Impact of mixing and resting times on the droplet size distribution and the petroleum hydrocarbons' concentration in diluted bitumen-based water-accommodated fractions (WAFs).

The preparation of Water-accommodated Fractions (WAFs) and chemically enhanced WAFs (CEWAFs) are essential for evaluating oil toxicity. The Chemical Response to Oil Spills: Ecological Research Forum (CROSERF) method was widely adopted, with variables (e.g., mixing time, oil loading, etc.) being continuously changed among research groups, which limits the cooperation in this area. Herein, we conducted WAF and CEWAF experiments using two loadings of diluted bitumen (Dilbit): 1 g/L and 10 g/L. For the CEWAF, the dispersant to oil ratio was 1:20. We investigated the impact of three mixing durations (18 h, 42 h, and 66 h) and two resting times (6 h and 24 h) on the droplet size distribution (DSD) and accommodated oil concentration. This would be highly beneficial for analyzing toxicity from oil spills, especially when considering the toxic effect of both suspended oil droplets and dissolved hydrocarbons. The DSD results and oil chemistry analysis showed that at a low oil loading concentration (1 g/L), both WAFs and CEWAFs had the same DSD, with an average d50 (volume median diameter) of 3.38 ± 0.70 µm and 3.85 ± 0.63 µm, respectively. At a high oil loading concentration (10 g/L), the WAFs had an average d50 of 3.69 ± 0.52 µm, showing no correlation with mixing and resting time. The DSD of CEWAFs increased significantly at 42 h mixing and 24 h resting time, with oil concentration reaching equilibrium after 42 h mixing. Therefore, WAFs appears to require only 18 h mixing and 6 h resting, while it is recommended to have 42 h mixing and 24 h resting for CEWAFs at high dilbit oil loading concentrations.

Prostaglandins prevent acetaminophen induced embryo toxicity in zebrafish (Danio rerio).

Previous research in our laboratory showed that acetaminophen (ACE) induced embryonic mortality and abnormalities in zebrafish. Here, we examined the dose response of ACE (0.05-50 µg L-1) in zebrafish embryos. Concentrations as low as 0.1 µg L-1 significantly increased abnormalities, and all test concentrations significantly increased mortality rates. In mammals, ACE inhibits cyclooxygenase (COX) enzymes to decrease prostaglandin production. Here we report COX activity and expression of the cox-1, cox-2a, and cox-2b genes in zebrafish embryos. COX activity was significantly inhibited by specific mammalian cox-1 (SC-560) and cox-2 (DuP-697) inhibitors in unexposed and ACE-exposed embryos. COX activity declined with development time. Maternal transcripts of all cox genes were found at 1 -h post fertilization and embryonic expression began in gastrulation or early segmentation. Co-exposure of ACE and prostaglandin E2 abolished the ACE-induced effects. This strongly supports that ACE elicits embryo toxicity in zebrafish though the same molecular mechanism of action of their therapeutic effects in mammals.

Effects of chronic, parental pharmaceutical exposure on zebrafish (Danio rerio) offspring.

In this study we explored how parental exposure to pharmaceuticals influences reproduction in offspring. Adult zebrafish (Danio rerio) were exposed for 6 weeks to 10 µgL(-1) of carbamazepine (CBZ) and gemfibrozil (GEM), two commonly prescribed drugs. Embryos were collected, reared in clean water until sexual maturity and then assessed for reproductive output, courtship, sperm function and organ histology. While 34% of the control pairs produced clutches, only 11% of the fish with CBZ exposed parents or 17% of the fish with GEM exposed parents produced clutches. Reciprocal crosses indicated that exposure in males had more profound reproductive effects. When a control F1 male was crossed with either a F1 female whose parents were CBZ or GEM exposed; no differences were observed in embryo production compared to controls. However, when a control F1 female was crossed with either a CBZ or GEM F1 male, 50% less embryos were produced. Male courtship was reduced in both CBZ and GEM F1 fish but the deficits in courtship displays were drug specific. Compared to control males, the sperm from GEM F1 males had shorter head lengths and midpieces whereas sperm from CBZ F1 males had longer midpieces. Although it remains unclear how specifically these morphological differences influenced sperm velocity, the sperm from GEM F1 males and from CBZ F1 males swam faster than the sperm of control F1 at 20s post activation. No significant differences were observed in the histology of the liver, kidney and gonads across treatment groups. These data are important as they show that chronic, low dose pharmaceutical exposure of parental fish is sufficient to cause significant reproductive effects in offspring.

Chronic, low concentration exposure to pharmaceuticals impacts multiple organ systems in zebrafish.

Pharmaceuticals are found in both receiving and drinking water due to their persistent release in waste-water effluents, raising concerns for environmental and human health. Chronic, aqueous exposure of zebrafish (Danio rerio) to environmentally relevant concentrations of acetaminophen (ACE), venlafaxaine (VEN) (10µgL(-1)), carbamazepine (CBZ) and gemfibrozil (GEM) (0.5 and 10µgL(-1)) decreased reproductive output. Atretic oocytes and altered ovarian histology were seen in female zebrafish exposed to CBZ and GEM, suggesting a direct effect on oocyte development that may account for the reduced fecundity. Apoptosis within the theca and granulosa cells was identified in exposed female zebrafish with atretic oocytes by TUNEL positive staining. The incidence of follicular apoptosis was nearly 2-fold higher in exposed females than the controls. All compounds significantly altered kidney proximal tubule morphology but there was no difference in the incidence of apoptotic cells within the kidney between control and exposed in either males or females. Liver histology was altered by ACE and GEM exposure. Parental exposure to pharmaceuticals did not increase developmental abnormalities, hatching success, or mortality in embryos. Yet, direct exposure of embryos to ACE increased developmental abnormalities and mortality; exposure to 0.5µgL(-1) of all pharmaceuticals increased mortality. CBZ decreased plasma 11-ketotestosterone concentrations in males and females. Overall, these data suggest that low concentration, chronic exposure of fish to pharmaceuticals impacts fish development as well as multiple organ systems in adult fish, leading to effects on reproduction and histology of liver and kidney. These results are significant in understanding the consequences of chronic, low concentration pharmaceutical exposure to fish and suggest that exposed populations are at risk of negative impacts to reproduction and health.

Chronic effects of exposure to a pharmaceutical mixture and municipal wastewater in zebrafish.

Pharmaceuticals and personal care products (PPCPs) are discharged in municipal wastewater. Effects in aquatic organisms exposed to individual pharmaceuticals in the laboratory have raised concerns regarding the environmental impacts of PPCPs, yet environmental exposures are always to complex mixtures. In this study, adult zebrafish (Danio rerio) showed significantly decreased embryo production after a 6 week exposure to a pharmaceutical mixture (MIX; 0.5 and 10µgL(-1)) of acetaminophen, carbamazepine, gemfibrozil and venlafaxine and to diluted wastewater effluent (WWE; 5% and 25%). Atretic oocytes and altered ovarian histology were significantly increased in female zebrafish exposed to both concentrations of MIX or WWE, which indicates a direct effect on oocyte development that may account for reduced embryo production. Apoptosis within the thecal and granulosa cell layers was identified in female zebrafish with atresia. Exposures to MIX or WWE at both concentrations severely altered kidney proximal tubule morphology, but no histological impacts on other organs were observed. Exposure of embryos to MIX or WWE at the high concentration significantly increased the incidence of developmental abnormalities. Embryo mortality was elevated with exposure to the high concentration of MIX. These studies indicate that chronic exposure of fish to pharmaceutical mixtures and wastewater impacts reproduction and induces histopathological changes, similar to what we have previously seen with single compound exposures. These data suggest that fish populations exposed to pharmaceuticals discharged in wastewater are at risk of negative impacts to reproductive capacity and health.

Dietary iron alters waterborne copper-induced gene expression in soft water acclimated zebrafish (Danio rerio).

Metals like iron (Fe) and copper (Cu) function as integral components in many biological reactions, and, in excess, these essential metals are toxic, and organisms must control metal acquisition and excretion. We examined the effects of chronic waterborne Cu exposure and the interactive effects of elevated dietary Fe on gene expression and tissue metal accumulation in zebrafish. Softwater acclimated zebrafish exposed to 8 microg/l Cu, with and without supplementation of a diet high in Fe (560 vs. 140 mg Fe/kg food) for 21 days demonstrated a significant reduction in liver and gut Cu load relative to waterborne Cu exposure alone. Gene expression levels for divalent metal transport (DMT)-1, copper transporter (CTR)-1, and the basolateral metal transporter ATP7A in the gills and gut increased when compared with controls, but the various combinations of Cu and high-Fe diet revealed altered levels of expression. Further examination of the basolateral Fe transporter, ferroportin, showed responses to waterborne Cu exposure in the gut and a significant increase with Fe treatment alone in the liver. Additionally, we examined metallothionein 1 and 2 (MT1 and MT2), which indicated that MT2 is more responsive to Cu. To explore the relationship between transcription and protein function, we examined both CTR-1 protein levels and gill apical uptake of radiolabeled Cu64, which demonstrated decreased Cu uptake and protein abundance in the elevated Cu treatments. This study shows that high dietary Fe can significantly alter the genetic expression pattern of Cu transporters at the level of the gill, liver, and gastrointestinal tract.