Novel aspects of uptake patterns, metabolite formation and toxicological responses in Salmon exposed to the organophosphate esters—Tris(2-butoxyethyl)- and tris(2-chloroethyl) phosphate
Graphical abstract
Introduction
Organophosphate esters (OPEs) such as tris(2-butoxyethyl) and tris(2-cloroethyl) phosphate have been increasingly applied to replace halogenated flame retardants such as poly-brominated diphenyl ethers (PBDEs) that are almost phased out of production due to their proven toxicity, bioaccumulative and persistent properties (Hung et al., 2015). OPEs account for about 15% of global flame-retardants and are applied mostly in plastics, electronic parts, textiles and rubber products. OPEs are released from these materials and are spread into environment with partially unknown consequences for ecosystems and human health (Reemtsma et al., 2008a, Reemtsma et al., 2008b; Leonards et al., 2011a, Leonards et al., 2011b). In order to address the environmental and human health risks of OPEs, there is a need for scientific data on their occurrence and fate in the environment and biota. TCEP was shown to produce reproductive effects with potential deleterious consequences for fertility in animals and humans (Liu et al., 2012; Jin et al., 2013; Ta et al., 2014). Particularly, individual steps of steroidogenic pathways were influenced by TCEP in juvenile salmon (Arukwe et al., 2016). Other OPEs such as tris(1,3-dichloro-2-propyl) phosphate (TDCPP) produced developmental effects in zebrafish during embryogenesis (Dishaw et al., 2014), reduced circulating thyroxin (T4) levels in zebrafish larvae (Kim et al., 2015), interfered with the expression of thyroid hormone (TH)-responsive genes in cultured chicken embryos (Farhat et al., 2013) and modulated steroid hormone levels in H295R human adrenocortical carcinoma cells (Liu et al., 2012).
In mammals, bis(2-chloroethyl) carboxymethyl phosphate, bis(2-chloroethyl) hydrogen phosphate and bis(2-chloroethyl)-2-hydroxyethyl phosphate glucuronides were identified as urinary metabolites of TCEP (European Union Risk Assessment Report, 2009). These findings suggest metabolic pathways that involve oxidative and hydrolytic reactions, as well as glucuronidation through phase-II metabolism (ibid). Reported toxicological effects such as neurological dysfunctions, adverse reproductive effects, and endocrine disruptive and systemic responses (European Union Risk Assessment Report, 2009; Liu et al., 2012), prompted the classification of halogenated phosphate esters as risky substances in the Priority List of the Council Regulation (EEC, Council Regulation, 1993). On the other hand, non-halogenated OPEs, such as TBOEP are more hydrophobic than chlorinated OPEs (Fries and Puttmann, 2003; Qian et al., 2014). Thus, their spread in the environment is more associated with particle transport, precipitation and sorption to carbon-rich surfaces (Xu et al., 2017). The degradation of TBOEP in air and water is slow compared to biodegradation by microbes. Thus, based on available data, accumulation in aquatic organisms will not be expected (European Union Risk Assessment Report, 2009). In animal experiments, repeated exposure of TBOEP at high doses produced alterations in the liver and nervous system, with females showing higher susceptibility to toxic effects than males (Xu et al., 2017). TBOEP has been classified as a moderately toxic compound to aquatic organisms as shown for developing zebrafish (Ma et al., 2016). In salmon, low to no effects on steroid hormone synthesis in brain and kidney were observed (Arukwe et al., 2016). Data on in vivo uptake kinetics and metabolism of TBOEP in aquatic animals has not been previously reported. Further, despite the widespread occurrence of OPEs in the aquatic environment (van der Veen and de Boer, 2012), data on their bioaccumulation, metabolism and non-lethal effects on aquatic biota, especially fish, are limited to non-existent. Risk assessment has mainly focused on human health and the application of in vitro systems such as human liver microsomes or S9 liver fractions (van den Eede et al., 2013, van den Eede et al., 2015a, van den Eede et al., 2015b) or the excretion of metabolites in human urine samples (Reemtsma et al., 2011).
A recent study that compared biota transfer of nine OPEs, reported TBOEP levels in both benthic and pelagic food web organisms in the range of 17 μg/kg ww (sculpin: Myoxocephalus scorpius) and 27 μg/kg ww (pouting: Trisopterus luscus) as the highest average concentrations (Brandsma et al., 2015). TCEP concentration was much lower, showing respective benthic and pelagic food web levels of 1 and 1.6 μg/kg ww in goby and herring (Brandsma et al., 2015). The toxicity and bioaccumulation of emerging contaminants (ECs) depend on their uptake, elimination and metabolism kinetics. Estimating bioaccumulation of ECs is crucial for food safety and ecosystem biomagnification perspectives. For risk assessment, a chemical bioaccumulation factor (BAF) is essential, either through experimentally based value or by estimation, based on the octanol-water partition coefficients (Kow) (TGD, 2003). Therefore, the aims of this study were to investigate the uptake, bioaccumulation, metabolism and metabolite formation in juvenile Atlantic salmon exposed to waterborne TBOEP and TCEP. Given the compound differences between these two OPEs, we hypothesized that exposure of juvenile salmon to TBOEP and TCEP will produce compound-specific differences in uptake and bioaccumulation patterns, resulting to potential formation of hydroxylated (OH) metabolites. TBOEP and TCEP were selected for the present study because they are commonly measured in a wide range of environmental and biota samples (both aquatic and terrestrial organisms) and with associated toxicity responses.
Section snippets
Chemicals and reagents
Tris(2-butoxyethyl) phosphate (94%) (TBOEP; [CH3(CH2)3OCH2CH2O]3P(O)) and Tris(2-chloroethyl) phosphate (97%) (TCEP; (ClCH2CH2O)3P(O)), from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Trizol™ reagent was purchased from Gibco-Invitrogen Life Technologies (Carlsbad, CA, USA). Direct-zol™ RNA MiniPrep RNA isolation kit was purchased from Zymo Research Corporation (Irvine, CA, USA). iScript cDNA Synthesis Kit and iTaq SYBR Green Supermix with ROX were purchased from BioRad Laboratories
Uptake and bioaccumulation patterns
Measured concentration of TCEP and TBOEP in salmon muscle, Cfish, exposed to nominal water concentration of 0.04, 0.2 and 1 mg/L for 7 days were in the range of 9.7 ± 2.2, 23.8 ± 6.3, 263.6 ± 34.1 and 4.3 ± 3.2, 161.4 ± 139.9 and 954.6 ± 69.5 μg/kg fresh weight (fw), respectively (Table 2). When the measured muscle concentrations were calculated on dry weight (dw) basis, TCEP was measured at 41.5 ± 11.4, 101.6 ± 28.5 and 1052.9 ± 236.3 μg/kg dw, while TBOEP was 19.3 ± 14.8-, 645.5 ± 520.2 and
Discussion
We have studied the uptake, metabolism and toxicological effects of waterborne halogenated and non-halogenated OPEs (TCEP and TBOEP, respectively) in juvenile salmon. In general, we show that both OPEs accumulated in fish muscle with estimated higher BCF for TBOEP, compared to TCEP. Further, metabolites were only detected in TBOEP exposure, showing that TCEP was resistant to metabolism in salmon. Seven phase I biotransformation TBOEP metabolites were detected, showing that hydroxylation, ether
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