Comparative sensitivity of aquatic invertebrate and vertebrate species to wastewater from an operational coal mine in central Queensland, Australia.

Coal excavation and refinement processes generate substantial volumes of contaminated effluent that may be detrimental to aquatic ecosystems. As such, understanding the impacts of coal mine water releases on aquatic animals and ecosystems is essential for effectively managing and protecting neighboring environments. Such information will ultimately be applied towards developing ongoing monitoring strategies that are protective of native wildlife. Despite intensive mining operations in Australia, few studies have documented toxicity associated with coal mine wastewater (CMW) on native species. To address existing knowledge gaps, we investigated acute toxicity (48-96h) using eight native invertebrate species and sub-chronic effects (2 week) using three vertebrate species following exposure to wastewater from two dams (CMW1 and CMW2) located at an open-cut coal mine licensed to discharge into the Fitzroy catchment (Queensland, Australia). Wastewater from these sites is characterized by elevated conductivity, pH, sulfates as well as relatively high total and dissolved metal(loid)s (including As, Al, B, Cu, Mn, Ni, Se and Zn). Acute exposures revealed cladocerans (Daphnia carinata) and planarians (Dugesia sp.) to be the most sensitive species, exhibiting significant mortality after 48 and 96h exposure to CMW2, respectively. Neither wastewater was found to elicit acute toxicity in vertebrates, but a range of sub-lethal morphological effects were observed following the sub-chronic exposures. The overall response pattern was characterized by decreased condition factor and hepatosomatic index in the fish Hypseleotris compressa and Pseudomugil signifier, and in Limnodynastes peronii tadpoles. Tadpoles were generally more sensitive compared to the two fish species. Differences in responses were observed amongst CMW1 and CMW2, which likely relates to differences in physico-chemical properties between sites. Our results have identified several candidate vertebrate and invertebrate species that show promise for ongoing monitoring of water quality and toxicity risk in Central Queensland, Australia.

Behaviour, development and metal accumulation in striped marsh frog tadpoles (Limnodynastes peronii) exposed to coal mine wastewater.

Coal mining generates large quantities of complex effluent, and this often contains high levels of dissolved solids, suspended solids, metals, hydrocarbons, salts and other compounds. Substantial volumes of mine wastewater are periodically discharged into the environment, through both planned and accidental releases, and this raises concerns about the potential for adverse impacts on aquatic wildlife. There have been few attempts to explore sub-lethal effects of coal mine wastewater on amphibians compared to other organisms, and this is particularly true for Australian species. To address existing knowledge gaps, we exposed striped marsh frog (Limnodynastes peronii) tadpoles to 25, 50 and 100% coal mine wastewater collected from two holding dams (CMW1 and CMW2) located at an open cut mine in Central Queensland, Australia. The exposure lasted for four weeks, after which survival, growth and development, swimming behaviour, and concentrations of metals and metalloids in tail and liver tissues were assessed. Physico-chemical parameters varied considerably between sites, with higher turbidity, nutrients, total and dissolved organic carbon, alkalinity and arsenic (As) concentrations at CMW1, and higher conductivity, salinity, dissolved solids, hardness and sulfate levels at CMW2. There was no mortality in controls and less than 5% mortality in CMW1 treatments, whereas survival was significantly decreased in tadpoles exposed to CMW2 with 40 and 55% mortality in the 50 and 100% treatments, respectively. Development was significantly delayed in 100% CMW1 wastewater, but tadpole size (growth) was not influenced by the exposure. Hepatosomatic indices were significantly increased in tadpoles exposed to 25 and 50% CMW1 but not the 100% treatment group. Exposed tadpoles (predominantly those exposed to CMW1) exhibited increased activity after very short-term exposure (24h), but this did not persist as animals approached metamorphic climax. At the end of the experiment, tadpoles exposed to both wastewaters had elevated levels of selenium (Se), cobalt (Co) and As in tail and liver tissue compared to controls. Manganese (Mn) levels were also elevated in livers and tails of CMW2 exposed tadpoles. Hepatic tissue accumulated 8-9 times higher concentrations of Co, Mn and Se compared to tail tissue, irrespective of treatments. Future research is warranted to explore possible relationships between metal bioaccumulation, morpho-physiological effects during development, and subsequent higher-level outcomes related to individual performance and population fitness.

Effects of coal mine wastewater on locomotor and non-locomotor activities of empire gudgeons (Hypseleotris compressa).

Coal mining represents an important industry in many countries, but concerns exist about the possible adverse effects of minewater releases on aquatic animals and ecosystems. Coal mining generates large volumes of complex wastewater, which often contains high concentrations of dissolved solids, suspended solids, metals, hydrocarbons, salts and other compounds. Traditional toxicological testing has generally involved the assessment of acute toxicity or chronic toxicity with longer-term tests, and while such tests provide useful information, they are poorly suited to ongoing monitoring or rapid assessment following accidental discharge events. As such, there is considerable interest in developing rapid and sensitive approaches to environmental monitoring, and particularly involving the assessment of sub-lethal behavioural responses in locally relevant aquatic species. We therefore investigated behavioural responses of a native Australian fish to coal mine wastewater, to evaluate its potential use for evaluating sub-lethal effects associated with wastewater releases on freshwater ecosystems. Empire gudgeons (Hypseleotris compressa) were exposed to wastewater from two dams located at an open cut coal mine in Central Queensland, Australia and activity levels were monitored using the Multispecies Freshwater Biomonitor® (LimCo International GmbH). A general decrease in locomotor activity (i.e., low frequency movement) and increase in non-locomotor activity (i.e., high frequency movement including ventilation and small fin movement) was observed in exposed fish compared to those in control water. Altered activity levels were observable within the first hour of exposure and persisted throughout the 15-d experiment. Results demonstrate the potential for using behavioural endpoints as tools for monitoring wastewater discharges using native fish species, but more research is necessary to identify responsible compounds and response thresholds, and to understand the relevance of the observed effects for populations in natural receiving environments.

Locomotor and behavioural responses of empire gudgeons (Hypseleotris compressa) exposed to coal mine wastewater.

Coal mining generates large quantities of complex effluent and may pose a threat to aquatic wildlife. Despite this, few studies have explored the consequences of exposure to mine wastewater on aquatic organisms, and this is particularly true for the Australian environment. We investigated sub-lethal behavioural responses in a native Australian fish exposed to wastewater from two releasing dams (CMW1 and CMW2) located at an open cut coal mine in Central Queensland. Swimming activity and movement of empire gudgeons (Hypseleotris compressa) were assessed during a two-week exposure using video-tracking software. Increased activity was observed in exposed fish after 7 and 14 days. Specifically, we found a significant increase in the mean velocity and mobility of fish exposed to CMW1 treatments. Exposed fish also spent on average 23% more time in the peripheral zone compared to controls after 14-d exposures. A similar response pattern was observed in fish exposed to CMW2, but differences between treated and control fish did not generally reach statistical significance. Alterations to normal swimming activity and movement patterns can be indicative of a stress response in fish, and could subsequently lead to negative population-level impacts by increasing the conspicuousness of exposed individuals to predators, or by altering foraging abilities. More research is warranted to explore relationships between behavioural and physiological outcomes, including endocrine disruption, and subsequent population-level outcomes in aquatic organisms at risk of exposure to coal process-affected water.

Chronic unpredictable mild stress decreases BDNF and NGF levels and Na(+),K(+)-ATPase activity in the hippocampus and prefrontal cortex of mice: antidepressant effect of chrysin.

Our working hypothesis is that brain neurotrophins and brain Na(+),K(+)-ATPase may be strongly associated with the occurrence of depression in animals subjected to chronic unpredictable mild stress (CUMS). Still, we believe that chrysin, a natural and bioactive flavonoid found in honey and some plants, can provide satisfactory effects on antidepressant therapy. Thus, we aimed to evaluate the effect of CUMS on brain-derived neurotropic factor (BDNF) and nerve growth factor (NGF) levels as well as the Na(+),K(+)-ATPase activity in the hippocampus and prefrontal cortex of female mice. We also aimed to examine the effect of a 28-day oral treatment with chrysin (5 or 20mg/kg) in female mice subjected to CUMS, comparing to the effect of fluoxetine. Results showed that CUMS applied for 28days induced a decrease in BDNF and NGF levels as well as in the Na(+),K(+)-ATPase activity. CUMS also promoted a depressive status in the swimming forced test (FST), in the sucrose preference test, and in corticosterone levels. Chrysin (20mg/kg) and fluoxetine also occasioned the up-regulation of BDNF and NGF levels in non-stressed mice and in mice subjected to CUMS. CUMS decreased non-protein thiol (NPSH) levels and increased reactive oxygen species (ROS) levels. In response to these changes, the glutathione reductase (GR), glutathione peroxidase (GPx) and catalase (CAT) activities were increased in mice exposed to CUMS. Chrysin and fluoxetine treatments protected against all these alterations, suggesting the involvement of the antioxidant function in the antidepressant effect of chrysin and fluoxetine. In conclusion, CUMS decreased BDNF and NGF levels as well as the Na(+),K(+)-ATPase activity in mice. Chrysin presented antidepressant effect in mice on behavioral, neurotrophic and biochemistry parameters equivalent to fluoxetine. Furthermore, we suggest that the up-regulation of BDNF and NGF levels is a mechanism possibly involved in the antidepressant effect of chrysin in mice.

Neuroprotective effects of swimming training in a mouse model of Parkinson's disease induced by 6-hydroxydopamine.

Parkinson's disease (PD) is characterized by progressive dopamine (DA) depletion in the striatum. Exercise has been shown to be a promising non-pharmacological approach to reduce the risk of neurodegeneration diseases. This study was designed to investigate the potential neuroprotective effect of swimming training (ST) in a mouse model of PD induced by 6-hydroxydopamine (6-OHDA) in mice. The present study demonstrated that a 4-week ST was effective in attenuating the following impairments resulting from 6-OHDA exposure: (i) depressive-like behavior in the tail suspension test; (ii) increase in the number of falls in the rotarod test; (iii) impairment on long-term memory in the object recognition test; (iv) increase of the reactive species and interleukin 1-beta (IL-1ß) levels; (v) inhibition of the glutathione peroxidase (GPx) activity; (vi) rise of the glutathione reductase (GR) and glutathione S-transferase (GST) activities and vii) decrease of DA, homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) levels. The mechanisms involved in this study are the modulation of GPx, GR and GST activities as well as IL-1ß level in a PD model induced by 6-OHDA, protecting against the decrease of DA, DOPAC and HVA levels in the striatum of mice. These findings reinforce that one of the effects induced by exercise on neurodegenerative disease, such as PD, is due to antioxidant and anti-inflammatory properties. We suggest that exercise attenuates cognitive and motor declines, depression, oxidative stress, and neuroinflammation induced by 6-OHDA supporting the hypothesis that exercise can be used as a non-pharmacological tool to reduce the symptoms of PD.

Variable growth responses of water thyme (Hydrilla verticillata) to whole-cell extracts of Cylindrospermopsis raciborskii.

Static-renewal ecotoxicity trials monitored growth of Hydrilla verticillata in conjunction with exposure to Cylindrospermopsis raciborskii whole-cell extracts containing the cyanotoxin cylindrospermopsin (CYN). Maximum exposure concentrations were 400 microg L(-1) CYN over 14 days. The responses of Hydrilla to the treatments were variable according to the toxin concentrations and lengths of exposure. Plant deaths, chlorosis, and necrosis were not recorded from treated plants. However, Hydrilla experienced significant growth stimulation and redistribution of plant resources in conjunction with exposure to the whole-cell extracts. Root production was particularly impacted. The results of this study imply that root production could aid in reducing C. raciborskii cell concentrations and CYN toxicity. Results of chlorophyll analyses differed, indicating that CYN in whole-cell extracts might exert complex effects on photosynthesis. This is the first study to describe the responses of an aquatic macrophyte following exposure to C. raciborskii whole-cell extracts containing the cyanotoxin cylindrospermopsin.

Multiple-organ toxicity resulting from cylindrospermopsin exposure in tadpoles of the cane toad (Bufo marinus).

Histological examinations were made of cane toad (Bufo marinus) tadpoles after exposure to freeze-thawed Cylindrospermopsis raciborskii whole cell extracts and live C. raciborskii cultures containing sublethal concentrations of the blue-green algal toxin, cylindrospermopsin (CYN). Toxin exposure resulted in tissue injuries to multiple organs, with particular severity noted in the liver, intestine, nephric ducts and gill epithelia. The extent of cellular damage was similar across trials exposing tadpoles to aqueous and cell-bound toxins, despite unequal toxin concentrations being present in each. It was concluded that the presence of cell-bound toxin, which may be directly ingested via grazing, plays a crucial role in the exertion of histological effects in B. marinus. This work provides baseline information regarding the ecotoxicity of CYN toward amphibians. The range of cellular effects noted in CYN-exposed tadpoles suggests that toxic C. raciborskii blooms could represent considerable health risks to amphibian populations and indicate potentially far-reaching ecological impacts of toxic C. raciborskii blooms.

Mortality and toxin bioaccumulation in Bufo marinus following exposure to Cylindrospermopsis raciborskii cell extracts and live cultures.

Cylindrospermopsis raciborskii is a cyanobacterium responsible for the production of the toxin, cylindrospermopsin (CYN). Tadpoles of the cane toad Bufo marinus were exposed to freeze-thawed whole cell extracts or live cultures of C. raciborskii containing maximum CYN concentrations of 400 microg L-1 or 232 microg L-1, respectively. Exposure to live culture treatment solutions resulted in up to 66% mortality of B. marinus, whereas tadpoles exposed to whole cell extracts containing similar toxin concentrations survived. Decreases in relative growth rates and time spent for swimming were recorded from tadpoles during both types of exposure regimes. Bioconcentration of CYN was not evident following exposure to whole cell extracts containing extracellular toxin. In contrast exposure to live cultures, which contained cell-bound toxin, resulted in maximum average tissue concentrations of 895 microg free-CYN kg-1 fresh weight. This is the first investigation of C. raciborskii exposure effects and toxin bioaccumulation in the developmental stages of an amphibian.

Influence of intracellular toxin concentrations on cylindrospermopsin bioaccumulation in a freshwater gastropod (Melanoides tuberculata).

Scant information is available regarding the bioaccumulation of cylindrospermopsin (CYN) in aquatic organisms, particularly in invertebrates. This study examined toxin bioconcentration and bioaccumulation in the aquatic snail, Melanoides tuberculata, following exposure to freeze-thawed whole cell extracts and a live Cylindrospermopsis raciborskii culture containing CYN. Both bioconcentration and bioaccumulation were evident, but exposure to toxin in the freeze-thawed solutions resulted in minor tissue contamination compared with that resulting from live C. raciborskii exposure. Thus, whilst CYN uptake resulted from both extracellular and intracellular exposures, the availability of intracellular toxin was critical in affecting tissue CYN values. M. tuberculata did not bioconcentrate CYN into the shell. Bioaccumulation of the analog deoxy-CYN was also recorded. Knowledge of intracellular toxin concentrations may be critical in evaluating the bioaccumulation, ecological and human health risks associated with contaminated systems.

The effects of the ciliate Paramecium cf. caudatum Ehrenberg on toxin producing Cylindrospermopsis isolated from the Fitzroy River, Australia.

The large ciliate Paramecium cf. caudatum Ehrenberg was found to be a successful grazer of toxin producing Cylindrospermopsis in the laboratory. The feeding rate increased with increasing cell concentration to 1367 cell animal hr-1 at 4.1 x 10(5) cells mL-1 but declined slightly at cell concentrations greater than this. Preliminary studies on the effects of this grazing on toxin concentrations in cultures of both straight and coiled forms of Cylindrospermopsis resulted in the production of different amounts of the toxin cylindrospermopsin in the different isolates. Differences in toxin production were also found between cultured and field populations from the lower Fitzroy River indicating that toxin production may be influenced by a suite of genetic and environmental factors. The proven ability of this ciliate to graze toxic Cylindrospermopsis provides some insight into interactions that may be able to control some toxic blooms in semiarid Australian conditions.