Do physical habitat complexity and predator cues influence the baseline and stress-induced glucocorticoid levels of a mangrove-associated fish?

As human populations continue to expand, increases in coastal development have led to the alteration of much of the world's mangrove habitat, creating problems for the multitude of species that inhabit these unique ecosystems. Habitat alteration often leads to changes in habitat complexity and predation risk, which may serve as additional stressors for those species that rely on mangroves for protection from predators. However, few studies have been conducted to date to assess the effects of these specific stressors on glucocorticoid (GC) stress hormone levels in wild fish populations. Using the checkered puffer as a model, our study sought to examine the effects of physical habitat complexity and predator environment on baseline and acute stress-induced GC levels. This was accomplished by examining changes in glucose and cortisol concentrations of fish placed in artificial environments for short periods (several hours) where substrate type and the presence of mangrove roots and predator cues were manipulated. Our results suggest that baseline and stress-induced GC levels are not significantly influenced by changes in physical habitat complexity or the predator environment using the experimental protocol that we applied. Although more research is required, the current study suggests that checkered puffers may be capable of withstanding changes in habitat complexity and increases in predation risk without experiencing adverse GC-mediated physiological effects, possibly as a result of the puffers' unique morphological and chemical defenses that help them to avoid predation in the wild.

Assessment of biomarkers for contaminants of emerging concern on aquatic organisms downstream of a municipal wastewater discharge.

Contaminants of emerging concern (CECs), including pharmaceuticals, personal care products and estrogens, are detected in wastewater treatment plant (WWTP) discharges. However, analytical monitoring of wastewater and surface water does not indicate whether CECs are affecting the organisms downstream. In this study, fathead minnows (Pimephales promelas) and freshwater mussels Pyganodon grandis Say, 1829 (synonym: Anodonta grandis Say, 1829) were caged for 4 weeks in the North Saskatchewan River, upstream and downstream of the discharge from the WWTP that serves the Edmonton, AB, Canada. Passive samplers deployed indicated that concentrations of pharmaceuticals, personal care products, an estrogen (estrone) and an androgen (androstenedione) were elevated at sites downstream of the WWTP discharge. Several biomarkers of exposure were significantly altered in the tissues of caged fathead minnows and freshwater mussels relative to the upstream reference sites. Biomarkers altered in fish included induction of CYP3A metabolism, an increase in vitellogenin (Vtg) gene expression in male minnows, elevated ratios of oxidized to total glutathione (i.e. GSSG/TGSH), and an increase in the activity of antioxidant enzymes (i.e. glutathione reductase, glutathione-S-transferase). In mussels, there were no significant changes in biomarkers of oxidative stress and the levels of Vtg-like proteins were reduced, not elevated, indicating a generalized stress response. Immune function was altered in mussels, as indicated by elevated lysosomal activity per hemocyte in P. grandis caged closest to the wastewater discharge. This immune response may be due to exposure to bacterial pathogens in the wastewater. Multivariate analysis indicated a response to the CECs Carbamazepine (CBZ) and Trimethoprim (TPM). Overall, these data indicate that there is a 1 km zone of impact for aquatic organisms downstream of WWTP discharge. However, multiple stressors in municipal wastewater make measurement and interpretation of impact of CECs difficult since water temperature, conductivity and bacteria are also inducing biomarker responses in both fish and mussels.

Assessment of energetic costs of AhR activation by ß-naphthoflavone in rainbow trout (Oncorhynchus mykiss) hepatocytes using metabolic flux analysis.

Exposure to environmental contaminants such as activators of the aryl hydrocarbon receptor (AhR) leads to the induction of defense and detoxification mechanisms. While these mechanisms allow organisms to metabolize and excrete at least some of these environmental contaminants, it has been proposed that these mechanisms lead to significant energetic challenges. This study tests the hypothesis that activation of the AhR by the model agonist ß-naphthoflavone (ßNF) results in increased energetic costs in rainbow trout (Oncorhynchus mykiss) hepatocytes. To address this hypothesis, we employed traditional biochemical approaches to examine energy allocation and metabolism including the adenylate energy charge (AEC), protein synthesis rates, Na(+)/K(+)-ATPase activity, and enzyme activities. Moreover, we have used for the first time in a fish cell preparation, metabolic flux analysis (MFA) an in silico approach for the estimation of intracellular metabolic fluxes. Exposure of trout hepatocytes to 1µM ßNF for 48h did not alter hepatocyte AEC, protein synthesis, or Na(+)/K(+)-ATPase activity but did lead to sparing of glycogen reserves and changes in activities of alanine aminotransferase and citrate synthase suggesting altered metabolism. Conversely, MFA did not identify altered metabolic fluxes, although we do show that the dynamic metabolism of isolated trout hepatocytes poses a significant challenge for this type of approach which should be considered in future studies.

A multi-assay screening approach for assessment of endocrine-active contaminants in wastewater effluent samples.

Environmental agencies must monitor an ever increasing range of contaminants of emerging concern, including endocrine disrupting compounds (EDCs). An alternative to using ultra-trace chemical analysis of samples for EDCs is to test for biological activity using in vitro screening assays, then use these assay results to direct analytical chemistry approaches. In this study, we used both analytical approaches and in vitro bioassays to characterize the EDCs present in treated wastewater from four wastewater treatment plants (WWTPs) in Ontario, Canada. Estrogen-mediated activity was assessed using a yeast estrogenicity screening (YES) assay. An in vitro competitive binding assay was used to assess capacity to interfere with binding of the thyroid hormone, thyroxine (T4) to the recombinant human thyroid hormone transport protein, transthyretin (i.e. hTTR). An in vitro binding assay with a rat peroxisome proliferator responsive element transfected into a rainbow trout gill cell line was used to evaluate binding and subsequent gene expression via the peroxisome proliferator activated receptor (PPAR). Analyses of a suite of contaminants known to be EDCs in extracts from treated wastewater were conducted using either gas chromatography with mass spectrometry (GC-MS) or liquid chromatography with tandem mass spectrometry (LC-MS/MS). Estrogenic activity was detected in the YES assay only in those extracts that contained detectable amounts of estradiol (E2). There was a positive relationship between the degree of response in the T4-hTTR assay and the amounts of polybrominated diphenyl ether (PBDE) congeners 47 and 99, triclosan and the PBDE metabolite, 4-OH-BDE17. Several wastewater extracts gave a positive response in the PPAR assay, but these responses were not correlated with the amounts of any of the EDCs analyzed by LC-MS/MS. Overall, these data indicate that a step-wise approach is feasible using a combination of in vitro testing and instrumental analysis to monitor for EDCs in wastewater and other environmental matrixes.

Assessment of nanosilver toxicity during zebrafish (Danio rerio) development.

Nanomaterials (NMs) including silver nanoparticles (AgNPs) are incorporated into an increasing number of consumer and medical products. However, the potential toxicity of AgNPs to aquatic organisms is largely unknown. This study characterizes the effects of AgNPs on zebrafish (Danio rerio) development. The effects of silver ions (Ag(+)) and AgNPs were examined at equivalent Ag concentrations, which ranged from 0.03 to 1.55 µg mL(-1) total Ag. The Ag(+) was more toxic than AgNPs but both lead to death and delayed hatching in surviving embryos. Both silver types depleted glutathione levels but generally did not affect antioxidant enzymes activities. In addition to silver some of the embryos were also exposed to cysteine, which generally reduced the toxicity of both silver types. This study demonstrates that AgNPs and Ag(+) are capable of inducing toxicity in zebrafish embryos including the induction of oxidative stress.

Environmental risk assessment for the serotonin re-uptake inhibitor fluoxetine: Case study using the European risk assessment framework.

The serotonin re-uptake inhibitor fluoxetine was selected for an environmental risk assessment, using the most recent European guideline (EMEA 2006) within the European Union (EU)-funded Environmental Risk Assessment of Pharmaceuticals (ERAPharm) project due to its environmental persistence, acute toxicity to nontarget organisms, and unique pharmacokinetics associated with a readily ionizable compound. As a widely prescribed psychotropic drug, fluoxetine is frequently detected in surface waters adjacent to urban areas because municipal wastewater effluents are the primary route of entry to aquatic environments. In Phase I of the assessment, the initial predicted environmental concentration of fluoxetine in surface water (initial PEC(SW)) reached or exceeded the action limit of 10 ng/L, when using both a default market penetration factor and prescription data for Sweden, Germany, and the United Kingdom. Consequently, a Phase II risk assessment was conducted in which green algae were identified as the most sensitive species with a NOEC of <0.6 microg/L. From this value, a predicted no effect concentration for surface waters (PNEC(SW)) of 0.012 microg/L was derived. The PEC/PNEC ratio was above the trigger value of 1 in worst-case exposure scenarios indicating a potential risk to the aquatic compartment. Similarly, risks of fluoxetine for sediment-dwelling organisms could not be excluded. No risk assessment was conducted for the terrestrial compartment due to a lack of data on effects of fluoxetine on soil organisms. The need for a separate risk assessment for the main metabolite of fluoxetine, norfluoxetine, was not conducted because of a lack of fate and effect studies. Based on published data, fluoxetine and norfluoxetine appeared to have a low to moderate bioaccumulation potential, which should be confirmed in formal studies according to OECD guidelines. Exposure assessments for fluoxetine according to the current framework rely heavily on K(OC) and K(OW) values. This approach is problematic, because fluoxetine is predominantly a cationic substance at environmental pH values. Consequently, the fate of fluoxetine (and other ionic substances) cannot be predicted using partition coefficients established for nonionic compounds. Further, published estimates for partition coefficients of fluoxetine vary, resulting in considerable uncertainties in both the exposure and environmental risk assessments of fluoxetine.