Effect‐directed analysis of Elizabeth River porewater: Developmental toxicity in zebrafish (Danio rerio)

Abstract

In the present study, effect-directed analysis was used to identify teratogenic compounds in porewater collected from a Superfund site along the Elizabeth River estuary (VA, USA). Zebrafish (Danio rerio) exposed to the porewater displayed acute developmental toxicity and cardiac teratogenesis, presumably because of elevated sediment levels of polycyclic aromatic hydrocarbons (PAHs) from historical creosote use. Pretreatment of porewater with several physical and chemical particle removal methods revealed that colloid-bound chemicals constituted the bulk of the observed toxicity. Size-exclusive chromatography and normal-phase high-performance liquid chromatography were used to fractionate Elizabeth River porewater. Acute toxicity of porewater extracts and extract fractions was assessed as the pericardial area in embryonic zebrafish. The most toxic fraction contained several known aryl hydrocarbon receptor (AhR) agonists (e.g., 1,2-benzofluorene and 1,2-benzanthracene) and cytochrome P450 A1 (CPY1A) inhibitors (e.g., dibenzothiophene and fluoranthene). The second most toxic fraction contained known AhR agonists (e.g., benzo[a]pyrene and indeno[1,2,3-cd]pyrene). Addition of a CYP1A inhibitor, fluoranthene, increased toxicity in all active porewater fractions, suggesting synergism between several contaminants present in porewaters. The results indicate that the observed acute toxicity associated with Elizabeth River porewater results from high concentrations of AhR agonistic PAHs and mixture effects related to interactions between compounds co-occurring at the Elizabeth River site. However, even after extensive fractionation and chemical characterization, it remains plausible that some active compounds in Elizabeth River porewater remain unidentified.

The value of zebrafish as an integrative model in effect-directed analysis - a review

“…Natural product studies aimed to identify bioactive compounds for pharmacological applications, investigating mostly plant extracts [19,[34][35][36][37][38][39][40][41][42][43][44][45] but also extracts of bacteria [46], cyanobacteria and algae [47], seaweed [48] and marine organisms [49]. Environmental toxicology studies aimed to identify the toxic compounds in various environmental samples, including marine and fluvial sediments [50][51][52], soil [53], cyanobacteria and algae [54,55], industrial effluent [33], rubber tyre leachates [32], oil sand process waters [56,57] and river pore water [58]. Finally, fish skin extracts were investigated in a behavioural sciences study [59].…”

“…In the EDA studies, there was submission of the respective solvents [51,53,56,57] or of HPLC-grade water [58] through the same or part of the procedures that were applied to samples (i.e. sample preparation, extraction, fractionation).…”

“…Assessment of teratogenesis and developmental effects was done in studies that identified the bioactive compounds from microalgae, cyanobacteria and plant [41,54,55], river pore water [58] and developmental toxicants in soil [53]. Most studies evaluated traditionally assessed morphological endpoints, while one investigation of plant fractions focused on ectopic tail formation [41].…”

“…For Environmental toxicology Sediment extracts [50], industrial effluents [33], rubber tyre leachates [32], oil sand process waters [57] Natural products Cyanobacteria and algae extracts [47], seaweed hydrolysates [48] Acute and developmental toxicity Lethal and morphological endpoints Environmental toxicology Sediment extracts [51], soil extracts [53], microalgae extracts [54], cyanobacteria extracts [55], river pore water extract [58] Natural products Bacteria extracts [46] Developmental toxicity Phenotypic endpoints Natural products Plant extracts [41] Anti-or pro-angiogenesis ISV formation and/or function in wild-type or fli1:EGFP zebrafish Natural products Plant extracts [19,[36][37][38][42][43][44] Anti-angiogenesis and anti-inflammatory ISV outgrowth in fli1:EGFP, leukocyte migration after tail transection Natural products Plant extracts [40] Glucose uptake Uptake of fluorescein-tagged glucose bioprobe…”

“…Characteristic phenotypical effects were identified for specific fractions [53], also on a dose-dependent manner [54,55]. Two studies also investigated if additive or synergistic effects occurred between different fractions [55] or between aryl hydrocarbon receptor (AhR) agonists by co-exposure to a CYP1A inhibitor [58].…”