation is species-dependent in earthworms [25,26]. Thus, the bioaccumulation of DecaBDE in earthworms could also be affected by the species. However, limited information has been reported for other earthworms such as Pheretima guillelmi, a typical geophagous earthworm that processes large amounts of soil during its feeding and burrowing activities in soil. In addition to accumulation, earthworms also significantly impact the distribution, partition and degradation of xenobiotics [27,28]. For example, the presence of earthworms could decrease the formation of bound residues of isoproturon, dicamba and atrazine by

up to four-fold [19]; accordingly, the coexistence of earthworms and microorganisms increases the potential risks to ecological systems. As previously reported, several bacteria (i.e., Lysinibacillus fusiformis strain DB-1, Sphingomonas strain SS3 and B. xenovorans LB400) have been isolated and used for the remediation of DecaBDE contamination [29–32]. However, whether the distribution and partition of DecaBDE in the bodies of worms and the soil are affected by microorganisms, such as degrading strains, remains unknown. In this study, we aimed to elucidate the fate of DecaBDE and its transport in an earthworm-soil system with and without a DecaBDE-degrading microbial strain. Soil was amended with 14 Clabelled DecaBDE and a DecaBDE-degrading strain, Rhodococcus erythropolis, and suitable extraction and radio-autographic imaging techniques were used to determine DecaBDE transport and accumulation. 2. Materials and methods 2.1. Chemicals Decabromodiphenyl ether (14 C-DecaBDE)was uniformly radiolabeled with 14 C on one of its benzene rings and purified in the laboratory according to a previously published method [33,34]. High-performance liquid chromatography (Waters, Milford, MA, U.S.A.) analyses coupled with liquid scintillation counting (LSC, TriCarb2910 TR, Perkin-Elmer, and Turku, U.S.A.) showed that both the radiochemical and the chemical purity of the synthesized 14 CDecaBDE was >98%. A stock solution of 14 C-DecaBDE was prepared in tetrahydrofuran at a concentration of 474 mg/L and a final specific activity of 1.25 mCi/mmol. 2.2. Soil A typical paddy soil (the top 0–10 cm) was collected from Hangzhou, Zhejiang Province, China. The soil was characterized as fluvio-marine yellow loamy soil with a pH of 7.02, a total organic carbon content of 3.05%, a cation exchange capacity of 10.83 cmol kg−1 , and a particle distribution of 8% clay, 71% silt and 21% sand. After air drying and sieving (<2 mm), the soil was kept in the dark at room temperature until use. 2.3. Experimental organism Adult earthworms (Pheretima guillelmi) with an average body weight of approximately 3.25 g were purchased from an earthworm culture farm in Jirong, Jiangsu, China. The earthworms were acclimated in clean soil with a water-holding capacity (WHC) of 60% in the laboratory for seven days at room temperature (24–25 ◦ C) before use. 2.4. Microorganism The methods used for the enrichment, isolation, and identification of the strain HNS209 are available in the Supporting information. The HNS209-1 strain can efficiently transform more than 20% of DecaBDE within seven days when using DecaBDE as its sole carbon source (Fig. S1). The strain was characterized as Rhodococcus erythropolis based on its 16S rDNA (Fig. S2), cultivated aerobically at 30 ◦ C in Luria-Bertani (LB) broth and stored in 20% glycerol at −20 ◦ C. 2.5. Earthworm exposure experiments The earthworm exposure experiments were performed according to the method described by Liu et al. [35] with some

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modifications. Briefly, 500-g batches of soil were pre-incubated in the dark for 10 days at 30 ± 1 ◦ C and a WHC of 40% to activate the soil microbes. To exclude potential toxic effects on soil microorganisms, 0.5 mL of 14 C-DecaBDE stock solution was dispensed into each 10-g subsample in a 50-mL glass beaker. The spiked soil was then thoroughly mixed and left in a fume hood until the solvent was evaporated, and the premixed soil samples were further mixed with another 490 g of soil. The homogeneity of the 14 C-DecaBDE distribution in the soil was validated by determining the radioactivity from the combustion of triplicate soil samples (1.0 g) on an OX501 biological oxidizer (R. J. Harvey Instruments, Hillsdale, NJ, U.S.A.) at 900 ◦ C for 4 min. The initial concentration of DecaBDE was 4.7 ␮g g−1 (dry soil). The treated soil was then transferred to a 1-L flask, and the soilwater content was adjusted to a WHC of 60% by the addition of water. The 14 C-DecaBDE exposure experiment was then initiated by placing 12 adult earthworms into the flasks. All of the exposure experiments followed the OECD 207; the earthworms were weighed and placed between two wet filter papers for 24 h to allow gut clearance [13]. The incubation flasks were attached to an air flow-through apparatus with a series of airtight flasks containing adsorbent solutions. In the upstream section, as shown in Fig. 1, one flask containing 50 mL of 5 M NaOH was used to eliminate CO2 ; one flask containing 30 mL of distilled water was included to humidify the air; and one empty flask was used to prevent backflow. In the downstream section, a total of four flasks were included: one empty flask was used to prevent backflow; one containing 20 mL of ethylene glycol and 0.05 M H2 SO4 to trap alkaline volatile products and two containing 20 mL of 0.2 M NaOH to adsorb mineralized 14 CO2 and acidic volatiles. The experiment was performed in a climate chamber with a 16-h light:8-h dark cycle at 25 ± 1 ◦ C. Water loss from the soil during the experiment was determined by weighing, and this loss was then compensated by the addition of an equivalent amount of distilled water. The adsorbed solutions were periodically replaced with fresh solutions. The sampled adsorption solutions were mixed with cocktail II (the composition of cocktail II is available in the Supporting information) and analyzed by ultra-low-level liquid scintillation counting (ULLSC Quatalus-1220, Perkin-Elmer, and Turku, U.S.A). The samples were maintained in the dark for 24 h prior to the measurement of radioactivity to eliminate chemiluminescence. Four different soil treatments were included in the exposure experiment: active soil (AS); sterile soil (SS); active soil with the strain (ASS), and sterile soil with the strain (SSS). For the treatments with the microbial agent, HNS209-1 cells were harvested by centrifugation (1, 2000 rpm, 4 ◦ C, and 10 min) and washed twice with 0.85% (w/w) sodium chloride solution, and approximately 0.15g of HNS209-1 cells with sterile water was then added when needed.

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was readjusted to 25 mL, a 1-mL aliquot of extract was collected to measure the 14 C-ER in the earthworms. At each sampling time, 10-g aliquots of soil (dry weight) were sampled and extracted with acetonitrile (20 mL, thrice). After three additions of 10 mL of 2-propanol for 30 min, the sample was extracted in an ultrasonic bath with 40 mL of hexane/MTBE (1:1; v/v). After each extraction, the sample was mechanically shaken for 10 min and then centrifuged at 8000 rpm for 5 min in an Eppendorf 5840R centrifuge (Eppendorf, Hamburg, Germany). All of the extracts were combined, and the volume was reduced to 50 mL. One milliliter of the extract solutions was mixed with cocktail I (the composition is available in the Supporting information) to measure the DecaBDE concentration in the earthworms and soil by LSC, and the radioactive portions of the extracts were defined as the ER. The soil/earthworm samples remaining after the last step of the extraction were air-dried in a fume hood and combusted on an OX501 biological oxidizer at 900 ◦ C for 4 min to generate 14 CO2 . The emerged 14 CO2 was trapped in cocktail I and then measured for radioactivity by LSC to derive the fraction of 14 C-BR. The lipid content was determined from the extracts of three earthworms using a previously developed gravimetric method [35]. 2.7. HPLC-LSC analysis All of the remaining solutions were concentrated to dryness (approximate 5 mL) under a vacuum using a rotary evaporator at 35 ◦ C. The concentrated and washed solutions were merged and passed through 0.45-␮m filter, and the organic extracts were evaporated to near dryness under nitrogen and then recovered in a 1.0-mL aliquot of tetrahydrofuran. The 1.0-mL extract was centrifuged at 12,000 rpm for 10 min, and the supernatant was then passed through a 0.22-␮m filter and stored at 4 ◦ C until analysis. A 20-␮L aliquot of the sample extracted from the soil was injected into an HPLC system equipped with a Waters 2695 multisolvent delivery unit, a Waters 2998 photodiode array (PDA) detector (Waters, Milford, MA, U.S.A.) equipped with a Diamonsil C18 column (5 ␮m, 250 × 4.6 mm, Dikma Technologies, Lake Forest, CA, U.S.A.) and a C18 protection column (5 ␮m, 30 × 4.6 mm, Dikma Technologies). The HPLC conditions were described previously [38]. The column temperature was maintained at 30 ± 1 ◦ C. In the mobile phase, methanol-water (95:5, v:v) was used as an isocratic eluent at a flow rate of 1.0 mL min−1 [39], and the detector was set to 226 nm. The post-column eluent was collected by a Waters Faction Collector III (Waters, Milford, MA, U.S.A.) in 20-mL scintillation vials. Each post-column eluent fraction was collected by minutes and measured for radioactivity using a LSC after the addition of 10 mL of cocktail I. 2.8. Radioautographic imaging

2.6. Extraction and analysis Both the soil and the earthworms were periodically sampled for analysis. The sampled worms were placed between two wet filter papers for 24 h to allow gut clearance and then extracted using a previously reported method [24,36] with slight modifications. Preliminary experiments and published papers [20,22,37] showed that 24 h is sufficient for gut clearance. Briefly, the earthworms were freeze-dried and pulverized using a mortar, and an aliquot of approximately 0.2 g of tissue was mixed with 2 mL of 2-propanol for 30 min and then extracted thrice in an ultrasonic bath with 10 mL of hexane/methyl tert-butyl ether (MTBE) (1:1; v/v). After the ultrasonic bath, the sample was placed in a static state to allow equilibration for another 30 min. After extraction, the solution was filtered, combined and reduced by a rotary evaporator (Eyela SB-1000, Eyela, Tokyo, Japan) at 40 ◦ C. After the volume

After exposure to 14 C-DecaBDE for 35 days, four worms were sampled and subjected to whole-body autoradiography analysis. The worms were thoroughly rinsed with water and placed between two wet filter papers for 24 h to allow gut clearance. The earthworms were then air-dried in a fume hood and subjected to whole-body autoradiography analysis using a Bioimaging Analyzer System (Fuji BAS 1800, Fuji Photo Film, Tokyo, Japan), as described by Wang et al. [40]. 2.9. Statistical analysis The bioaccumulation data were fitted to the following twocompartment model described by Landrum et al. [41]: Cet =

 ks Cs  −t e − e−kd t kd − 

(1)

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Fig. 1. Experimental setup for the earthworm incubation experiments.

In Eq. (1), Cet is the 14 C concentration in the earthworms at uptake time t (Bq g−1 of organism wet weight); Cs is the initial radioactivity in the soil (Bq g−1 of dry soil); ks is the uptake rate constant (g of dry soil g−1 of organism); kd is the elimination rate constant (d−1 ); and  is the rate constant for the decrease in the bioavailability of the compound (d−1 ). The biota-soil accumulation factor (BSAF) was used to evaluate the accumulation of DecaBDE in Pheretima guillelmi. This factor was normalized based on the soil organic carbon and earthworm lipid contents and calculated according to Eq. (2): BSAF =

Ce /flipid Cs /foc

(2)

where Ce and Cs are the total radioactivity concentrations in the earthworms [Bq (g dry earthworm)−1 ] and soil [Bq (g dry soil)−1 ], respectively, flipid is the lipid content in the earthworms (%), and foc is the organic carbon content in the soil (%). All of the samples were collected in triplicate, and the arithmetic means and standard errors (means ± SEM) were calculated by repeated measurements. Significance was determined by one-way ANOVA at ␣ = 0.05 using SPSS 20.0 (IBM SPSS Statistics, Armonk, NY, U.S.A.), and graphs and the averaged values were fitted and generated using Origin 9.0 (MicroCal Software, Northampton, MA, U.S.A.). 3. Results and discussion 3.1. Degradation and mineralization of DecaBDE in aerobic soil Throughout the 35-day incubation period, the traps from all of the treatments (with and without the strain) showed negligible radioactivity for volatile products or cumulative 14 CO2 production. In addition, the radioactivity analysis after HPLC-LSC fractionation showed that DecaBDE did not undergo appreciable degradation during this incubation period; specifically, approximately 98% of the radioactivity from the HPLC elution was recovered as the parent compound. Furthermore, the parent compound (DecaBDE) presented the only radioactivity and peaked at a retention time of 32 min after HPLC-LSC analysis. The observed results generally demonstrated that DecaBDE is stable in aerobic soil for an incubation term of ≤35 d, and these results are generally consistent with previously published reports that found no significant biological transformation of DecaBDE in test groups coexisting with Pb [42]. Similar relatively long DecaBDE degradation half-lives have been previously observed [43]; in fact, a previous study found a

pseudo-first-order rate of 1 × 10−3 and an estimated half-life of approximately 700 d. It is known that 14 C-DecaBDE is labile based on its chemical structure, and this compound tends to readily react with nucleophiles and could be reduced by hydride reagents, such as sodium borohydride. However, due to the great hydrophobicity of DecaBDE (with an estimated log Kow value of approximately 10), it is generally considered to be very persistent in the environmental matrix, which corresponds to a low freely dissolved concentration in aqueous solutions and limited availability for biological/chemical transformation [23,44]. The extremely low mineralization rate also indicates that the DecaBDE benzoate ring is resistant to cleavage, which is consistent with the results of previous studies [9,42,45]. In the past decades, numerous studies have been conducted to understand the degradation or metabolism of PBDEs [32,45,46], and degradation products, such as methylated, methoxylated, debrominated and hydroxylated PBDEs, have been observed [9,47,48]. However, none of the reported products result from transformation due to cleavage of the benzoate ring, and this finding is also in agreement with the negligible mineralization rate observed in this study. The present study also found that the introduction of a DecaBDEdegrading strain did not significantly contribute to the degradation or mineralization of DecaBDE. It is known that benzoate rings are the only carbon sources for the tested strain when DecaBDE is used as its sole carbon source, and cleavage of the benzoate ring should be included in the microbial degradation process. Therefore, we hypothesized that the observed inhibition of DecaBDE degradation/mineralization is very likely due to a strong adsorption of DecaBDE by soil particles [4], corresponding to the inhibition of microbial degradation [32]. This inhibition also indicates that the biodegradation ability of an artificially isolated strain might decrease due to adsorption by soil particles following application to the soil or field. Similar results have been reported previously [29,49], indicating that the effectiveness of a bioremediation method for DecaBDE contamination in aerobic soil should be combined with other treatments. 3.2. Formation of bound residues in soil The 14 C-DecaBDE residues remaining in the soil were further recognized as extractable and bound residues by extreme solvent extraction and subsequent bio-combustion. As shown in Fig. 2, more than 97% of the total radioactivity in the soil was recovered as ER, and the fraction of BR was consistently <2.5%, regardless of the treatment. In general, 14 C-DecaBDE tended to rapidly form bound

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Fig. 2. Dynamics of bound and extractable radioactivity of 14 C-DecaBDE in (A) active soil (AS), (B) sterile soil (SS), (C) active soil supplemented with the strain (ASS), and (D) sterile soil supplemented with the strain (SSS). The values are the means with standard deviations (n = 3).

residues after its application to the soil (2 d), and the fraction of bound radioactivity remained steady at a rate of approximately 1% during the first 10 days of incubation with all of the treatments (Fig. 2). After 10 days, some limited effects of BR formation by the microbes were observed, with the 14 C-BR level gradually increasing to 2.50%, 2.47%, 2.33% and 1.71% with the AS, SS, ASS and SSS treatments, respectively, as shown in Fig. 2. The strong adsorption, low degradation rate and limited bound residue formation of DecaBDE in aerobic soil generally demonstrated that the interaction between soil and DecaBDE likely occurs through mechanisms such as van der Walls forces and hydrophobic partitioning, which are relatively more reversible by solvent extraction than stronger covalent bonding or sequestration [43]. We also conducted a parallel experiment to elucidate the effect of earthworm activities on the formation of BR of DecaBDE. As shown in Fig. S3, only 0.45% of the introduced 14 C-DecaBDE formed BR in the soil without earthworms, which is statistically markedly lower (p < 0.05) than that found in the treatment with earthworms (with BR values ranging from 1.17% to 2.5%). The results indicated that the earthworm activities could enhance BR formation of DecaBDE in soil. We hypothesize that this finding could be attributed to an increase in the microbial group numbers, a change in the microbial colonies, and an increase in the soil organic matter content induced by the earthworms [50,51].

3.3. Accumulation of 14 C-DecaBDE in earthworms All of the earthworms survived the exposure experiment, and the 14 C-DecaBDE originally spiked in the soil was found to be gradually adsorbed by the earthworms over time. As shown in Table 1 and Fig. 3, 14 C-DecaBDE was rapidly adsorbed and accumulated by the earthworms. After exposure to 14 C-DecaBDE for two days, the total accumulated radioactivity in the earthworms subjected to the AS, SS, ASS and SSS treatments reached 125, 111, 122 and 99 Bq g−1 , respectively, accounting for 72–82% of the peak values (Fig. S5). From the second day until the end of the incubation, the corresponding values gradually increased to 163, 155, 153, and 147 Bq g−1 , respectively. Because no metabolites were detected in this experiment, the concentration of radioactivity in the earthworm could be equivalent to the mass concentration of DecaBDE, as shown in Table S1, which corresponds to a final concentrations of 3.06-3.39 ␮g g−1 in the earthworms (dry weight) at the end of the exposure period (Table S1). The accumulated concentration of DecaBDE in the earthworms fit the two-compartment accumulation model described in Eq. (1) (r2 ≥ 0.87, Table 1), and the curve is shown in Fig. S4. The e−␭t term could be used to describe the amount of 14 C-DecaBDE that became biologically unavailable. A previously published study found that soil aging had no effect on DecaBDE due to its low mobility in the soil [22]; thus, the decreasing bioavailability of the compound becomes

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Table 1 Distribution of 14 C-DecaBDE radioactivity in earthworms and soil during the incubation period (Bq g−1 dry weight). Treatment

Distribution

0

5

10

15

25

35

active soil (AS)

soil earthworm

227.53 0.00

216.72 ± 13.70 132.30 ± 7.44

212.54 ± 7.38 143.14 ± 10.34

209.37 ± 12.92 146.50 ± 14.73

206.80 ± 6.88 152.30 ± 3.62

199.89 ± 8.05 162.97 ± 5.18

active soil with strain (ASS)

soil earthworm

227.53 0.00

209.60 ± 12.34 128.79 ± 5.88

203.62 ± 11.41 134.40 ± 12.52

202.46 ± 11.38 136.86 ± 20.24

195.96 ± 16.94 146.95 ± 10.78

188.89 ± 8.56 153.45 ± 16.11

sterile soil (SS)

soil earthworm

227.53 0.00

211.35 ± 2.57 115.80 ± 12.72

205.13 ± 3.58 122.86 ± 9.13

204.30 ± 3.80 134.68 ± 5.18

202.38 ± 6.39 142.16 ± 12.08

199.36 ± 6.36 155.13 ± 9.68

soil earthworm

227.53 0.00

210.67 ± 8.07 102.84 ± 15.98

205.85 ± 7.82 114.17 ± 7.22

199.70 ± 8.38 117.03 ± 6.05

188.31 ± 7.91 132.22 ± 11.21

183.18 ± 3.36 147.02 ± 24.29

– sterile soil with strain (SSS)

Fig. 3. Accumulation of bound and extractable radioactivity in earthworms exposed to 14 C-DecaBDE in (A) active soil (AS), (B) sterile soil (SS), (C) active soil supplemented with the strain (ASS), (D) sterile soil supplemented with the strain (SSS). The values are the means with standard deviations (n = 3).

Table 2 Fitting parameters of the two-compartment model for the accumulation of 14 CDecaBDE residues in the earthworm Pheretima guillelmi during different soil treatments. Treatments

ks

kd

r2

active soil (AS) active soil with strain (ASS) sterile soil (SS) sterile soil with strain (SSS)

0.57 ± 0.10 0.60 ± 0.14 0.45 ± 0.13 0.37 ± 0.12

0.87 ± 0.16 0.97 ± 0.23 0.75 ± 0.50 0.67 ± 0.23

0.971 0.963 0.913 0.88

negligible. The earthworm 14 C-DecaBDE uptake rate from the soil (ks ) and the elimination rate (kd ) of the total residues from the earthworms were obtained and are shown in Table 2. The ks and kd measurements were averaged to obtained values of 0.37–0.60 and 0.67–0.97, respectively, and the fact that the kd values were signifi-

cantly greater than the ks values demonstrated that the depuration of 14 C-DecaBDE was faster than its accumulation in the earthworm body. This result suggested that the accumulation of DecaBDE by geophagous earthworms (Pheretima guillelmi) was mainly through the ingestion of soil particles while processing large amounts of soil. This result was consisted with that reported Wang, Ji, Jiang and Chen [25], who suggested that contamination uptake by Metaphire guillelmi (M. guillelmi) was largely affected by gut processes. The BSAF were calculated using the concentrations of 14 CDecaBDE normalized relative to the earthworm lipid contents and the soil organic carbon contents. As shown in Table 3, the mean BSAF values for DecaBDE were 0.19–0.31, and similar BSAF values have been obtained for DecaBDE and its chlorinated counterparts in soils of different types in previous studies [22,23,26,52]. The results demonstrated that although the DecaBDE in soil is adsorbable

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Fig. 4. Schematic radioautographic imaging of the earthworm after exposure to 14 C-DecaBDE for 35 days.

Table 3 Biota-soil accumulation factors (BSAF) for DecaBDE in earthworms (Pheretima guillelmi) during the incubation period. The values are the means with standard deviations (n = 3). Day

Active soil (AS)

Active soil with strain (ASS)

Sterile soil (SS)

Sterile soil with strain (SSS)

2 5 8 10 15 20 25 30 35

0.24 ± 0.01 0.25 ± 0.02 0.27 ± 0.03 0.27 ± 0.02 0.28 ± 0.02 0.28 ± 0.01 0.29 ± 0.06 0.28 ± 0.05 0.31 ± 0.01

0.23 ± 0.00 0.24 ± 0.02 0.24 ± 0.03 0.25 ± 0.02 0.26 ± 0.01 0.28 ± 0.03 0.28 ± 0.02 0.28 ± 0.01 0.29 ± 0.01

0.21 ± 0.00 0.22 ± 0.02 0.22 ± 0.01 0.23 ± 0.01 0.25 ± 0.05 0.27 ± 0.04 0.27 ± 0.02 0.28 ± 0.05 0.29 ± 0.02

0.19 ± 0.01 0.19 ± 0.01 0.20 ± 0.03 0.22 ± 0.01 0.22 ± 0.00 0.23 ± 0.01 0.25 ± 0.02 0.26 ± 0.02 0.28 ± 0.01

for terrestrial organisms, it does not tend to be bioaccumulated (a compound with a BSAF value of <3 is typically considered non-bioaccumulative) [53]. However, the BSAF values can vary in different soils or at different DecaBDE concentrations. For instance, Nyholm et al. [22] found BSAF values of 0.03, 0.4 and 1.6 for soils spiked with DecaBDE at high, medium and low levels, respectively. Our results showed that the BSAF in the earthworm Pheretima guillelmi was close to the level found for E. fetida in soil contaminated with 100 ng g−1 DecaBDE. This result indicated that the bioaccumulation ability of geophagous earthworms in DecaBDE-contaminated soil might be greater than that of E. fetida. The use of 14 C radiolabeling enabled the detection of bound residues in the worms, and the results showed that after adsorption, 14 C-DecaBDE tended to rapidly form bound residues in the worm body (Fig. S5), accounting for 20–31% of the total accumulated radioactivity at the beginning of the incubation (2 d) and rapidly reaching its peak value within five days (25–29%). In general, quite a considerable amount of the adsorbed 14 C-DecaBDE was present as bound residues in the worm body, and this fraction could reach 31%. Consequently, the conventional solvent extraction method based

only on extractable concentrations may result in underestimations and lead to inaccurate evaluations of the bioaccumulation/risks of PBDEs. Thus, this method should be further compensated by an analysis of bound residue formation. No statistically significant differences were observed among the soils with different microbial actives, indicating that the microorganism had a limited effect on the bioavailability and bioaccumulation of 14 C-DecaBDE from the soil to the earthworms (p > 0.5). The limited effects of the microbes on bioavailability were consistent with the inhibition of DecaBDE degradation/mineralization, which was actually attributed to its strong adsorption to soil particles.

3.4. Distributions of 14 C-DecaBDE in earthworm tissues After exposure to 14 C-DecaBDE in soils for 35 days, the distribution of 14 C radioactivity in earthworm tissues was illustrated through quantitative whole-body autoradiography. As shown in Fig. 4, the 14 C-DecaBDE in the soil was adsorbed by the earthworm and distributed throughout all of its tissues, indicating the

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bioaccessibility of DecaBDE to soil invertebrates. According to the earthworm taxonomy recommended by Fernandez et al., the body of the earthworm is composed of a mouth, head, crop gizzard intestine, digestive system, body wall and tail (as shown in Fig. 4) [54]. The autoradiography demonstrated that most of the adsorbed 14 C-DecaBDE radioactivity was concentrated in the crop gizzard intestine followed by the digestive system and the head, and the lowest levels of radioactivity were observed in the tail and body wall (Fig. 4). According to Jager et al., earthworms are exposed to DecaBDE in soils both via dermal contact with soil water and via soil ingestion and subsequent uptake from the gut [55]. In this study, the 14 C-DecaBDE distribution in worm tissues, which followed the pattern of crop gizzard > digestive system > head > tail > body wall, demonstrated that the uptake of DecaBDE by the worms was mainly through ingestion and gut digestion rather than adsorption through the body wall. These results are consistent with those reported in a previously published paper that also suggested that the bioaccumulation of atrazine by P. guillelmi is largely affected by gut processes [25]. The observed result was reasonable and consistent with the compound’s large molecular size and high hydrophobicity, which results in a very low concentration in the soil water and inaccessibility to soil organisms [44].

4. Conclusion In general, this study provides important data regarding the fates of DecaBDE in a typical soil-terrestrial organism system with and without a DecaBDE-degrading microbial strain and the potential transfer of DecaBDE through the terrestrial food chain. The results demonstrate that DecaBDE does not tend to be degraded or mineralized, and the bound residue formation in aerobic soil was higher in the presence of earthworms (p < 0.05), a finding that could be attributed to increases in the quantity of soil microbes. Most of the DecaBDE in the soil existed as extractable residues (> 95%), suggesting that the strong adsorption of DecaBDE to the soil was mainly through mechanisms such as Van der Walls forces and hydrophobic partitioning instead of stronger covalent bonding or sequestration. Facilitated by 14 C labeling and auto-radiographing, the present study revealed the first illustration of the distribution of 14 C-DecaBDE in earthworms (Pheretima guillelmi). The distribution of 14 C-DecaBDE in the earthworm body followed the pattern of crop gizzard > digestive system > head > tail > body wall, indicating that the uptake of DecaBDE by these earthworms is mainly through ingestion. Quite a considerable amount of the DecaBDE residues in the worm are found as bound residues (up to 31%), suggesting that the conventional assessment method, which is based only on extractable concentrations, underestimates the total concentration of the accumulated compound. No significant contribution of the soil microbes to the fate/transformation of DecaBDE in the soil-earthworm system was observed, even in the presence of a strain capable of degrading DecaBDE when it is used as the sole carbon source. The present study demonstrates that the conventional assessment system for bioaccumulation and risk, which is based on extractable concentrations, may underestimate the risks and should therefore be improved. The results also reveal that the DecaBDE-contaminated soil particles adsorbed and distributed in the geophagous earthworm digestive system should receive more attention and require further study.

Acknowledgments This work was financially supported by the National Natural Science Foundation of China (Nos. 21207113 and 21577120) and the China Postdoctoral Science Foundation.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhazmat.2016.09. 041.

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