Developing simple indicators of nitrogen and phosphorus removal in constructed stormwater wetlands.

Quantifying pollutant removal by stormwater wetlands requires intensive sampling which is cost-prohibitive for authorities responsible for a large number of wetlands. Wetland managers require simple indicators that provide a practical means of estimating performance and prioritising maintenance works across their asset base. We therefore aimed to develop vegetation cover and metrics derived from monitoring water level, as simple indicators of likely nutrient pollutant removal from stormwater wetlands. Over a two-year period, we measured vegetation cover and water levels at 17 wetlands and used both to predict nitrogen (N) and phosphorus (P) removal. Vegetation cover explained 48 % of variation in total nitrogen (TN) removal; with a linear relationship suggesting an approximate 9 % loss in TN removal per 10 % decrease in vegetation cover. Vegetation cover is therefore a useful indicator of TN removal. Further development of remotely-sensed data on vegetation configuration, species and condition will likely improve the accuracy of TN removal estimates. Total phosphorus (TP) removal was not predicted by vegetation cover, but was weakly related to the median water level which explained 25 % of variation TP removal. Despite weak prediction of TP removal, metrics derived from water level sensors identified faults such as excessive inflow and inefficient outflow, which in combination explained 50 % of the variation in the median water level. Monitoring water levels therefore has the potential to detect faults prior to loss of vegetation cover and therefore TN removal, as well as inform the corrective action required.

Empirical evidence of climate change and urbanization impacts on warming stream temperatures.

Climate change and urbanization threaten streams and the biodiversity that rely upon them worldwide. Emissions of greenhouse gases are causing air and sea surface temperatures to increase, and even small areas of urbanization are degrading stream biodiversity, water quality and hydrology. However, empirical evidence of how increasing air temperatures and urbanization together affect stream temperatures over time and their relative influence on stream temperatures is limited. This study quantifies changes in stream temperatures in a region in South-East Australia with an urban-agricultural-forest landcover gradient and where increasing air temperatures have been observed. Using Random Forest models we identify air temperature and urbanization drive increasing stream temperatures and that their combined effects are larger than their individual effects occurring alone. Furthermore, we identify potential mitigation measures useful for waterway managers and policy makers. The results show that both local and global solutions are needed to reduce future increases to stream temperature.

Species-level dataset is required for setting biodiversity conservation priorities for freshwater macroinvertebrates in Melbourne streams.

Family-level identification of freshwater macroinvertebrates is often used to monitor the health of streams due to the lower cost and higher accuracy of identification compared to identifying species. While data on the presence of families from stream monitoring programs can also be used for biodiversity conservation planning, the ability of family-level datasets to accurately reflect regional biodiversity patterns for freshwater macroinvertebrates in Australia remains untested. This study compares family-level and species-level datasets for freshwater insects identified using morphological features and collected over 16 years from 140 sites in Greater Melbourne, Australia. Similar to the results of other studies, our results show a strong positive relationship between family- and species-level taxon richness. However, using the planning software Marxan to compare conservation priorities in our study region, we found that a data analysis of the family-level dataset underestimated the minimum sampling effort required to accurately reflect species diversity. It also identified sub-optimal conservation priority sites and overlooked regionally rare species. We recommend that aquatic macroinvertebrate monitoring programs aimed at understanding regional biodiversity patterns and conservation priorities should routinely include species-level identification, which is now becoming feasible with advances in molecular methods.

Riparian buffers: Disrupting the transport of E. coli from rural catchments to streams.

High levels of E. coli and associated faecal microbes in waterways as a result of agricultural and residential land use can pose environmental, human health, and economic risks. This study aims to understand the impacts of land use, climatic variables, and riparian buffers on in-stream E. coli concentrations. Flow, temperature, and E. coli were monitored during three sampling campaigns within eleven independent catchments. These catchments have varying land use and extents of riparian buffer coverage. Results showed that catchments with predominantly agricultural and residential land uses (average = 349.7 MPN/100 mL) had higher E. coli concentrations than predominantly forested catchments (average = 111.8 MPN/100 mL). However, there were no statistically significant differences in E. coli concentrations between the agricultural and residential land uses. Riparian buffers appear to reduce E. coli concentrations in streams, as indicated by significant negative correlations between in-stream E. coli concentrations with the riparian buffer areal coverage (Pearson's r = -0.95, Spearman's ρ = -0.90) and the ratio of buffer length to stream length (Pearson's r = -0.87, Spearman's ρ = -0.90). We find that riparian buffers potentially disrupt transport pathways that govern E. coli movement, which in-turn can affect the concentration-discharge relationship. This reinforces the importance of protecting and restoring riparian buffers along drainage lines in agricultural and rural-residential catchments to improve downstream microbial water quality.

Understanding and managing the interactive impacts of growth in urban land use and climate change on freshwater biota: A case study using the platypus (Ornithorhynchus anatinus).

Globally, urban expansion and climate change interact to threaten stream ecosystems and are accelerating the loss of aquatic biodiversity. Waterway managers urgently need tools to understand the potential combined impacts of urbanization and climate change and to identify effective mitigating management interventions for protecting freshwater biota. We address this challenge using the semi-aquatic mammal the platypus (Ornithorhynchus anatinus) as a focal species. We developed high-resolution environmental spatial data for stream networks and spatially explicit habitat suitability models (HSMs) to explore the impact of threats and to identify the combination of management actions most likely to maintain or improve habitat suitability over the next 50 years in greater Melbourne, Australia. We developed and evaluated platypus HSMs (males-and-females and females-only) including validation using an independent environmental DNA data set. Platypus occurred more commonly in larger, cooler streams with greater catchment-weighted discharge, following periods of greater stream flow. They were positively associated with near-stream forest cover and negatively associated with annual air temperature and urban stormwater runoff. Extensive reductions in suitable platypus habitat are predicted to occur under urbanization and climate change scenarios, with the greatest threat expected from reduced streamflows. This emphasizes the importance of maintaining flow regimes as part of conserving platypus in the region; however, substantial additional benefit is predicted by concurrent riparian revegetation and urban stormwater management efforts (that also have the potential to contribute to the streamflow objectives). Provision of adequate streamflows in a future with increasing water demands and water security requirements will likely require creative integrated water management solutions. Our high-resolution stream network and HSMs have allowed predictions of potential range-shifts due to urban expansion and climate change impacts at management-relevant scales and at the whole-of-landscape scale. This has enabled systematic strategic planning, priority action planning and target setting in strategic policy development.

Multispecies models reveal that eDNA metabarcoding is more sensitive than backpack electrofishing for conducting fish surveys in freshwater streams.

Environmental DNA (eDNA) sampling can provide accurate, cost-effective, landscape-level data on species distributions. Previous studies have compared the sensitivity of eDNA sampling to traditional sampling methods for single species, but similar comparative studies on multispecies eDNA metabarcoding are rare. Using hierarchical site occupancy detection models, we examined whether key choices associated with eDNA metabarcoding (primer selection, low-abundance read filtering and the number of positive water samples used to classify a species as present at a site) affect the sensitivity of metabarcoding, relative to backpack electrofishing for fish in freshwater streams. Under all scenarios (teleostei and vertebrate primers; 0%, 0.1% and 1% read filtering thresholds; one or two positive samples required to classify species as present), we found that eDNA metabarcoding is, on average, more sensitive than electrofishing. Combining vertebrate and teleostei markers resulted in higher detection probabilities relative to the use of either marker in isolation. Increasing the threshold used to filter low-abundance reads decreased species detection probabilities but did not change our overall finding that eDNA metabarcoding was more sensitive than electrofishing. Using a threshold of two positive water samples (out of five) to classify a species as present typically had negligible effects on detection probabilities compared to using one positive water sample. Our findings demonstrate that eDNA metabarcoding is generally more sensitive than electrofishing for conducting fish surveys in freshwater streams, and that this outcome is not sensitive to methodological decisions associated with metabarcoding.

Testing the adaptive advantage of a threatened species over an invasive species using a stochastic population model.

Introduced species are a major threat to freshwater biodiversity. Often eradication is not feasible, and management must focus on reducing impacts on native wildlife. This requires an understanding of how native species are affected but also how environmental characteristics influence population dynamics of both invasive and native species. Such insights can inform how to manipulate systems in order to take advantage of life-history traits native species possesses that invaders do not. The highly invasive fish, Gambusia holbrooki, has been implicated in the decline of many freshwater fish and amphibians. In south-eastern Australia, one of these is the threatened native fish, Galaxiella pusilla. As G. pusilla can survive periods without surface water, this presents an opportunity for adaptive management, given G. holbrooki lack these adaptations. We develop a stochastic population model to explore the impact of G. holbrooki on G. pusilla and test the feasibility of both natural and management-induced drying to protect this species. Our results support recent empirical studies showing G. holbrooki are a serious threat to G. pusilla persistence, especially through impacts on larval survival. While persistence is more likely in water bodies that frequently dry out, even optimal natural drying regimes may be insufficient when impacts from G. holbrooki are high. However, management-induced drying may allow persistence of G. pusilla in sites inhabited by both species. Given our model outcomes, the biology of these species and the habitats they occupy, we recommend maintaining or restoring aquatic and riparian vegetation and natural drying regimes to protect G. pusilla from G. holbrooki, in addition to undertaking management-induced drying of invaded water bodies. Our results provide insights into how the effects of G. holbrooki may be mitigated for other native species, which is important given this species is perhaps the most pervasive invader of freshwater ecosystems. We conclude with a discussion of the potential for using disturbance processes in the management of invasive species more broadly in freshwater and terrestrial systems.

Salmonella from a Microtidal Estuary Are Capable of Invading Human Intestinal Cell Lines.

Faecal contamination poses health risks for the recreational users of urban estuaries. However, our understanding of the potential pathogenicity of faecal microbes in these environments is limited. To this end, a study was conducted to understand the spatial and seasonal distribution of Salmonella in water and sediments of the Yarra River estuary, Melbourne, Australia. Among 210 samples in total, culturable Salmonella were recovered from 27%, 17%, and 19% of water, bank, and bed sediment samples, respectively. The combined detection increased from 15% in winter to 32% in summer (p < 0.05) indicating seasonal variation as potential part of public health risk assessments. Further, pathogenic potential of the Salmonella isolates was characterised via the quantification of attachment and invasion capacity using human epithelial colorectal cell line Caco-2 on a subset of isolates (n = 62). While all of these isolates could attach and invade Caco-2 cells, 52% and 13% of these showed greater attachment and invasiveness, respectively, than the corresponding mean values for S. Typhimurium ATCC14028 control. Isolates from winter were on average more invasive (seven out of eight isolates with the highest invasiveness recovered from the colder sampling period) than the isolates from summer, and Salmonella collected during summer showed lower invasion (p < 0.05) compared with the control. Similar low invasion compared with the same control was observed for isolates recovered from bank sediment (p < 0.05). While the higher prevalence in summer may imply higher risks during these peak recreational periods, it is essential that this information is used in combination with quantitative microbial risk assessments to fully understand the health risks posed by Salmonella in microtidal estuaries.

Campylobacter in an Urban Estuary: Public Health Insights from Occurrence, HeLa Cytotoxicity, and Caco-2 Attachment Cum Invasion.

Aquatic recreation in urban estuaries worldwide is often restricted by fecal pollution. Variability in the occurrence of fecal pathogens and their differential virulence potentials within these estuaries may result in variable public health risks. To address this hypothesis, Campylobacter were isolated from the Yarra River estuary, Australia and then characterized via HeLa cell cytotoxicity and attachment to and the invasion of Caco-2 monolayers. Overall, 54% (n=216) of estuarine samples (water and sediment combined) yielded biochemically confirmed culturable Campylobacter; higher detection was recorded in water (92%, n=90) than in the bank and bed sediments combined (27%, n=126). The seasonality of occurrence was not significant. HeLa cell cytotoxicity revealed that estuarine Campylobacter had low cytotoxin titers; the 95% confidence interval (CI) ranged between 61 and 85, which was markedly lower than the mean value (~386) for the C. jejuni 11168 reference pathogenic strain. The Caco-2 attachment of estuarine Campylobacter isolates (n=189) revealed that the 95%CI for the attachment efficiency of the test strains ranged between 0.09 and 0.1%, with only 3.7% having a higher efficiency than the 5th percentile value for C. jejuni 11168. None of the estuarine strains exhibited Caco-2 invasion capabilities. In contrast to the common assumption during quantitative microbial/risk assessments (QMRAs) that all environmental strains are pathogenic, the present results revealed that Campylobacter within the Yarra River estuary had very low virulence potential. Since this is the first study to use human epithelial cell lines to characterize estuary-borne pathogens, these results generate valuable insights for a better understanding of the public health risks in urban estuaries that will underpin more robust QMRAs.

Metabolite Changes in an Estuarine Annelid Following Sublethal Exposure to a Mixture of Zinc and Boscalid.

Environmental pollutants such as heavy metals and fungicides pose a serious threat to waterways worldwide. Toxicological assessment of such contaminants is usually conducted using single compound exposures, as it is challenging to understand the effect of mixtures on biota using standard ecotoxicological methods; whereas complex chemical mixtures are more probable in ecosystems. This study exposed Simplisetia aequisetis (an estuarine annelid) to sublethal concentrations of a metal (zinc) and a fungicide (boscalid), both singly and as a mixture, for two weeks. Metabolomic analysis via gas and liquid chromatography-mass spectrometry was used to measure the stress response(s) of the organism following exposure. A total of 75 metabolites, including compounds contributing to the tricarboxylic acid cycle, the urea cycle, and a number of other pathways, were identified and quantified. The multiplatform approach identified distinct metabolomic responses to each compound that differed depending on whether the substance was presented singly or as a mixture, indicating a possible antagonistic effect. The study demonstrates that metabolomics is able to elucidate the effects and mode of action of contaminants and can identify possible outcomes faster than standard ecotoxicological endpoints, such as growth and reproduction. Metabolomics therefore has a possible future role in biomonitoring and ecosystem health assessments.

Using metabolomics to assess the sub-lethal effects of zinc and boscalid on an estuarine polychaete worm over time.

INTRODUCTION: Zinc is a heavy metal commonly detected in urban estuaries around Australia. Boscalid is a fungicide found in estuaries, both in water and sediment, it enters the system predominantly through agricultural run-off. Zinc is persistent while boscalid breaks down, with a half-life of 108 days. Both contaminants are widely distributed and their effects on ecosystems are not well understood. OBJECTIVES: The aim of this study was to determine the metabolite changes in Simplisetia aequisetis (an estuarine polychaete) following laboratory exposure to a sub-lethal concentration of zinc or boscalid over a 2-week period. METHODS: Individuals were collected at six time points over a 2-week period. Whole polychaete metabolites were extracted and quantified using a multi-platform approach. Polar metabolites were detected using a semi-targeted GC-MS analysis and amine containing compounds were analysed using a targeted LC-MS analysis. Total lipid energy content was also analysed for Simplisetia aequisetis. RESULTS: The pathways that responded to zinc and boscalid exposure were alanine, aspartate and glutamate metabolism (AAG); glycine, serine and threonine metabolism (GST) and metabolites associated with the tricarboxylic acid cycle (TCA). Results showed that changes in total abundance of some metabolites could be detected as early as 24-h exposure. Changes were detected in the metabolites before commonly used total lipid energy assays identified effects. CONCLUSION: A multi-platform approach provided a holistic overview of the metabolomic response to contaminants in polychaetes. This approach shows promise to be used in biomonitoring programs to provide early diagnostic indicators of contamination and exposure.

Understanding spatiotemporal variability of in-stream water quality in urban environments - A case study of Melbourne, Australia.

To support sustainable urban planning and the design of water pollution mitigation strategies, the spatial and temporal trends of water quality in urban streams needs to be further understood. This study analyses over ten years of surface water quality data from 53 upstream catchments (20 of them predominated by a single type of land use) and two lowland sites across Greater Melbourne, Australia. We evaluated the impact of various catchment characteristics, especially urban land uses, on spatial and temporal urban water quality trends. Here, we focused on common urban pollutants: total suspended solids (TSS), total phosphorous (TP), total nitrogen (TN), zinc (Zn), copper (Cu) and nickel (Ni). Site median nutrient and heavy metal concentrations were negatively correlated with the catchment's elevation and its average annual rainfall. Further analysis shows that such trends were driven by the geographical pattern of Melbourne - i.e. low-laying sites tend to have less rainfall and be more urbanised. Only median concentrations of heavy metals (Zn and Cu) were correlated to catchment imperviousness. Further characterising of the urban environment was done into specific land uses (residential, industrial and commercial), yet median concentrations of all pollutants were not significantly correlated with land uses. This is because simple metrics, such as land use proportions, do not adequately reflect the significant variability in pollution sources that can exist even within the same land use type. Indeed, our temporal analysis found that the water quality difference between catchments with similar land uses is likely caused by their site-specific pollutant sources (construction and illegal discharge) and environmental management actions (wastewater management actions) regardless of similarities in land use. A 3-stage urbanisation cycle (development, operation and renewal) is suggested to further explain the urban water quality variance, but more data from small areas of an urban catchment is required to directly understand the unique impact of each urbanisation stage on water quality.

Can non-destructive DNA extraction of bulk invertebrate samples be used for metabarcoding?

BACKGROUND: High throughput DNA sequencing of bulk invertebrate samples or metabarcoding is becoming increasingly used to provide profiles of biological communities for environmental monitoring. As metabarcoding becomes more widely applied, new reference DNA barcodes linked to individual specimens identified by taxonomists are needed. This can be achieved through using DNA extraction methods that are not only suitable for metabarcoding but also for building reference DNA barcode libraries. METHODS: In this study, we test the suitability of a rapid non-destructive DNA extraction method for metabarcoding of freshwater invertebrate samples. RESULTS: This method resulted in detection of taxa from many taxonomic groups, comparable to results obtained with two other tissue-based extraction methods. Most taxa could also be successfully used for subsequent individual-based DNA barcoding and taxonomic identification. The method was successfully applied to field-collected invertebrate samples stored for taxonomic studies in 70% ethanol at room temperature, a commonly used storage method for freshwater samples. DISCUSSION: With further refinement and testing, non-destructive extraction has the potential to rapidly characterise species biodiversity in invertebrate samples, while preserving specimens for taxonomic investigation.

Development of a passive sampler based on a polymer inclusion membrane for total ammonia monitoring in freshwaters.

A passive sampler for determining the time-weighted average total ammonia (i.e. molecular ammonia and the ammonium cation) concentration (C TWA) in freshwaters, which incorporated a polymer inclusion membrane (PIM) as a semi-permeable barrier separating the aqueous source solution from the receiving solution (i.e. 0.8 mol L(-1) HCl), was developed for the first time. The PIM was composed of dinonylnaphthalene sulfonic acid (DNNS) as a carrier, poly (vinyl chloride) (PVC) as a base polymer and 1-tetradecanol as a modifier. Its optimal composition was found to be 35 wt% commercial DNNS, 55 wt% PVC and 10 wt% 1-tetradecanol. The effect of environmental variables such as the water matrix, pH and temperature were also studied using synthetic freshwaters. The passive sampler was calibrated under laboratory conditions using synthetic freshwaters and exhibited a linear response within the concentration range 0.59-2.8 mg L(-1) NH4(+) (0.46-2.1 mg N L(-1)) at 20 °C. The performance of the sampler was further investigated under field conditions over 7 days. A strong correlation between spot sampling and passive sampling was achieved, thus providing a proof-of-concept for the passive sampler for reliably measuring the C(TWA) of total ammonia in freshwaters, which can be used as an indicator in tracking sources of faecal contamination in stormwater drains.

A review of Galaxiella pusilla (Mack) (Teleostei: Galaxiidae) in south-eastern Australia with a description of a new species.

The dwarf galaxias, Galaxiella pusilla (Mack), is a small, threatened freshwater fish from coastal south-eastern Australia. Recent genetic studies, using multiple nuclear and mitochondrial DNA markers, found substantial differences between populations in western Victoria and south Australia ('west region') compared to eastern Victoria, Flinders Island, and Tasmania ('east region') that suggest the presence of a cryptic species. Morphological measurements and meristic counts from multiple populations within each region were undertaken to investigate potential differences between regions. Several characters, found to discriminate between individuals in the regions and to be diagnostic for two taxa, were used to describe a new species, Galaxiella toourtkoourt, for the west region. This is only the second species in the Galaxiidae to exhibit sexual dimorphism. The original description of Galaxiella pusilla, based on five specimens, is revised following examination of a large number of individuals. Both species are considered nationally threatened and are categorised as 'endangered'; the revised distribution of G. pusilla s.s. is reduced by approximately 60%. A number of inconsistencies in the most recent revision of the genus Galaxiella are also corrected.

Microsatellite loci and the complete mitochondrial DNA sequence characterized through next generation sequencing and de novo genome assembly for the critically endangered orange-bellied parrot, Neophema chrysogaster.

A suite of polymorphic microsatellite markers and the complete mitochondrial genome sequence was developed by next generation sequencing (NGS) for the critically endangered orange-bellied parrot, Neophema chrysogaster. A total of 14 polymorphic loci were identified and characterized using DNA extractions representing 40 individuals from Melaleuca, Tasmania, sampled in 2002. We observed moderate genetic variation across most loci (mean number of alleles per locus = 2.79; mean expected heterozygosity = 0.53) with no evidence of individual loci deviating significantly from Hardy-Weinberg equilibrium. Marker independence was confirmed with tests for linkage disequilibrium, and analyses indicated no evidence of null alleles across loci. De novo and reference-based genome assemblies performed using MIRA were used to assemble the N. chrysogaster mitochondrial genome sequence with mean coverage of 116-fold (range 89 to 142-fold). The mitochondrial genome consists of 18,034 base pairs, and a typical metazoan mitochondrial gene content consisting of 13 protein-coding genes, 2 ribosomal subunit genes, 22 transfer RNAs, and a single large non-coding region (control region). The arrangement of mitochondrial genes is also typical of Avian taxa. The annotation of the mitochondrial genome and the characterization of 14 microsatellite markers provide a valuable resource for future genetic monitoring of wild and captive N. chrysogaster populations. As found previously, NGS provides a rapid, low cost and reliable method for polymorphic nuclear genetic marker development and determining complete mitochondrial genome sequences when only a fraction of a genome is sequenced.