Sara M. Long

Ecological evidence links adverse biological effects to pesticide and metal contamination in an urban Australian watershed

Summary: Aquatic ecosystems near urban areas are often ecologically impaired, but causative factors are rarely identified. Effects may be revealed by considering multiple lines of evidence at different levels of biological organization. Biological impairment is evident in the urban section of the Upper Dandenong Creek Catchment (Victoria, Australia). We assessed whether episodic sewage spills or other pollutants were the cause of poor ecological condition in the stream. The evidence evaluated included chemical and invertebrate assessments, caging studies of mudsnails Potamopyrgus antipodarum, antioxidant biomarkers and endocrine disruption-related endpoints in fish (Carassius auratus and Gambusia holbrooki) and toxicological studies with chironomids (Chironomus tepperi). A combination of metals and pesticides is likely to be affecting the aquatic fauna across all biological levels, with macroinvertebrate communities, P. antipodarum and C. tepperi populations and C. auratus individuals all ecologically impaired. Adverse alterations to aquatic fauna were consistently seen in Bungalook Creek and persisted downstream of this confluence into Dandenong Creek. In addition, chemical assessments and toxicity identification evaluation (TIEs) resulted in several point sources of both metals and pesticides being identified as origins of impairment. This contrasted with an expectation that adverse effects were likely to be associated with sewer-related pollution. As a consequence, target areas and specific pollutants were identified for remediation instead of an expensive sewer upgrade. Synthesis and applications. The results demonstrate that it is important to investigate biological effects in different taxa, in both the laboratory and field, to understand which stressors are causing adverse effects on faunal assemblages. When adverse effects are seen across multiple levels of biological organization and caused by the same pollutant from an identifiable source, there is a clear

The use of respiratory enzymes as biomarkers of petroleum hydrocarbon exposure in Mytilus edulis planulatus

The effect of exposure to petroleum hydrocarbons via the water column and through contaminated sediment upon changes in respiratory enzymes in the common mussel (Mytilus edulis planulatus) was investigated. Mussels were exposed to three concentrations of the water-accommodated fraction (WAF) of Bass Strait crude oil, for 24, 48, and 96 h. In a second study mussels were exposed to three concentrations of crude oil-contaminated sediment for 2 weeks and 1, 2, 4 and 6 months. Activities of citrate synthase (CS) and lactate dehydrogenase (LDH) were measured in the gills. In mussels exposed to WAF, a significant decrease in CS activity was observed over time (P<0.05), whereas treatment did not cause a significant change in CS activity (P>0.05); neither treatment nor time had an effect on LDH activity. Exposure to contaminated sediment did not have a significant effect on CS activity, however, time had a significant effect on CS activity (P<0.05). Both time and treatment had an effect on LDH activity (P<0.05). Results demonstrated that changes in gill CS and LDH are not sensitive biomarkers of petroleum hydrocarbon exposure in M. edulis planulatus.

Acute toxicity of crude and dispersed oil to Octopus pallidus (Hoyle, 1885) hatchlings.

There is an increasing risk of a major oil spill in Australian waters over the next 20 years but there have been few studies on the impact of oil spills, and subsequent remedial action, on native Australian fauna. Octopus pallidus is a native Australian octopus species found in south-eastern Australia. The aim of the experiment was to investigate the effects of acute exposure to crude and dispersed crude oil and 4-chlorophenol (a reference toxicant) on recently hatched O. pallidus by calculating the 48-h LC50. Water-accommodated fraction (WAF) of Bass Strait crude oil was prepared using a ratio of one part crude oil to nine parts filtered seawater and mixing for 23 h. Dispersed-WAF was prepared using a ratio of one part Corexit 9527 to 50 parts crude oil and an oil to water ratio of one to nine and mixing for 23 h. Mean (SE) 48 h LC50 values were 0.39 (0.04), 1.83 (0.64) and 0.89 (0.08) ppm for WAF, dispersed-WAF and 4 chlorophenol, respectively. These results demonstrate that addition of the chemical dispersant Corexit 9527 to WAF does not increase the toxicity of WAF to O. pallidus hatchlings.

Combined chemical (fluoranthene) and drought effects on lumbricus rubellus demonstrate the applicability of the independent action model for multiple stressor assessment

The combined effect of a chemical (fluoranthene) and a nonchemical stress (reduced soil moisture content) to the widely distributed earthworm Lumbricus rubellus were investigated in a laboratory study. Neither fluoranthene (up to 500 microg/g) nor low soil moisture (15% below optimal) had a significant effect on the survival of the exposed worms, but a significant effect on reproduction (cocoon production rate) was found for both stressors (p < 0.001 in both cases). The response of cocoon production to each stressor could be well described by a logistic model; this suggested that the joint effects may be applicable to description using the independent action (IA) model that is widely used in pharmacology and chemical mixture risk assessment. Fitting of the IA model provided a good description of the combined stressor data (accounting for 53.7% of total variation) and was the most parsimonious model describing joint effect (i.e., the description of the data was not improved by addition of further parameters accounting for synergism or antagonism). Thus, the independent action of the two responses was further supported by measurement of internal fluoranthene exposure. The chemical activity of fluoranthene in worm tissue was correlated only with soil fluoranthene concentration and not with soil moisture content. Taken together these results suggest that the IA model can help interpret the joint effects of chemical and nonchemical stressors. Such analyses should, however, be done with caution since the literature data set suggests that there may be cases where interactions between stressors result in joint effects that differ significantly from IA predictions.

Genes involved in cysteine metabolism of Chironomus tepperi are regulated differently by copper and by cadmium

Freshwater invertebrates are often exposed to metal contamination, and changes in gene expression patterns can help understand mechanisms underlying toxicity and act as pollutant-specific biomarkers. In this study the expressions of genes involved in cysteine metabolism are characterized in the midge Chironomus tepperi during exposures to sublethal concentrations of cadmium and copper. These metals altered gene expression of the cysteine metabolism differently. Both metals decreased S-adenosylhomocysteine hydrolase expression and did not change the expression of S-adenosylmethionine synthetase. Cadmium exposure likely increased cystathionine production by up-regulating cystathionine-β-synthase (CβS) expression, while maintaining control level cysteine production via cystathionine-γ-lyase (CγL) expression. Conversely, copper down-regulated CβS expression and up-regulated CγL expression, which in turn could diminish cystathionine to favor cysteine production. Both metals up-regulated glutathione related expression (γ-glutamylcysteine synthase and glutathione synthetase). Only cadmium up-regulated metallothionein expression and glutathione S-transferase d1 expression was up-regulated only by copper exposure. These different transcription responses of genes involved in cysteine metabolism in C. tepperi point to metal-specific detoxification pathways and suggest that the transsulfuration pathway could provide biomarkers for identifying specific metals.

A comparison of the effects of single and repeated exposure to an organophosphate insecticide on acetylcholinesterase activity in mammals

Exposure to organophosphate (OP) pesticides can occur in free-living mammals in treated areas. Risk to nontarget animals from OPs usually is assessed with acute exposure data, but exposure of wild animals is likely to be intermittent and chronic. We compared the effects of single or repeated (hourly and daily) exposure to dimethoate on acetylcholinesterase (AChE) activity in laboratory mice to assess the suitability of standard laboratory tests for assessing risk. Mice were exposed either to a single dose (10 or 30 mg/kg) or to short-term repeated (three hourly doses of 10 mg/kg) intraperitoneal doses of dimethoate, and brain and serum AChE activity were measured. No significant difference was found in the degree of inhibition of AChE activity following acute and short-term repeated exposure. In a second experiment, mice were given three daily doses of 10 or 20 mg/kg of dimethoate, and both AChE activity and hepatic cytochrome P450 enzyme activity were measured. Daily exposure resulted in a dose-dependent decline in brain and serum AChE activity, and inhibition increased progressively with successively repeated exposures. However, this effect was relatively small compared to the effect of dose. Cytochrome P450 enzyme activity (CYP2B) was inhibited in the dimethoate-dosed mice. Our results indicate that acute dose-response toxicity studies are suitable models for predicting the likely occurrence of adverse effects from either short- or longer-term exposure of wild mammals to anticholinesterase compounds. Likely differences in exposure pattern between the laboratory and the natural environment are unlikely to bias the predictive power of these studies significantly.

Effects of two commonly used fungicides on the amphipod Austrochiltonia subtenuis.

Fungicides are used widely in agriculture and have been detected in adjacent rivers and wetlands. However, relatively little is known about the potential effects of fungicides on aquatic organisms. The present study investigated the effects of 2 commonly used fungicides, the boscalid fungicide Filan® and the myclobutanil fungicide Systhane™ 400 WP, on life history traits (survival, growth, and reproduction) and energy reserves (lipid, protein, and glycogen content) of the amphipod Austrochiltonia subtenuis under laboratory conditions, at concentrations detected in aquatic environments. Amphipods were exposed to 3 concentrations of Filan (1 μg active ingredient [a.i.]/L, 10 μg a.i./L, and 40 μg a.i./L) and Systhane (0.3 μg a.i./L, 3 μg a.i./L, and 30 μg a.i./L) over 56 d. Both fungicides had similar effects on the amphipod at the organism level. Reproduction was the most sensitive endpoint, with offspring produced in controls but none produced in any of the fungicide treatments, and total numbers of gravid females in all fungicide treatments were reduced by up to 95%. Female amphipods were more sensitive than males in terms of growth. Systhane had significant effects on survival at all concentrations, whereas significant effects of Filan on survival were observed only at 10 μg a.i./L and 40 μg a.i./L. The effects of fungicides on energy reserves of the female amphipod were different. Filan significantly reduced amphipod protein content, whereas Systhane significantly reduced the lipid content. The present study demonstrates wide-ranging effects of 2 common fungicides on an ecologically important species that has a key role in trophic transfer and nutrient recycling in aquatic environments. These results emphasize the importance of considering the long-term effects of fungicides in the risk assessment of aquatic ecosystems. Environ Toxicol Chem 2017;36:720-726.

Comparisons of metabolic and physiological changes in rats following short term oral dosing with pesticides commonly found in food

¹H Nuclear Magnetic Resonance spectroscopy has been used to profile urinary metabolites in male Fischer F344 rats in order to assess the metabolic changes induced by oral exposure to two benzimidazole fungicides (carbendazim and thiabendazole) and two bipyridyllium herbicides (chlormequat and mepiquat). Exposure levels were selected to be lower than those expected to cause overt signs of toxicity. We then compared the sensitivity of the metabolomics approach to more traditional methods of toxicity assessment such as the measurement of growth and organ weights. Separate, acute exposure experiments were conducted for each pesticide to identify potential metabolic markers of exposure across four doses (and a control). Growth, organ weights and feeding/drinking rates were not significantly affected by any compounds at any dose levels tested. In contrast, metabolic responses were detected within 8 and 24h for chlormequat and mepiquat, and after 24h for carbendazim and thiabendazole. These results demonstrate the potential for the use of metabolomics in food toxicity testing.

A multi-platform metabolomics approach demonstrates changes in energy metabolism and the transsulfuration pathway in Chironomus tepperi following exposure to zinc

Measuring biological responses in resident biota is a commonly used approach to monitoring polluted habitats. The challenge is to choose sensitive and, ideally, stressor-specific endpoints that reflect the responses of the ecosystem. Metabolomics is a potentially useful approach for identifying sensitive and consistent responses since it provides a holistic view to understanding the effects of exposure to chemicals upon the physiological functioning of organisms. In this study, we exposed the aquatic non-biting midge, Chironomus tepperi, to two concentrations of zinc chloride and measured global changes in polar metabolite levels using an untargeted gas chromatography-mass spectrometry (GC-MS) analysis and a targeted liquid chromatography-mass spectrometry (LC-MS) analysis of amine-containing metabolites. These data were correlated with changes in the expression of a number of target genes. Zinc exposure resulted in a reduction in levels of intermediates in carbohydrate metabolism (i.e., glucose 6-phosphate, fructose 6-phosphate and disaccharides) and an increase in a number of TCA cycle intermediates. Zinc exposure also resulted in decreases in concentrations of the amine containing metabolites, lanthionine, methionine and cystathionine, and an increase in metallothionein gene expression. Methionine and cystathionine are intermediates in the transsulfuration pathway which is involved in the conversion of methionine to cysteine. These responses provide an understanding of the pathways affected by zinc toxicity, and how these effects are different to other heavy metals such as cadmium and copper. The use of complementary metabolomics analytical approaches was particularly useful for understanding the effects of zinc exposure and importantly, identified a suite of candidate biomarkers of zinc exposure useful for the development of biomonitoring programs.

Contaminant (PAHs, OCs, PCBs and trace metals) concentrations are declining in axial tissue of sand flathead (Platycephalus bassensis) collected from an urbanised catchment (Port Phillip Bay, Australia)

Concentrations of PAHs, OCs, PCBs and trace metals were determined in the white muscle of sand flathead Platycephalus bassensis collected at 6 locations in Port Phillip Bay during 2015. No PAHs, OCs or PCBs were detected in the white muscle of sand flathead at any of the locations, however measurable levels of As, Cu, Hg, Se and Zn were detected at all sites. Only As and Hg exhibited regional difference in white muscle concentrations, with As present only in a non-toxic organic form and Hg measured at levels that are comparable to levels reported in reference sites in other studies. All contaminants detected in the white muscle of sand flathead collected in Port Phillip Bay in 2015 were below Australian Food Standards guideline values, and by world standards, the Port Phillip Bay sand flathead population is considered minimally contaminated. Furthermore, tissue contaminant concentrations appear to be decreasing over time.