N-(1,3-Dimethylbutyl)-N′-phenyl-p-phenylenediamine-quinone (6PPD-quinone), a transformation product of the rubber tire antioxidant 6PPD, has recently been identified as the chemical responsible for urban runoff mortality syndrome in coho salmon, with a median lethal concentration (LC50) of <0.1 μg/L. Subsequent studies have failed to confirm comparable sensitivity in other fish species. Here, we investigated the acute toxicity of 6PPD-quinone to rainbow trout, brook trout, Arctic char, and white sturgeon. Fish were exposed under static renewal conditions, and exposure concentrations were verified analytically. Mortalities in brook trout occurred between 1.2 and 20 h, while mortalities began after 7 h and spanned 60 h in rainbow trout. The LC50s in brook trout (24 h) and rainbow trout (72 h) were 0.59 and 1.00 μg/L, respectively. Both species showed characteristic symptoms (increased ventilation, gasping, spiraling, and loss of equilibrium) shortly before death. No mortalities were observed after exposure of either char or sturgeon for 96 h at measured concentrations as high as 14.2 μg/L. This is the first study to demonstrate the acute toxicity of 6PPD-quinone to other fishes of commercial, cultural, and ecological importance at environmentally relevant concentrations and provides urgently needed information for environmental risk assessments of this contaminant of emerging concern.

N-(1,3-Dimethylbutyl)-N′-phenyl-p-phenylenediaminequinone (6PPD-quinone), a rubber tire oxidation product found in road runoff, is highly and acutely toxic to selected salmonids including coho salmon, brook trout, and rainbow trout but not other fish species and invertebrates studied to date. Sensitive species displayed increased ventilation and gasping, suggesting a possible impact on respiration. Here, adherent cell lines RTL-W1 and RTgill-W1 were exposed to 5− 80 μg/L 6PPD-quinone, and cytotoxicity, oxygen consumption rate (OCR), and biotransformation of 6PPD-quinone were measured to assess the ability of 6PPD-quinone to uncouple mitochondrial respiration in vitro. RTL-W1 cells were not sensitive to 6PPD-quinone, and exposure did not result in significant impacts on cytotoxicity or OCR. In contrast, RTgill-W1 cells demonstrated decreased cell viability at 80 μg/L and a 2-fold increase in OCR at 20 μg/L. Effects appear to be partly driven by toxicokinetic differences where incubation of RTL-W1 cells with 6PPD-quinone led to almost quantitative conversion of 6PPD-quinone into a suspected hydroxy-metabolite, which was not observed in RTgill-W1 cells. Exposure studies with primary cultures of rainbow trout gill cells indicated that 6PPD-quinone increased OCR by uncoupling the mitochondrial electron transport chain. Together, these findings suggest that 6PPD-quinone toxicity might be driven by a tissue-specific disruption of mitochondrial respiration.

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There is an urgent demand for more efficient and ethical approaches in ecological risk assessment. Using 17αethinylestradiol (EE2) as a model compound, this study established an embryo benchmark dose (BMD) assay for rainbow trout (RBT; Oncorhynchus mykiss) to derive transcriptomic points-of-departure (tPODs) as an alternative to live-animal tests. Embryos were exposed to graded concentrations of EE2 (measured: 0, 1. 13, 1.57, 6.22, 16.3, 55.1, and 169 ng/L) from hatch to 4 and up to 60 days post-hatch (dph) to assess molecular and apical responses, respectively. Whole proteome analyses of alevins did not show clear estrogenic effects. In contrast, transcriptomics revealed responses that were in agreement with apical effects, including excessive accumulation of intravascular and hepatic proteinaceous fluid and significant increases in mortality at 55.1 and 169 ng/L EE2 at later time points. Transcriptomic BMD analysis estimated the median of the 20th lowest geneBMD to be 0.18 ng/L, the most sensitive tPOD. Other estimates (0.78, 3.64, and 1.63 ng/L for the 10th percentile geneBMD, first peak geneBMD distribution, and median geneBMD of the most sensitive over-represented pathway, respectively) were within the same order of magnitude as empirically derived apical PODs for EE2 in the literature. This 4-day alternative RBT embryonic assay was effective in deriving tPODs that are protective of chronic effects of EE2.

There is increasing pressure to develop alternative ecotoxicological risk assessment approaches that do not rely on expensive, time-consuming, and ethically questionable live animal testing. This study aimed to develop a comprehensive early life stage toxicity pathway model for the exposure of fish to estrogenic chemicals that is rooted in mechanistic toxicology. Embryo-larval fathead minnows (FHM; Pimephales promelas) were exposed to graded concentrations of 17α-ethinylestradiol (water control, 0.01% DMSO, 4, 20, and 100 ng/L) for 32 days. Fish were assessed for transcriptomic and proteomic responses at 4 days post-hatch (dph), and for histological and apical end points at 28 dph. Molecular analyses revealed core responses that were indicative of observed apical outcomes, including biological processes resulting in overproduction of vitellogenin and impairment of visual development. Histological observations indicated accumulation of proteinaceous fluid in liver and kidney tissues, energy depletion, and delayed or suppressed gonad development. Additionally, fish in the 100 ng/L treatment group were smaller than controls. Integration of omics data improved the interpretation of perturbations in early life stage FHM, providing evidence of conservation of toxicity pathways across levels of biological organization. Overall, the mechanism-based embryo-larval FHM model showed promise as a replacement for standard adult live animal tests.

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The epithelial cell layer that lines the gills of fish controls paracellular permeation of chemicals through tight junctions. The integrity of tight junctions can be affected by inflammation, which likely affects the bioavailability of chemicals.Here, the inflammation of the rainbow trout gill cell line RTgill-W1 was induced via exposure to bacterial lipopolysaccharides (LPS). Cells were then coexposed to extracts of oil sands processaffected water (OSPW), which contain complex mixtures of chemicals. After 24 h of exposure, cells exposed to LPS showed a reduction in transepithelial electrical resistance, an indicator of tight junction integrity. Quantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis determined that abundances of transcripts of genes coding for tight junction proteins were significantly less in cells exposed to 20, 50, or 100 mg L −1 LPS. Chemical analysis revealed increased permeation of constituents of OSPW across epithelia at all studied LPS concentrations. These in vitro findings were confirmed in vivo in rainbow trout exposed to LPS and OSPW for 48 h, which resulted in greater accumulation of chemicals relative to that for fish exposed to OSPW alone. Our results demonstrated that inflammation and disruption of tight junctions could lead to greater uptake of potentially harmful chemicals from the environment, which has implications for mixture risk assessment.

Focus articles are part of a regular series intended to sharpen understanding of current and emerging topics of interest to the scientific community.