Fate and O-methylating detoxification of Tetrabromobisphenol A (TBBPA) in two earthworms (Metaphire guillelmi and Eisenia fetida)

Highlights

• Considerable amounts of bound residues of TBBPA formed rapidly in the earthworms.

• M. guillelmi accumulated more TBBPA via gut than E. fetida via skin uptake.

• M. guillelmi had a higher potential to transform TBBPA than did E. fetida.

• O-methylation was a metabolic pathway for TBBPA detoxification in the earthworms.

Abstract

Tetrabromobisphenol A (TBBPA) is the world's most widely used brominated flame retardant but there is growing concern about its fate and toxicity in terrestrial organisms. In this study, two ecologically different earthworms, Metaphire guillelmi and Eisenia fetida, were exposed to soil spiked with ¹⁴C-labeled TBBPA for 21 days. M. guillelmi accumulated more TBBPA than E. fetida, evidenced by a 2.7-fold higher ¹⁴C-uptake rate and a 1.3-fold higher biota-soil accumulation factor. Considerable amounts of bound residues (up to 40% for M. guillelmi and 18% for E. fetida) formed rapidly in the bodies of both earthworms. ¹⁴C accumulated mostly in the gut of M. guillemi and in the skin of E. fetida, suggesting that its uptake by M. guillelmi was mainly via gut processes whereas in E. fetida epidermal adsorption predominated. The TBBPA transformation potential was greater in M. guillelmi than in E. fetida, since only 5% vs. 34% of extractable ¹⁴C remained as the parent compound after 21 days of exposure. Besides polar metabolites, the major metabolites in both earthworms were TBBPA mono- and dimethyl ethers (O-methylation products of TBBPA). Acute toxicity assessments using filter paper and natural soil tests showed that the methylation metabolites were much less toxic than the parent TBBPA to both earthworms. It indicated that earthworms used O-methylation to detoxify TBBPA, and M. guillelmi exhibited the higher detoxification ability than E. fetida. These results imply that if only the free parent compound TBBPA is measured, not only bioaccumulation may be underestimated but also its difference between earthworm species may be misestimated. The species-dependent fate of TBBPA may provide a better indicator of the differing sensitivities of earthworms to this environmental contaminant.

Introduction

Tetrabromobisphenol A (TBBPA) is widely used as an additive or reactive component in polymers and electronics and accounts for ∼60% of the total brominated flame retardant (BFR) market (Cruz et al., 2015). TBBPA has been frequently detected in the environment, including its bioaccumulation in aquatic organisms, plants, and humans (Cariou et al., 2008, Covaci et al., 2009, Cruz et al., 2015, Li et al., 2011, Liu et al., 2016, Sun et al., 2014). However, there is little information on its occurrence in terrestrial animals. Despite its more extensive application, relatively low concentrations of TBBPA generally at ppb level have been measured in soils, sediments and biota (EFSA, 2011, Luigi et al., 2015, Morris et al., 2004), and occasionally the concentrations at ppm level were observed in contaminated sites (Covaci et al., 2009, Liu et al., 2016), such that there has been less concern about TBBPA than other major BFRs (Cruz et al., 2015).

Conventional assessment methods based on solvent extraction may have greatly underestimated the real accumulation of TBBPA as they consider only the extractable fraction of the parent compound and ignore the fact that TBBPA can form non-extractable (bound) residues in soil/sediment and biota. Li et al., 2015a, Li et al., 2015b reported that bound ¹⁴C accounted for >50% of the initially applied ¹⁴C-TBBPA in soil and sludge. Bound residues of organic compounds can also be formed by covalent binding to the cellular components of organisms (Shan et al., 2010). For example, many organic pollutants rapidly formed large amounts of bound residues in earthworms (Belden et al., 2011, Huang et al., 2017, Liu et al., 2015, Sarrazin et al., 2009, Shan et al., 2010). In addition, because TBBPA is chemically reactive it undergoes biotransformation, and several biotransformation pathways have been described (An et al., 2011, George and Häggblom, 2008, Li et al., 2014, Li et al., 2015a, Li et al., 2015b, Liu et al., 2013, Sun et al., 2014). A major transformation pathway is the O-methylation of TBBPA under oxic conditions to form TBBPA mono- and dimethyl ethers (MeO-TBBPA and diMeO-TBBPA, respectively). In fact, diMeO-TBBPA was detected at even higher levels than TBBPA in mussels (Watanabe et al., 1983) and sediments (Sellström and Jansson, 1995), although its origin in the mussels and sediments is unclear. Studies of the toxicity of TBBPA metabolites are important to better understand the mechanisms of TBBPA toxicity in terrestrial and other organisms and to accurately evaluate the environmental risks of TBBPA. Previous reports demonstrated differences in the toxicity of the debromination and methylation metabolites of TBBPA vs. the parent compound in aquatic organisms (Debenest et al., 2010, McCormick et al., 2010), but whether this is also the case in terrestrial organisms is unknown. While it seems likely that the fate and metabolism of organic compounds will determine their specific toxicity in organisms, this has yet to be demonstrated for TBBPA.

Earthworms occupy an important position in the terrestrial ecosystem and are thus a frequently used bioindicator for soil pollution risk assessments. The bioaccumulation and toxicity of xenobiotics are often studied in epigeic Eisenia fetida, a standard test species (OECD, 2004, OECD, 2010). However, conclusions based only on the results from E. fetida are unlikely to reflect the true risk of soil pollutants because this earthworm species is not typically found in mineral soils and its sensitivity to pollutants is very different from that of other ecological earthworm groups (Chen et al., 2017, Fourie et al., 2007, Pelosi et al., 2013, Qiu et al., 2014). Thus, soil-dwelling, local earthworm species may be better indicators of soil health. Metaphire guillelmi is an anecic and geophagous species widely found in China and the internal behavior of several chemicals in its body strongly differed from that in E. fetida (Chen et al., 2017, Li et al., 2016, Wang et al., 2014). In a previous study we showed that M guillelmi used strategies different from those of E. fetida with respect to the uptake and subcellular distribution of cadmium, such that it was much more sensitive to cadmium toxicity than E. fetida (Chen et al., 2017). M. guillelmi also differed from E. fetida in its uptake and depuration of organic pollutants, as demonstrated for hexabromocyclododecane (HBCD) (Li et al., 2016) and atrazine (Wang et al., 2014). Thus, parallel studies in M. guillelmi and E. fetida will provide important information on species-specific sensitivity to TBBPA and the respective underlying mechanisms.

In this work, we used the radio-tracer ¹⁴C-TBBPA to investigate the uptake kinetics of TBBPA in M. guillelmi and E. fetida. By following the tissue distribution of the radioactivity in both species, we were able to identify the different routes of TBBPA uptake. In addition, we characterized the metabolites of TBBPA and determined the kinetics of their formation in the two earthworm species to establish the relevant metabolic pathways. Finally, we evaluated the toxicity of TBBPA and its main metabolites in the earthworm bodies. Our results will provide insights into the internal mechanisms of TBBPA toxicity dependent on earthworm species.