Elsevier

Aquatic Toxicology

Volume 196, March 2018, Pages 146-153
Aquatic Toxicology

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

https://doi.org/10.1016/j.aquatox.2018.01.014Get rights and content

Abstract

Given the compound differences between tris(2-butoxyethyl)- and tris(2-cloroethyl) phosphate (TBOEP and TCEP, respectively), we hypothesized that exposure of juvenile salmon to TBOEP and TCEP will produce compound-specific differences in uptake and bioaccumulation patterns, resulting in potential formation of OH-metabolites. Juvenile salmon were exposed to waterborne TCEP or TBOEP (0.04, 0.2 and 1 mg/L) for 7 days. The muscle accumulation was measured and bioconcentration factor (BCF) was calculated, showing that TCEP was less accumulative and resistant to metabolism in salmon than TBOEP. Metabolite formations were only detected in TBOEP-exposed fish, showing seven phase I biotransformation metabolites with hydroxylation, ether cleavage or combination of both reactions as important metabolic pathways. In vitro incubation of trout S9 liver fraction with TBOEP was performed showing that the generated metabolite patterns were similar to those found in muscle tissue exposed in vivo. However, another OH-TBOEP isomer and an unidentified metabolite not present in in vivo exposure were observed with the trout S9 incubation. Overall, some of the observed metabolic products were similar to those in a previous in vitro report using human liver microsomes and some metabolites were identified for the first time in the present study. Toxicological analysis indicated that TBOEP produced less effect, although it was taken up faster and accumulated more in fish muscle than TCEP. TCEP produced more severe toxicological responses in multiple fish organs. However, liver biotransformation responses did not parallel the metabolite formation observed in TBOEP-exposed fish.

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

References (47)

  • S. Kim et al.

    Thyroid disruption by triphenyl phosphate, an organophosphate flame retardant, in zebrafish (Danio rerio) embryos/larvae, and in GH3 and FRTL-5 cell lines

    Aquat. Toxicol.

    (2015)
  • X. Liu et al.

    Endocrine disruption potentials of organophosphate flame retardants and related mechanisms in H295R and MVLN cell lines and in zebrafish

    Aquat. Toxicol.

    (2012)
  • Z. Ma et al.

    Effects of tris (2-butoxyethyl) phosphate (TBOEP) on endocrine axes during development of early life stages of zebrafish (Danio rerio)

    Chemosphere

    (2016)
  • D.J. McGoldrick et al.

    Organophosphate flame retardants and organosiloxanes in predatory freshwater fish from lcations across Canada

    Environ. Pollut.

    (2014)
  • M.C. Pietrogrande et al.

    Decoding of complex isothermal chromatograms recovered from space missions: identification of molecular structure

    J. Chromatogr. A

    (2003)
  • T. Reemtsma et al.

    Organophosphorus flame retardants and plasticizers in water and air I. Occurrence and fate

    Trends Anal. Chem.

    (2008)
  • T. Reemtsma et al.

    Determination of 14 monoalkyl phosphates, dialkyl phosphates and dialkyl thiophosphates by LC-MS/MS in human urinary samples

    Sci. Total Environ.

    (2011)
  • N. Ta et al.

    Toxicity of TDCPP and TCEP on PC12 cell: changes in CAMKII, GAP43, tubulin and NF-H gene and protein levels

    Toxicol. Lett.

    (2014)
  • N. van den Eede et al.

    First insights in the metabolism of phosphate flame retardants and plasticizers using human liver fractions

    Toxicol. Lett.

    (2013)
  • N. van den Eede et al.

    In vitro biotransformation of tris(2-butoxyethyl) phosphate (TBOEP) in human liver and serum

    Toxicol. Appl. Pharmacol.

    (2015)
  • N. van den Eede et al.

    Age as a determinant of phosphate flame retardant exposure of the Australian population and identification of novel urinary PFR metabolites

    Environ. Int.

    (2015)
  • N. van den Eede et al.

    Kinetics of tris (1-chloro-2-propyl) phosphate (TCIPP) metabolism in human liver microsomes and serum

    Chemosphere

    (2016)
  • I. van der Veen et al.

    Phosphorus flame retardants: properties, production, environmental occurrence, toxicity and analysis

    Chemosphere

    (2012)
  • Cited by (21)

    • Parental whole life-cycle exposure to tris (2-chloroethyl) phosphate (TCEP) disrupts embryonic development and thyroid system in zebrafish offspring

      2022, Ecotoxicology and Environmental Safety
      Citation Excerpt :

      With the gradual ban on halogenated flame retardants, organophosphate esters (OPEs) have been increasingly applied as proper substitutes and widely applied as additives in plastics, electronics, lubricants, lacquers, textiles and rubber products (Marklund et al., 2003). It is estimated that the production of OPEs accounts for about 15 % of global flame retardants (Arukwe et al., 2018). OPEs are added into manufactured materials through physical mixing rather than chemical bonding, therefore they can be easily migrated into the surrounding environment via abrasion, leaching and volatilization (Pantelaki and Voutsa, 2018).

    • Occurrence, seasonal variation, potential sources, and risks of organophosphate esters in a cold rural area in Northeast China

      2022, Science of the Total Environment
      Citation Excerpt :

      Organismal exposure to OPEs in the environment has numerous adverse health risks. For example, Cl-OPEs, including tris(2-chloroethyl) phosphate (TCEP) and tris(1-chloro-2-propyl) phosphate (TCPP), are potentially carcinogenic (Kim et al., 2017), whereas the aryl-OPE triphenyl phosphate (TPhP) and alkyl-OPEs tris(2-butoxyethyl) phosphate (TBEP) and tri-n-butyl phosphate (TnBP) are suspected neurotoxins with adverse effects on metabolism (Arukwe et al., 2018; Du et al., 2016; Greaves and Letcher, 2017; Zhang et al., 2014). Hence, understanding the occurrence, fate, and potential risks of OPEs in the environment is of paramount importance.

    View all citing articles on Scopus
    View full text