Soil uranium concentration at Ranger Uranium Mine Land Application Areas drives changes in the bacterial community.

Soil microorganisms may respond to metal stress by a shift in the microbial community from metal sensitive to metal resistant microorganisms. We assessed the bacterial community from low (2-20 mg kg-1), medium (200-400 mg kg-1), high (500-900 mg kg-1) and very high (>900 mg kg-1) uranium soils at Ranger Uranium Mine in northern Australia through pyrosequencing. Proteobacteria (28.85%) was the most abundant phylum at these sites, followed by Actinobacteria (9.31%), Acidobacteria (7.33%), Verrucomicrobia (2.11%), Firmicutes (2.02%), Chloroflexi (1.11%), Cyanobacteria (0.93%), Planctomycetes (0.82%), Bacteroidetes (0.46%) and Candidate_division_WS3 (Latescibacteria) (0.21%). However, 46.79% of bacteria were unclassified. Bacteria at low U soils differed from soils with elevated uranium. Bacterial OTUs closely related to Kitasatospora spp., Sphingobacteria spp. and Rhodobium spp. were only present at higher uranium concentrations and the bacterial community also changed with seasonal and temporal changes in soil uranium and physicochemical variables. This study using next generation sequencing in association with environmental variables at a uranium mine has laid a foundation for further studies of soil-microbe-metal interactions which may be useful for developing sustainable management and rehabilitation strategies. Furthermore, bacterial species associated with higher uranium may serve as useful indicators of uranium contamination in the wet-dry tropics.

Spatial and Temporal Microbial Patterns in a Tropical Macrotidal Estuary Subject to Urbanization.

Darwin Harbour in northern Australia is an estuary in the wet-dry tropics subject to increasing urbanization with localized water quality degradation due to increased nutrient loads from urban runoff and treated sewage effluent. Tropical estuaries are poorly studied compared to temperate systems and little is known about the microbial community-level response to nutrients. We aimed to examine the spatial and temporal patterns of the bacterial community and its association with abiotic factors. Since Darwin Harbour is macrotidal with strong seasonal patterns and mixing, we sought to determine if a human impact signal was discernible in the microbiota despite the strong hydrodynamic forces. Adopting a single impact-double reference design, we investigated the bacterial community using next-generation sequencing of the 16S rRNA gene from water and sediment from reference creeks and creeks affected by effluent and urban runoff. Samples were collected over two years during neap and spring tides, in the dry and wet seasons. Temporal drivers, namely seasons and tides had the strongest relationship to the water microbiota, reflecting the macrotidal nature of the estuary and its location in the wet-dry tropics. The neap-tide water microbiota provided the clearest spatial resolution while the sediment microbiota reflected current and past water conditions. Differences in patterns of the microbiota between different parts of the harbor reflected the harbor's complex hydrodynamics and bathymetry. Despite these variations, a microbial signature was discernible relating to specific effluent sources and urban runoff, and the composite of nutrient levels accounted for the major part of the explained variation in the microbiota followed by salinity. Our results confirm an overall good water quality but they also reflect the extent of some hypereutrophic areas. Our results show that the microbiota is a sensitive indicator to assess ecosystem health even in this dynamic and complex ecosystem.

Molecular screening of microbial communities for candidate indicators of multiple metal impacts in marine sediments from northern Australia.

Coastal sediments accumulate metals from anthropogenic sources and as a consequence industry is required to monitor sediment health. The total concentration of a metal does not necessarily reflect its potential toxicity or biological impact, so biological assessment tools are useful for monitoring. Rapid biological assessment tools sensitive enough to detect relatively small increases in metal concentrations would provide early warning of future ecosystem impact. The authors investigated in situ populations of Archaea and Bacteria as potential tools for rapid biological assessment in sediment at 4 northern Australian coastal locations over 2 yr, in both wet and dry seasons. The 1 M HCl-extractable concentrations of metals in sediment were measured, and Archaeal and Bacterial community profiles were obtained by next-generation sequencing of sediment deoxyribonucleic acid (DNA). Species response curves were used to identify several taxonomic groups with potential as biological indicators of metal impact. Spatial variation, sediment grain size, water depth, and dissolved oxygen also correlated with microbial population shifts. Seasonal variation was less important than geographic location. Metal-challenge culture trials supported the identification of metal-resistant and -sensitive taxa. In situ Archaea and Bacteria are potentially sensitive indicators for changes in bioavailable concentrations of metals; however, the complexity of the system suggests it is important to identify metal-specific functional genes that may be informed by these sequencing surveys, and thus provide a useful addition to identity-based assays.

Characterisation of microcontaminants in Darwin Harbour, a tropical estuary of northern Australia undergoing rapid development.

The detection of microcontaminants in aquatic environments raises concerns about their potential to exert ecotoxicological effects and impact human health. In contrast to freshwater habitats, little information is available on environmental concentrations in urban estuarine and marine environments. This study investigated an extensive range of organic and inorganic microcontaminants in the Darwin Harbour catchment, a tropical estuary in northern Australia undergoing rapid urbanisation and industrial development. We sampled wastewater effluent and surface water from seven sites in Darwin Harbour for pharmaceuticals and personal care products, alkylphenols, hormones, pesticides, herbicides and metals. In vitro bioassays were used to estimate the (anti)estrogenic and (anti)androgenic activities of samples. Seventy-nine of 229 organic microcontaminants analysed were detected at concentrations ranging from 0.01 to 20 µg/L, with acesulfame, paracetamol, cholesterol, caffeine, DEET and iopromide detected at the highest concentrations in wastewater effluent (20 µg/L, 17 µg/L, 11 µg/L, 11 µg/L, 10 µg/L and 7.6 µg/L, respectively). Levels of estrogenic activity ranged from estradiol equivalency quotients (EEQs) of <0.10 to 6.29±0.16 ng/L while levels of androgenic activity ranged from dihydrotestosterone equivalency quotients (DHTEQs) of <3.50 to 138.23±3.71 ng/L. Environmental concentrations of organic microcontaminants were comparable to ranges reported from aquatic environments worldwide with sewage effluent discharges representing the dominant source of entry into Darwin Harbour. The measured concentration range of DEET was higher than ranges reported in previous studies.

Land application of mine water causes minimal uranium loss offsite in the wet-dry tropics: Ranger Uranium Mine, Northern Territory, Australia.

Ranger Uranium Mine (RUM) is situated in the wet-dry tropics of Northern Australia. Land application (irrigation) of stockpile (ore and waste) runoff water to natural woodland on the mine lease is a key part of water management at the mine. Consequently, the soil in these Land Application Areas (LAAs) presents a range of uranium (U) and other metals concentrations. Knowledge of seasonal and temporal changes in soil U and physicochemical parameters at RUM LAAs is important to develop suitable management and rehabilitation strategies. Therefore, soil samples were collected from low, medium, high and very high U sites at RUM LAAs for two consecutive years and the effect of time and season on soil physicochemical parameters particularly U and other major solutes applied in irrigation water was measured. Concentrations of some of the solutes applied in the irrigation water such as sulphur (S), iron (Fe) and calcium (Ca) showed significant seasonal and temporal changes. Soil S, Fe and Ca concentration decreased from year 1 to year 2 and from dry to wet seasons during both years. Soil U followed the same pattern except that we recorded an increase in soil U concentrations at most of the RUM LAAs after year 2 wet season compared to year 2 dry season. Thus, these sites did not show a considerable decrease in soil U concentration from year 1 to year 2. Sites which contained elevated U after wet season 2 also had higher moisture content which suggests that pooling of U containing rainwater at these sites may be responsible for elevated U. Thus, U may be redistributed within RUM LAAs due to surface water movement. The study also suggested that a decrease in U concentrations in LAA soils at very high U (>900 mg kg(-1)) sites is most likely due to transport of particulate matter bound U by surface runoff and U may not be lost from the surface soil due to vertical movement through the soil profile. Uranium attached to particulate matter may reduce its potential for environmental impact. These findings suggest that U is effectively adsorbed by the soils and thus land application may serve as a useful tool for U management in the wet-dry tropics of northern Australia.

The relationship between early growth and survival of hatchling saltwater crocodiles (Crocodylus porosus) in captivity.

Hatchling fitness in crocodilians is affected by "runtism" or failure to thrive syndrome (FTT) in captivity. In this study, 300 hatchling C. porosus, artificially incubated at 32°C for most of their embryonic development, were raised in semi-controlled conditions, with growth criteria derived for the early detection of FTT (within 24 days). Body mass, four days after hatching (BM4d), was correlated with egg size and was highly clutch specific, while snout-vent length (SVL4d) was much more variable within and between clutches. For the majority of hatchlings growth trajectories within the first 24 days continued to 90 days and could be used to predict FTT affliction up to 300 days, highlighting the importance of early growth. Growth and survival of hatchling C. porosus in captivity was not influenced by initial size (BM4d), with a slight tendency for smaller hatchlings to grow faster in the immediate post-hatching period. Strong clutch effects (12 clutches) on affliction with FTT were apparent, but could not be explained by measured clutch variables or other factors. Among individuals not afflicted by FTT (N = 245), mean growth was highly clutch specific, and the variation could be explained by an interaction between clutch and season. FTT affliction was 2.5 times higher among clutches (N = 7) that hatched later in the year when mean minimum air temperatures were lower, compared with those clutches (N = 5) that hatched early in the year. The results of this study highlight the importance of early growth in hatchling C. porosus, which has implications for the captive management of this species.

The diversity and abundance of polychaetes (Annelida) are altered in sediments impacted by alumina refinery discharge in the Northern Territory, Australia.

We collected polychaete diversity and abundance data at a range of impacted and reference sites near an alumina refinery in Melville Bay, northern Australia. The aims were to measure the impact of sediment modified by the alumina refinery discharge on polychaete communities and secondly to gather baseline data from which to measure future changes. Polychaete communities in both soft-bottom habitats and subtidal areas adjacent to mangrove forests were studied. We also developed and deployed an artificial substratum device to sample polychaetes associated with hard-substrate habitats. For each habitat, polychaete community composition was different between impacted and reference sites and at multiple time points. The impact of future changes either from bioremediation or management practices can be measured against these baseline data. Indicator species analysis was used to identify polychaete species that were significantly different at the locations tested, and we discuss their potential as indicator species.

Fungi outcompete bacteria under increased uranium concentration in culture media.

As a key part of water management at the Ranger Uranium Mine (Northern Territory, Australia), stockpile (ore and waste) runoff water was applied to natural woodland on the mine lease in accordance with regulatory requirements. Consequently, the soil in these Land Application Areas (LAAs) presents a range of uranium concentrations. Soil samples were collected from LAAs with different concentrations of uranium and extracts were plated onto LB media containing no (0 ppm), low (3 ppm), medium (250 ppm), high (600 ppm) and very high (1500 ppm) uranium concentrations. These concentrations were similar to the range of measured uranium concentrations in the LAAs soils. Bacteria grew on all plates except for the very high uranium concentrations, where only fungi were recovered. Identifications based on bacterial 16S rRNA sequence analysis showed that the dominant cultivable bacteria belonged to the genus Bacillus. Members of the genera Paenibacillus, Lysinibacillus, Klebsiella, Microbacterium and Chryseobacterium were also isolated from the LAAs soil samples. Fungi were identified by sequence analysis of the intergenic spacer region, and members of the genera Aspergillus, Cryptococcus, Penicillium and Curvularia were dominant on plates with very high uranium concentrations. Members of the Paecilomyces and Alternaria were also present but in lower numbers. These findings indicate that fungi can tolerate very high concentrations of uranium and are more resistant than bacteria. Bacteria and fungi isolated at the Ranger LAAs from soils with high concentrations of uranium may have uranium binding capability and hence the potential for uranium bioremediation.

The transcriptome and proteome are altered in marine polychaetes (Annelida) exposed to elevated metal levels.

Polychaetes are often used in toxicological studies to understand mechanisms of resistance and for biomarker detection, however, we know of only a few genetic pathways involved in resistance. We found the marine polychaete Ophelina sp.1 (Opheliidae) in sediment containing high copper levels and investigated this phenomenon by measuring metal accumulation in the worms and changes in gene and protein expression. We sequenced the transcriptome of Ophelina sp.1 from both the impacted and reference sediments using 454-sequencing and analysed their proteomes using differential in gel electrophoresis (DIGE). We used the sequenced transcriptome to guide protein identification. Transcripts coding for the copper chaperone, Atox1, were up-regulated in the worms inhabiting the high copper sediment. In addition, genes coding for respiratory proteins, detoxification proteins and cytoskeletal proteins were significantly altered in metal-exposed worms; many of these changes were also detected in the proteome. This dual approach has provided a better understanding of heavy metal resistance in polychaetes and we now have a wider range of suitable indicator genes and proteins for future biomarker development.

The bacterial community associated with the marine polychaete Ophelina sp.1 (Annelida: Opheliidae) is altered by copper and zinc contamination in sediments.

Tolerant species of polychaete worms can survive in polluted environments using various resistance mechanisms. One aspect of resistance not often studied in polychaetes is their association with symbiotic bacteria, some of which have resistance to metals and may help the organism to survive. We used "next generation" 454 sequencing of bacterial 16S rRNA sequences associated with polychaetes from a copper- and zinc-polluted harbor and from a reference site to determine bacterial community structure. We found changes in the bacteria at the polluted site, including increases in the abundance of bacteria from the order Alteromonadales. These changes in the bacteria associated with polychaetes may be relatively easy to detect and could be a useful indicator of metal pollution.