Ecotoxicology

There is an acknowledged need for in vitro fish intestinal model to help understand dietary exposure to chemicals in the aquatic environment. The presence and use of such models is however largely restrictive due to technical difficulties in the culturing of enterocytes in general and the availability of appropriate established cell lines in particular. In this study, the rainbow trout (Oncorhynchus mykiss) intestinal derived cell line (RTgutGC) was used as a surrogate for the “gut sac” method. To facilitate comparison, RTgutGC cells were grown as monolayers (double-seeded) on permeable Transwell supports leading to a two-compartment intestinal model consisting of polarised epithelium. This two-compartment model divides the system into an upper apical (lumen) and a lower basolateral (portal blood) compartment. In our studies, these cells stained weakly for mucosubstances, expressed the tight junction protein ZO-1 in addition to E-cadherin and revealed the presence of polarised epithelium in addition to microvilli protrusions. The cells also revealed a comparable transepithelial electrical resistance (TEER) to the in vivo situation. Importantly, the cell line tolerated apical saline (1:1 ratio) thus mimicking the intact organ to allow assessment of uptake of compounds across the intestine. Following an exposure over 72 h, our study demonstrated that the RTgutGC cell line under sub-lethal concentrations of copper sulphate (Cu) and modified saline solutions demonstrated uptake of the metal with saturation levels comparable to short term ex situ gut sac preparations. Gene expression analysis revealed no significant influence of pH or time on mRNA expression levels of key stress related genes (i.e. CYP3A, GST, mtA, Pgp and SOD) in the Transwell model. However, significant positive correlations were found between all genes investigated suggesting a co-operative relationship amongst the genes studied. When the outlined characteristics of the cell line are combined with the division of compartments, the RTgutGC double seeded model represents a potential animal replacement model for ecotoxicological studies. Overall, this model could be used to study the effects and predict aquatic gastrointestinal permeability of metals and other environmentally relevant contaminants in a cost effective and high throughput manner.

Heavy metal contamination severely affects the aquatic environment and organisms. Copper (Cu) and cadmium (Cd) are two of the most common heavy metal contaminants that impair the survival, development, and reproduction of aquatic organisms. With the growth of agriculture and industry, there is a possibility of heavy metal pollution in Coregonus ussuriensis Berg’s water source. However, there are no published studies on the toxicity to C. ussuriensis. Acute toxicity experiments in C. ussuriensis revealed the 96-h median lethal concentrations of copper and cadmium to be 0.492 mg·L−1 (95% confidence interval: 0.452–0.529) and 1.548 mg·L−1 (95% confidence interval: 1.434–1.657), respectively, and safe concentrations of 4.92 µg·L−1 and 15.48 µg·L−1, respectively. C. ussuriensis was then treated for 96 h with Cu (20% of 96 h LC50), Cd (20% of 96 h LC50), and a combination of Cu and Cd (20% of Cu 96 h LC50 + 20% of Cd 96 h LC50). The histological damage caused by the three different exposure modes to the liver and gills of C. ussuriensis was verified using hematoxylin and eosin staining. All three exposure modes caused different degrees of vacuolization, nuclear consolidation, and necrosis in the liver tissue of C. ussuriensis and edema, hyperplasia, laminar fusion, and epithelial elevation in the gill tissue compared with the reference group. The severity of the damage increased with increasing exposure time. Anti-oxidant activity in the gill and liver tissues were measured using enzyme activity assay kits to reflect oxidative stress induced by copper and cadmium exposure alone and in combination. The enzyme activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH) were substantially higher than those in the reference groups. However, the activities of the enzymes decreased with increasing exposure time. Malondialdehyde (MDA) activity significantly increased during exposure in relation to that in the reference group. Analysis of immune gene expression in C. ussuriensis gill and liver tissues was executed using real-time inverse transcript polymerase chain response (RT-PCR). The expression levels of the pro-inflammatory cytokines interleukin one beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) were positively correlated with exposure time and were significantly upregulated with increasing exposure time. Metallothionein (MT) gene expression levels were significantly upregulated in the short term after exposure compared to the reference group but decreased with increasing exposure time. Our results indicate that exposure to aqueous copper and cadmium solutions, either alone or in combination, causes histopathological damage, oxidative stress, and immunotoxicity in C. ussuriensis gill and liver tissue. This study investigated the toxic effects of copper and cadmium on C. ussuriensis to facilitate the monitoring of heavy metals in water sources for healthy aquaculture.

A luxCDABE-based genetically engineered bacterial bioreporter (Escherichia coli ARL1) was used to detect bioavailable ionic mercury (Hg(II)) and investigate the effects of humic acids and ethylenediaminetetraacetic acid (EDTA) on the bioavailability of mercury in E. c oli. Results showed that the E. c oli ARL1 bioreporter was sensitive to mercury, with a detection limit of Hg(II) of 0.5 µg/L and a linear dose/response relationship up to 2000 µg Hg(II)/L. Humic acids and EDTA decreased the Hg(II)-induced bioluminescent response of strain ARL1, suggesting that the two organic ligands reduced the bioavailability of Hg(II) via complexation with Hg(II). Compared with traditional chemical methods, the use of E. c oli ARL1 is a cost-effective, rapid, and reliable approach for measuring aqueous mercury at very low concentrations and thus has potential for applications in field in situ monitoring.

Lead shot ingestion is the primary source of elevated lead exposure and poisoning in waterfowl and most other bird species. For some species (e.g. Common Loons, Gavia immer), lead sinker ingestion is a more frequent cause of lead poisoning. In freshwater environments where recreational angling activity and loon populations co-occur, lead poisoning from ingestion of small (<50 gram) lead sinkers or jigs accounts for 10–50% of recorded adult loon mortality, depending on the locations studied. Lead shot ingestion occurs in waterfowl, and in a wide variety of non-waterfowl species, including upland game birds, shorebirds, raptors, and scavengers. Where it has been explicitly studied in Canada and the US, lead poisoning mortality of bald (Haliacetus leucocephalus) and golden eagles (Aquila chrysactos) from eating prey animals with lead shot embedded in their tissues accounts for an estimated 10–15% of the recorded post-fledging mortality in these raptorial species. In addition to environments that experience hunting with lead shot, clay target shooting ranges, especially those in which the shotfall zones include ponds, marshes, lakes, rivers, beaches, or other aquatic-type environments, create a significant risk of shot ingestion and poisoning for waterbirds. Metallic lead pellets deposited onto soils and aquatic sediments are not chemically or environmentally inert, although tens or hundreds of years may be required for total breakdown and dissolution of pellets. Functional, affordable non-toxic alternatives to lead shot and sinkers are being currently produced, and additional such products are being developed. Several countries have successfully banned the use of small lead sinkers, and of lead shot for waterfowl and other hunting, also for clay target shooting, using a phasing-out process that gives manufactures, sellers, and users adequate time to adjust to the regulations.

To achieve the EU’s targets for reducing energy production from fossil fuels, the use of energy crops, such as Miscanthus × giganteus, is increasing resulting in a corresponding increase in waste ash from incineration. The chemical properties of Miscanthus ash (e.g. phosphorus and potassium content) may allow this waste material (currently landfilled) to be used as a fertiliser, but no information exists on the effect of the ash on the biological properties of soil. The main aim of this study was to determine the potential impact of Miscanthus ash on earthworms by assessing the effect on survival, change in biomass, reproduction and avoidance behaviour of the geophagous, soil dwelling earthworm, Aporrectodea caliginosa. Tests utilised a range of Miscanthus ash doses from 0 to 50 t ha−1 (0, 1, 2.5, 5, 10, 25, 50). Results showed that Miscanthus ash had no significant impact on A. caliginosa survival, biomass and reproduction, but negative trends were observed for biomass from 2.5 t ha−1 and for reproduction from 10 t ha−1. In contrast, a significant avoidance response was observed in the 25 and 50 t ha−1 treatment and according to ISO guideline 17512 there is a negative impact of the Miscanthus ash on soil habitat function at 25 t ha−1 and above as more than 80% of earthworms were in the control soil. It is suggested that this negative effect on soil habitat function could be attributed to a range of factors including the presence of heavy metals in the ash and a change in substrate pH, texture and/or osmotic stress. Further laboratory-based studies conducted over extended time periods with a more refined range of ash doses and associated field-based studies are required to validate the results and determine a more precise assessment of the threshold ash value inducing a loss of soil habitat function.