In Vitro Assessment of Endocrine Disrupting Potential of Naphthenic Acid Fractions Derived from Oil Sands-Influenced Water

Abstract

Oil sands-influenced process waters have been observed to cause reproductive effects and to induced CYP1A activity in fishes; however, little progress has been made in determining causative agents. Naphthenic acids (NAs) are the predominant organic compounds in process-affected waters, but due to the complexity of the mixture, it has been difficult to examine causal linkages in fishes. The aim of this study was to use in vitro assays specific to reproductive and CYP1A mechanisms to determine if specific acid extractable fractions of NAs obtained from oil sands-influenced water are active toward reproductive processes or interact with the Ah receptor responsible for CYP1A activity. NAs were extracted from aged oil sands-influenced waters by use of acid precipitation, and the mixture was fractionated into three acidic and one neutral fraction. The four fractions were examined for Ah receptor-mediated potency by use of the H4IIE-luc bioassay, effects on production of steroid hormones by use of the H295R steroidogenesis assay, and sex steroid receptor binding activity using the yeast estrogen screen and yeast androgen screen. The mixtures were characterized by high resolution mass spectrometry, (1)H nuclear magnetic resonance, and attenuated total reflectance infrared spectroscopy. The neutral fraction elicited Ah-receptor mediated activity after 24 h but not after 48 or 72 h. None of the fractions contained measurable levels of estrogen or androgen receptor agonists nor did they cause reductions in steroidogenesis. A number of fractions showed antiestrogenic or antiandrogenicity potency, with the neutral and main acidic fractions being the most potent. Neutral aromatic compounds are likely responsible for the CYP1A activity observed. Direct estrogenic, androgenic, or steroidogenic mechanisms are unlikely for NAs based on these results, but NAs act as potent antiandrogen or antiestrogens.

An evaluation of the endocrine disruptive potential of crude oil water accommodated fractions and crude oil contaminated surface water to freshwater organisms using in vitro and in vivo approaches

“…The bunker and refinery crude oil tested in the present study exhibited antiandrogenic action, as has been shown for Romanian crude oil, distillate marine grade oil, bilge oil, heavy nautical fuel oil [20], type-C heavy fuel oil [79], North Sea offshore oil platform-produced water [80,81], oil sands process-affected water [74], and hydraulic fracturing-impacted water [60,75]. In an effect-directed investigation, the constituents of North Sea-produced water were fractionated using high-performance liquid chromatography, allowing individual fractions to be screened for estrogenicity and androgenicity, together with chemical analysis [79].…”

“…The findings of Arcaro et al suggest that the antiestrogenicity was the result of both increased catabolism of β‐estradiol and antagonistic binding to ESRs, although it was not determined whether the interaction was specific to ESR1 or ESR2. Similarly, oil sands process–affected water was shown to be antiestrogenic in vitro , as was the case for water impacted by hydraulic fracturing . Although the exact compounds responsible for the antiestrogenicity observed in the present study remain unclear, it may have been caused by naphthenic acids, as well as certain alkyl phenols, PAHs, and PAH derivatives which are known to be antiestrogenic .…”

“…Similarly, naphthenic acids were found to be responsible for most of the antiandrogenic activity but, unlike with estrogenicity, PAHs contributed nearly equally (than naphthenic acids) and alkyl phenols to a lesser extent [80]. A number of other studies have also suggested the antiandrogenic potential of selected PAHs [2,79,82], naphthenic acids [59,74], and alkyl phenols [59]. The antiandrogenic action observed in the present study was therefore likely caused by PAHs, naphthenic acids, and alkyl phenols, as was the case with antiestrogenicity.…”

Toxicity of organic compounds from unresolved complex mixtures (UCMs) to primary fish hepatocytes

“…Interestingly, several studies have demonstrated that commercially available mixtures (OSPW, oil sand (OS), produced water (PW)) and single naphthenic acids may induce transcriptional and translation changes of ERα/β2, Vtg and Cyp19b in modified yeast strains with human ER (YES), primary rainbow trout hepatocytes, and zebrafish (Danio rerio) embryos (Gagné et al, 2012;Thomas et al, 2004;Wang et al, 2015a,b). In addition, mixtures and fractions of NAs and PW have displayed not only ER agonistic properties, but also displayed androgen-and ER antagonistic properties in vitro (Thomas et al, 2004(Thomas et al, , 2009Leclair et al, 2015). There are however uncertainties when comparing single fractionated compounds from UCMs e.g.…”

“…Numerous examples of non-polar UCM hydrocarbons and more polar UCM acids and hetero compounds, have been synthesised or purchased from commercial suppliers (Rowland et al, 2011b;Smith et al, 2001;West et al, 2014). Compounds such as NAs display acute toxicity (LC50) in fish at concentrations (25-75 mg/l) that is typically reported for NA mixtures in oil sands process-affected water (OSPW) (Scott et al, 2008;Dokholyan and Magomedov, 1984), whereas a range of modes of action (MOAs) including cytotoxicity, genotoxicity, immunotoxicity, and endocrine disruption has been proposed (Lacaze et al, 2014;Leclair et al, 2013Leclair et al, , 2015Thomas et al, 2009;Tollefsen et al 2012). The prospect of ecotoxicological testing and characterisation of the toxic MoA of these UCM-type components has thus arisen.…”