Sorption, degradation and microbial toxicity of chemicals associated with hydraulic fracturing fluid and produced water in soils.

Spills of hydraulic fracturing (HF) fluids and of produced water during unconventional gas extraction operations may cause soil contamination. We studied the degradation and microbial toxicity of selected HF chemical components including two biocides (methylisothiozolinone- MIT, chloromethylisothiozolinone- CMIT), a gel-breaker aid (triethanolamine -TEA), and three geogenic chemicals (phenol, m-cresol and p-cresol) in ultrapure water, HF fluid and produced water in five different soil types (surface and subsurface soils). The degradation of the two biocides (in soils treated with HF fluid or ultrapure water) and of the three geogenic chemicals (in soils treated with produced water) was rapid (in all cases DT50 values < 2 days in surface soils). In contrast, the loss of TEA was much slower in soils, especially in those treated with HF fluid (DT50 > 30 days). Sorption coefficients (Koc in L/Kg) in these soils ranged from 71 to 733 for TEA, 64-408 for MIT and 11-72 for CMIT. In terms of soil microbial toxicity, exposure to HF fluid and produced water reduced microbial respiration, albeit temporarily. The overall microbial activities in surface soils contaminated with produced water had fully recovered in most soils. In contrast, the HF fluid addition to soils completely inhibited the nitrification in all soils, with little recovery over the 60 day experimental period. In the case of produced water exposure, three out of five surface soils showed complete recovery in nitrification during the study period. The functional genes for nitrogen fixation (nifH) and carbon cycling (GA1) and microbial community composition (16 S rRNA) were significantly affected by HF fluid in some soils. Overall, the study shows that the HF fluid can have significant detrimental impact on soil microbial functions, especially on nitrogen cycling. More work is needed to identify the exact cause of microbial toxicity in soils contaminated with HF fluid.

The influence of salinity on the chronic toxicity of shale gas flowback wastewater to freshwater organisms.

The potential environmental risk associated with flowback waters generated during hydraulic fracturing of target shale gas formations needs to be assessed to enable management decisions and actions that prevent adverse impacts on aquatic ecosystems. Using direct toxicity assessment (DTA), we determined that the shale gas flowback wastewater (FWW) from two exploration wells (Tanumbirini-1 and Kyalla 117 N2) in the Beetaloo Sub-basin, Northern Territory, Australia were chronically toxic to eight freshwater biota. Salinity in the respective FWWs contributed 16% and 55% of the chronic toxicity at the 50% effect level. The remaining toxicity was attributed to unidentified chemicals and interactive effects from the mixture of identified organics, inorganics and radionuclides. The most sensitive chronic endpoints were the snail (Physa acuta) embryo development (0.08-1.1% EC10), microalga (Chlorella sp. 12) growth rate inhibition (0.23-3.7% EC10) and water flea (Ceriodaphnia cf. dubia) reproduction (0.38-4.9% EC10). No effect and 10% effect concentrations from the DTA were used in a species sensitivity distribution to derive "safe" dilutions of 1 in 300 and 1 in 1140 for the two FWWs. These dilutions would provide site-specific long-term protection to 95% of aquatic biota in the unlikely event of an accidental spill or seepage.

Using bioanalytical tools to detect and track organic micropollutants in the Ganga River near two major cities.

Major rivers in India are subject to ongoing impacts from urban drain discharges, most of which contain high levels of domestic and industrial wastewater and stormwater. The aim of the present study was to determine the levels of bioactive organic micropollutants at the discharge points of major urban drains in comparison to upstream and downstream sites. To achieve this, we employed a panel of in vitro bioanalytical tools to quantify estrogenic, androgenic, progestogenic, glucocorticoid and peroxisome proliferator-like activity in water extracts collected from two Indian cities in the Ganga Basin. Cytotoxicity of the water extracts in a human-derived cell line and the potential to cause oxidative stress in a fish cell line were also investigated. We found high levels of activity for all endpoints in samples directly receiving urban drain discharge and low levels at sites upstream from drain discharges. Estrogenicity was detected at levels equivalent to 10 ng/L 17ß-estradiol, representing a high likelihood of biomarker effects in fish. Sites located downstream from drain discharges exhibited low to intermediate activity in all assays. This study demonstrates the importance of managing urban drain discharges and the utility of applying bioanalytical tools to assess water quality.

Microplastics in municipal mixed-waste organic outputs induce minimal short to long-term toxicity in key terrestrial biota.

Sustainable alternatives to landfill disposal for municipal mixed wastes represents a major challenge to governments and waste management industries. In the state of New South Wales (NSW) Australia, mechanical biological treatment (MBT) is being used to reduce the volume and pathogen content of organic matter isolated from municipal waste. The product of this treatment, a compost-like output (CLO) referred to as mixed waste organic output (MWOO), is being recycled and applied as a soil amendment. However, the presence of contaminants in MWOO including trace organics, trace metals and physical contaminants such as microplastic fragments has raised concerns about potential negative effects on soil health and agriculture following land application. Here, we used multiple lines of evidence to examine the effects of land application of MWOO containing microplastics in three soils to a variety of terrestrial biota. Treatments included unamended soil, MWOO-amended soil and MWOO-amended soil into which additional high-density polyethylene (HDPE), polyethylene terephthalate (PET), or polyvinyl chloride (PVC) microplastics were added. Tests were conducted in soil media that had been incubated for 0, 3 or 9 months. Addition of microplastics had no significant negative effect on wheat seedling emergence, wheat biomass production, earthworm growth, mortality or avoidance behaviour and nematode mortality or reproduction compared to controls. There was also little evidence the microplastics affected microbial community diversity, although measurements of microbial community structure were highly variable with no clear trends.

De novo assembly and analysis of changes in the protein-coding transcriptome of the freshwater shrimp Paratya australiensis (Decapoda: Atyidae) in response to acid sulfate drainage water.

BACKGROUND: The atyid shrimp Paratya australiensis occurs in surface freshwater habitats throughout eastern Australia and has been used to study the ecotoxicology of contaminants such as pesticides and metals. The acidification of surface water that can occur after acid sulfate material in soils and sediments is oxidised and subsequently re-wetted is a serious environmental issue in coastal regions and inland riverine floodplains worldwide. Solubilisation of soil-associated minerals can result in high waterborne concentrations of mineral salts and dissolved metals, which together with low pH represent a potential threat to aquatic ecosystems in affected regions. The aims of the present study were to gain insight into stress responses induced by exposure to acid drainage water (ADW) in P. australiensis by determining changes in the abundance of protein-coding transcripts and to generate a comprehensive transcriptomic resource to facilitate further research into gene regulation or protein structure and function in this species. Adult P. australiensis were exposed for 24 h to undiluted ADW, 50 % ADW diluted in river water, or to river water as control, and high-throughput mRNA sequencing (RNA-Seq) conducted on whole-body tissues. A reference transcriptome was generated using de novo assembly and putative protein-coding regions were identified and annotated. Changes in transcript abundance in response to ADW exposure were determined by aligning reads to the reference transcriptome and quantifying coverage. RESULTS: A high proportion of arthropod benchmarking universal single-copy orthologues were present in the reference transcriptome. Functions associated with cuticle biosynthesis and oxidative stress were significantly enriched in the lists of transcripts exhibiting differential abundance in either direction after exposure to 50 % or 100 % ADW. Transcripts involved in osmoregulation exhibited decreased abundance following exposure to ADW. The transcriptome contained full-length coding sequences for numerous proteins known to be involved in environmental response pathways, including two putative metallothioneins, four glutathione peroxidases and 19 nuclear receptors. CONCLUSIONS: The results of the present study provide insight into stress response pathways induced in crustaceans by short-term exposure to multiple stressors present in ADW such as low pH, high salinity and dissolved metals, and represent a resource for future toxicogenomics and protein functional studies in P. australiensis.

In vitro nuclear receptor activity and in vivo gene expression analysis in Murray-Darling rainbowfish (Melanotaenia fluviatilis) after short-term exposure to fluoxetine.

Fluoxetine (FLX) is one of numerous pharmaceuticals found in treated municipal wastewater discharged to the environment. In the present study, we investigated the effects of short-term (96h) waterborne FLX exposure (1µg/L or 100µg/L) on the expression of selected genes in brain, liver, and gonads of female Murray-Darling rainbowfish (Melanotaenia fluviatilis), a small-bodied teleost of ecotoxicological relevance in the Australasia region. Plasma 17ß-estradiol (E2) levels were also determined. In the brain, no significant changes in mRNA levels were observed for the selected genes. In ovaries, 100µg/L FLX caused a 10-fold downregulation of aromatase A (cyp19a1a) mRNA and a 4-fold upregulation of estrogen receptor α (esr1) mRNA levels. In liver, mRNA levels for vitellogenin A (vtga) and choriogenin L (chgl) were downregulated by 50-fold and 18-fold compared with controls, respectively, in response to 100µg/L FLX. Concentrations of E2 in plasma were significantly lower than controls in response to 100µg/L FLX. This could be attributable to a decrease in estrogen biosynthesis as a result of the observed downregulation of cyp19a1a mRNA. To establish whether the observed changes in gene expression could be explained by the modulation of selected nuclear receptors by FLX, we employed panel of reporter gene assays in agonistic and antagonistic modes. Apart from minor activation of ERα after exposure to high concentrations (5µM), FLX did not activate or inhibit the nuclear receptors tested. Further study is required to determine whether the observed downregulation of ovarian aromatase expression and liver estrogen-regulated genes also occurs at environmentally relevant FLX concentrations over longer exposure periods.

Geogenic Factors as Drivers of Microbial Community Diversity in Soils Overlying Polymetallic Deposits.

This study shows that the geogenic factors landform, lithology, and underlying mineral deposits (expressed by elevated metal concentrations in overlying soils) are key drivers of microbial community diversity in naturally metal-rich Australian soils with different land uses, i.e., agriculture versus natural bushland. One hundred sixty-eight soil samples were obtained from two metal-rich provinces in Australia, i.e., the Fifield Au-Pt field (New South Wales) and the Hillside Cu-Au-U rare-earth-element (REE) deposit (South Australia). Soils were analyzed using three-domain multiplex terminal-restriction-fragment-length-polymorphism (M-TRFLP) and PhyloChip microarrays. Geogenic factors were determined using field-mapping techniques and analyses of >50 geochemical parameters. At Fifield, microbial communities differed significantly with geogenic factors and equally with land use (P < 0.05). At Hillside, communities in surface soils (0.03- to 0.2-m depth) differed significantly with landform and land use (P < 0.05). Communities in deeper soils (>0.2 m) differed significantly with lithology and mineral deposit (P < 0.05). Across both sites, elevated metal contents in soils overlying mineral deposits were selective for a range of bacterial taxa, most importantly Acidobacteria, Bacilli, Betaproteobacteria, and Epsilonproteobacteria. In conclusion, long-term geogenic factors can be just as important as land use in determining soil microbial community diversity.

Di-n-butyl phthalate causes estrogenic effects in adult male Murray rainbowfish (Melanotaenia fluviatilis).

Phthalic acid esters (PAEs) are a class of synthetic industrial chemicals commonly found in the aquatic environment worldwide. PAEs have been recognised as anti-androgens in male mammals but little is known about their endocrine disrupting effects in fish. This study investigated the effects of 7-day exposures to nominal (measured) concentrations of 125 (62), 250 (140), 500 (230) and 1,000 (383) µg/L of di-n-butyl phthalate (DnBP) on the biomarkers of reproduction in adult male Murray River rainbowfish (Melanotaenia fluviatilis) using molecular, biochemical and histological endpoints. None of the tested concentrations of DnBP had any effect on survival or the vital body indices of the fish. The sizes of spermatogonia, Type A and B spermatocytes and spermatids were significantly smaller relative to the controls after treatment with DnBP. This was accompanied by a significant increase in the proportion of spermatogonia in fish treated with 250-1,000 µg/L of DnBP in comparison to the unexposed fish. At the end of the exposure period, the expressions of the transcripts for the androgen receptors α and ß were significantly elevated in the livers of the fish treated with 500 and 1,000 µg/L of DnBP. In addition, there was also an increase in the circulating concentrations of vitellogenin in the plasma in the higher treatment groups. An induction in the activity of aromatase was noted in the brains of 1,000 µg/L DnBP-treated fish. This was accompanied by an increase in the hepatic expression of the genes (here and later, whenever the phrase gene expression is used as a synonym for gene transcription although it is acknowledged that it is also regulated, e.g., by translation, mRNA stability and protein stability) encoding for the oestrogen receptors α and ß and choriogenin L. Collectively, an increase in the proportion of spermatogonia in the testes, the upregulation of the genes for the oestrogen receptors and choriogenin in the liver, an induction in the brain aromatase activity and the increase in the circulating levels of plasma vitellogenin suggest that continuous exposures for 7 days to sub-acute concentrations of DnBP can adversely affect the reproductive health of the male Murray rainbowfish by an estrogenic mode of action.

Effects of the commercial antiandrogen flutamide on the biomarkers of reproduction in male Murray rainbowfish (Melanotaenia fluviatilis).

The endocrine responses in male Murray rainbowfish (Melanotaenia fluviatilis) were evaluated after exposures to biologically active concentrations of the nonsteroidal pharmaceutical, flutamide. Fish were exposed to nominal concentrations of 125 µg/L, 250 µg/L, 500 µg/L, and 1000 µg/L of flutamide for 7 d, after which plasma vitellogenin concentration; brain aromatase activity; and hepatic expression of the genes for vitellogenin, choriogenin, and androgen and estrogen receptors were assessed. Qualitative assessment of the testes of the fish exposed to flutamide exhibited hindrance in the transformation of spermatogonia to spermatozoa and increased testicular anomalies, such as multinucleated and pyknotic cells and interstitial fibrosis. An increase in the hepatosomatic index with respect to the controls was noted after treating the fish with flutamide at all concentrations. Vitellogenin was induced in plasma in the 1000 µg/L flutamide group. The activity of aromatase in the brain declined significantly after exposures to flutamide at all concentrations. Males exposed to 1000 µg/L of flutamide showed a downregulation in the genes encoding androgen receptors α and ß. The expression of the gene for the estrogen receptor α was induced and of vitellogenin was downregulated after treatment with 250 µg/L to 1000 µg/L of flutamide. The results suggest that 7-d exposures to 125 µg/L to 1000 µg/L flutamide can impair the reproductive endocrine system in male Murray rainbowfish at multiple levels by an antiandrogenic mode of action.

Influence of geogenic factors on microbial communities in metallogenic Australian soils.

Links between microbial community assemblages and geogenic factors were assessed in 187 soil samples collected from four metal-rich provinces across Australia. Field-fresh soils and soils incubated with soluble Au(III) complexes were analysed using three-domain multiplex-terminal restriction fragment length polymorphism, and phylogenetic (PhyloChip) and functional (GeoChip) microarrays. Geogenic factors of soils were determined using lithological-, geomorphological- and soil-mapping combined with analyses of 51 geochemical parameters. Microbial communities differed significantly between landforms, soil horizons, lithologies and also with the occurrence of underlying Au deposits. The strongest responses to these factors, and to amendment with soluble Au(III) complexes, was observed in bacterial communities. PhyloChip analyses revealed a greater abundance and diversity of Alphaproteobacteria (especially Sphingomonas spp.), and Firmicutes (Bacillus spp.) in Au-containing and Au(III)-amended soils. Analyses of potential function (GeoChip) revealed higher abundances of metal-resistance genes in metal-rich soils. For example, genes that hybridised with metal-resistance genes copA, chrA and czcA of a prevalent aurophillic bacterium, Cupriavidus metallidurans CH34, occurred only in auriferous soils. These data help establish key links between geogenic factors and the phylogeny and function within soil microbial communities. In particular, the landform, which is a crucial factor in determining soil geochemistry, strongly affected microbial community structures.

Bacterial communities associated with a mineral weathering profile at a sulphidic mine tailings dump in arid Western Australia.

We investigated bacterial community assemblages and functions down a hill slope contaminated by tailings from a volcanogenic massive sulphide mine in arid Western Australia. Weathering of waste rock, high in S and Fe, had resulted in a varying elemental dispersal down a face of the tailings hill. Bacterial community assemblage, characterised by PCR-DGGE fingerprinting, was significantly associated with electrical conductivity (E.C.) (ρ = 0.664; P < 0.01). Analysis of mobile salts showed that E.C. values were driven by ionic S, Zn, Cl and Al. The bacterial community assemblage was directly characterised across an E.C. gradient using an oligonucleotide microarray (PhyloChip). The dominant taxa at the site were Proteobacteria, Actinobacteria and Firmicutes; however, 37 phyla were detected. The most responsive taxa to variation in E.C. was Acidobacteria (negative correlation). Patterns of heterotrophic processes (BioLog analysis) were also best explained by variation in E.C. (ρ = 0.53; P < 0.01), showing a link between primary mineral weathering by lithotrophic bacteria and abiotic processes, and secondary biogeochemical processes by heterotrophic taxa. These data significantly broaden our knowledge of the bacteria present in metallomorphic ecosystems, establish that mobile phase elements are key drivers of community structure, and that primary biogeochemical cycling is directly influencing other geochemical interactions in the samples.

The effects of stubble retention and nitrogen application on soil microbial community structure and functional gene abundance under irrigated maize.

The effects of agronomic management practices on the soil microbial community were investigated in a maize production system in New South Wales, Australia. The site has been intensively studied to measure the impact of stubble management and N-fertilizer application on greenhouse gas emissions (CO(2) and N(2)O), N-cycling, pathology, soil structure and yield. As all of these endpoints can be regulated by microbial processes, the microbiology of the system was examined. Soil samples were taken after a winter fallow period and the diversity of the bacterial and fungal communities was measured using PCR-denaturing gradient gel electrophoresis. Stubble and N shifted the structure of bacterial and fungal communities with the primary driver being stubble addition on the fungal community structure (P<0.05 for all effects). Changes in C, N (total and NO(3)), K and Na, were correlated (P<0.05) with variation in the microbial community structure. Quantitative PCR showed that nifH (nitrogen fixation) and napA (denitrification) gene abundance increased upon stubble retention, whereas amoA gene numbers were increased by N addition. These results showed that the management of both stubble and N have significant and long-term impacts on the size and structure of the soil microbial community at phylogenetic and functional levels.