Assessing chronic toxicity of aluminium, gallium and molybdenum in tropical marine waters using a novel bioassay for larvae of the hermit crab Coenobita variabilis.

A novel bioassay is presented that allows for the estimation of the chronic toxicity of contaminants in receiving tropical marine environments. Relevant procedures to identify contaminants of concern and evaluate hazards associated with contamination in these environments have long remained inadequate. The 6-day bioassay is conducted using freshly hatched planktonic larvae of the hermit crab Coenobita variabilis and is targeted at generating environmentally relevant, chronic toxicity data. The developmental endpoint demonstrated consistently high control performance and was validated through the use of copper as a reference toxicant. In addition, the biological effects of aluminium, gallium and molybdenum were assessed. The endpoint expressed high sensitivity to copper (EC10 = 24 µg L-1) and moderate sensitivity to aluminium (EC10 = 312 µg L-1), whereas gallium and molybdenum elicited no obvious effects, even at high concentrations (EC10 > 6000 µg L-1), providing valuable information on the toxicity of these elements in tropical marine waters for derivation of water quality guidelines or testing of compliance limits.

Water quality guideline values for aluminium, gallium and molybdenum in marine environments.

Revised water quality guideline values (WQGVs) are presented for the metals aluminium (Al), gallium (Ga) and molybdenum (Mo) in receiving marine environments. These elements are commonly found in elevated concentrations in alumina refinery waste streams, yet current WQGVs fail to accurately assess the environmental risk. Here, chronic biological effects data we have generated over the course of several years were combined with toxicity data from the open literature to construct species sensitivity distributions (SSDs) which enabled the computation of revised WQGVs for Al, Ga and Mo in marine environments. These procedures are in accordance with internationally recommended derivation procedures, and newly computed WQGVs may be incorporated in regulatory frameworks aimed at sustainable exploitation of environmental resources and ongoing protection of the marine estate. Where the available datasets allowed such distinction, separate SSDs were constructed for temperate and tropical environments and zone-specific WQGVs derived. Extrapolated from the SSDs, WQGVs of 56 µg Al L-1, 800 µg Ga L-1 and 3.88 mg Mo L-1 (in the 0.45-µm filtered fraction) for 95% species protection were recommended for implementation in both temperate and tropical receiving environments. Currently, there is insufficient validation to separate the tropical from the temperate data and in most cases, application of the generic WQGVs is recommended.

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.

A protocol for identifying suitable biomarkers to assess fish health: A systematic review.

BACKGROUND: Biomarkers have been used extensively to provide the connection between external levels of contaminant exposure, internal levels of tissue contamination, and early adverse effects in organisms. OBJECTIVES: To present a three-step protocol for identifying suitable biomarkers to assess fish health in coastal and marine ecosystems, using Gladstone Harbour (Australia) as a case study. METHODS: Prior to applying our protocol, clear working definitions for biomarkers were developed to ensure consistency with the global literature on fish health assessment. First, contaminants of concern were identified based on the presence of point and diffuse sources of pollution and available monitoring data for the ecosystem of interest. Second, suitable fish species were identified using fisheries dependent and independent data, and prioritised based on potential pathways of exposure to the contaminants of concern. Finally, a systematic and critical literature review was conducted on the use of biomarkers to assess the health of fish exposed to the contaminants of concern. RESULTS/DISCUSSION: We present clear working definitions for bioaccumulation markers, biomarkers of exposure, biomarkers of effect and biomarkers of susceptibility. Based on emission and concentration information, seven metals were identified as contaminants of concern for Gladstone Harbour. Twenty out of 232 fish species were abundant enough to be potentially suitable for biomarker studies; five of these were prioritised based on potential pathways of exposure and susceptibility to metals. The literature search on biomarkers yielded 5,035 articles, of which 151met the inclusion criteria. Based on our review, the most suitable biomarkers include bioaccumulation markers, biomarkers of exposure (CYP1A, EROD, SOD, LPOX, HSP, MT, DNA strand breaks, micronuclei, apoptosis), and biomarkers of effect (histopathology, TAG:ST). CONCLUSION: Our protocol outlines a clear pathway to identify suitable biomarkers to assess fish health in coastal and marine ecosystems, which can be applied to biomarker studies in aquatic ecosystems around the world.

Assessing the chronic toxicity of copper and aluminium to the tropical sea anemone Exaiptasia pallida.

The world's most productive bauxite mines and alumina refineries are located in tropical or sub-tropical regions. The discharge water from alumina refineries can contain elevated aluminium (Al, <0.45µm fraction), from 30 to 1000µg/L. There is a need for additional information on the toxicity of Al to aquatic organisms to improve the environmental regulation and management of alumina refinery operations in tropical coastal regions. A 14-d chronic toxicity test was developed for the tropical sea anemone Exaiptasia pallida. Asexual reproduction and growth rates of E. pallida were assessed using the number of lacerates produced and oral disc diameter. The comparative sensitivity of E. pallida was assessed through exposure to a commonly-used reference toxicant, copper (Cu) at 28°C, with asexual reproduction toxicity estimates of 10% (EC10) and 50% (EC50) effect concentrations, calculated as 8.8µg/L (95% confidence limits (CL): 1-18µg/L) and 35µg/L Cu (95% CL: 30-39µg/L), respectively. Growth rate was a suitable additional endpoint (EC50=35µg/L Cu, 95% CL: 23-49µg/L). The EC10 and EC50 for Al (total fraction, based on reproduction) at 28°C were 817µg/L (95% CL: 440-1480µg/L) and 2270µg/L (95% CL: 1600-3900µg/L), respectively. The toxicity of Cu and Al was also assessed at 24°C and 31°C, representing average year-round water temperatures for sub-tropical and tropical Australian coastal environments. Changing the temperature from 28°C to 24°C or 31°C resulted in up to 45% less reproduction of anemones and increased their sensitivity to Cu (EC50s at 24°C=21µg/L, 95% CL: 17-26µg/L and at 31°C=23µg/L, 95% CL: 21-25µg/L). Sensitivity to Al was reduced at 24°C with an EC50 of 8870µg/L (95% CL: 6200-NC). An EC50 for Al at 31°C could not be calculated. This test is a reliable and sensitive addition to the suite of standardised tests currently developed for tropical marine species.

A novel bioassay using the barnacle Amphibalanus amphitrite to evaluate chronic effects of aluminium, gallium and molybdenum in tropical marine receiving environments.

A need exists for appropriate tools to evaluate risk and monitor potential effects of contaminants in tropical marine environments, as currently impact assessments are conducted by non-representative approaches. Here, a novel bioassay is presented that allows for the estimation of the chronic toxicity of contaminants in receiving tropical marine environments. The bioassay is conducted using planktonic larvae of the barnacle Amphibalanus amphitrite and is targeted at generating environmentally relevant, chronic toxicity data for water quality guideline derivation or compliance testing. The developmental endpoint demonstrated a consistently high control performance, validated through the use of copper as a reference toxicant. In addition, the biological effects of aluminium, gallium and molybdenum were assessed. The endpoint expressed high sensitivity to copper and moderate sensitivity to aluminium, whereas gallium and molybdenum exhibited no discernible effects, even at high concentrations, providing valuable information on the toxicity of these elements in tropical marine waters.

A chronic toxicity test for the tropical marine snail Nassarius dorsatus to assess the toxicity of copper, aluminium, gallium, and molybdenum.

Chronic toxicity test methods for assessing the toxicity of contaminants to tropical marine organisms are generally lacking. A 96-h chronic growth rate toxicity test was developed for the larval stage of the tropical dogwhelk, Nassarius dorsatus. Growth rates of N. dorsatus larvae were assessed following exposures to copper (Cu), aluminium (Al), gallium (Ga), and molybdenum (Mo). Exposure to Cu at 28 °C validated the sensitivity of the test method, with 10% (EC10) and 50% (EC50) effect concentrations of 4.2 µg/L and 7.3 µg/L Cu, respectively. The EC10 and EC50 values for Al (<0.45-µm filtered fraction) at 28 °C were 115 µg/L and 185 µg/L, respectively. The toxicity of Cu and Al was also assessed at 24 °C and 31 °C, representing average year-round water temperatures for subtropical and tropical Australian coastal environments. At 24 °C, the growth rate of control larvae was reduced by 52% compared with the growth rate at 28 °C and there was an increase in sensitivity to Cu (EC50 = 4.7 µg/L) but a similar sensitivity to Al (EC50 = 180 µg/L). At 31 °C the control growth rate increased by 35% from that measured at 28 °C and there was reduced sensitivity to both Cu and Al (EC50s = 8.5 µg/L and 642 µg/L, respectively). There was minimal toxicity resulting from Ga (EC50 = 4560 µg/L) and Mo (no effect at ≤7000 µg/L Mo). Environ Toxicol Chem 2016;35:1788-1795. © 2015 SETAC.

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.

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.

Aluminium, gallium, and molybdenum toxicity to the tropical marine microalga Isochrysis galbana.

There is a shortage of established chronic toxicity test methods for assessing the toxicity of contaminants to tropical marine organisms. The authors tested the suitability of the tropical microalga Isochrysis galbana for use in routine ecotoxicology and assessed the effects of 72-h exposures to copper (Cu, a reference toxicant), aluminium (Al), gallium (Ga), and molybdenum (Mo), key metals of alumina refinery discharge, on the growth of I. galbana at 3 temperatures: 24 °C, 28 °C, and 31 °C. The sensitivity of both I. galbana and the test method was validated by the response to Cu exposure, with 10% and 50% effect concentrations (EC10 and EC50) of 2.5 µg/L and 18 µg/L, respectively. The EC10 and EC50 values for total Al at 28 °C were 640 µg/L and 3045 µg/L, respectively. The toxicity of both Cu and Al at 24 °C and 31 °C was similar to that at 28 °C. There was no measurable toxicity from dissolved Ga exposures of up to 6000 µg/L or exposures to dissolved Mo of up to 9500 µg/L. Solubility limits at 28 °C for the dissolved fractions (<10 kDa) of Al, Ga, and Mo were approximately 650 µg/L Al, >7000 µg/L Ga, and >6000 µg/L Mo. In test solutions containing >650 µg/L total Al, dissolved and precipitated forms of Al were present, with precipitated Al becoming more dominant as total Al increased. The test method proved suitable for routine ecotoxicology, with I. galbana showing sensitivity to Cu but Al, Ga, and Mo exhibiting little to no toxicity to this species.

Saltwater intrusion history shapes the response of bacterial communities upon rehydration.

Saltwater intrusion (SWI) can result in the loss of dominant vegetation from freshwater habitats. In northern Australia, sea level is predicted to rise 17-50 cm by 2030-2070. This will exacerbate the impact of SWI, threatening Ramsar-listed habitats. Soil bacteria in these habitats play a significant role in biogeochemical cycling, regulating availability of essential nutrients such as nitrogen to vegetation. However, there is limited understanding as to how SWI will impact these soil bacteria. Floodplain soil samples were collected from the South Alligator River floodplain in Northern Australia from sites with contrasting histories of SWI. A SWI event was simulated over 7 days with treatments of saltwater and freshwater. Bacterial community composition before and after treatment were measured using next generation sequencing of bacterial DNA. Sites with no history of SWI showed no significant changes in community taxonomic composition following treatments, suggesting the community at these sites have broad functional capacity which may be due to their historic conditioning over many years. Sites with a history of SWI showed a significant response to both treatments. Following saltwater treatment, there was an increase in sulfate-reducing bacteria, which are known to have an impact on carbon and nitrogen cycling. We suggest that the impact of SWI causes a shift in the soil bacteria which alters the community to one which is more specialised, with implications for the cycling of essential elements and nutrients.

Identification of genes in the tomato big bud phytoplasma and comparison to those in sweet potato little leaf-V4 phytoplasma.

Genetic relatedness of phytoplasmas is commonly defined on the basis of differences in the highly conserved 16S rRNA gene, which may not resolve closely related phytoplasmas. An example of this is the closely related tomato big bud (TBB) and sweet potato little leaf strain V4 (SPLL-V4) phytoplasmas, which cannot easily be differentiated by their 16S rRNA gene sequences. This study aimed to identify genes on the TBB phytoplasma chromosome which could be used to examine genetic variation between these two closely related phytoplasmas. Random clones generated from TBB phytoplasma genomic DNA were sequenced and characterized by database analysis. Twenty-three genes were identified within 19 random clones, which contained approximately 18.0 kbp of TBB phytoplasma genomic DNA. Half of the TBB phytoplasma genes identified were involved in DNA replication, transcription and translation. The remaining TBB phytoplasma genes were involved in protein secretion, cellular processes and energy metabolism. Phylogenetic analysis of representative genes showed that the TBB phytoplasma grouped with the mycoplasmas with the exception of the TBB phytoplasma secA gene, which grouped with the onion yellows phytoplasma. PCR primers were designed based on the new genes and tested on isolates of the TBB and SPLL-V4 phytoplasmas. Most primers amplified a product from TBB and SPLL-V4 phytoplasma samples. When amplified products were subjected to RFLP analysis, the restriction patterns were the same as the respective original clones. This result confirmed that the same sequence had been amplified by PCR and showed that these isolates were indistinguishable using the new genes. This study showed that in fact the TBB and SPLL-V4 phytoplasmas are closely related even with the analysis of new genes. These new genes have, however, provided insight into the biology of the TBB and SPLL-V4 phytoplasmas.