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Zinc is an essential element for aquatic organisms, however, activities such as mining and refining, as well as zinc's ubiquitous role in modern society can contribute to elevated environmental concentrations of zinc. Water hardness is widely accepted as an important toxicity modifying factor for metals in aquatic systems, though other factors such as pH are also important. This study investigated the influence of increasing water hardness, at three different pH values (6.7, 7.6 and 8.3), on the chronic toxicity of zinc to the growth rate of a microalgae, Chlorella sp. Zinc toxicity decreased with increasing hardness from 5 to 93 mg CaCO3 L−1 at all three pH values tested. The 72 h growth rate inhibition EC50 values ranged from 6.2 μg Zn L−1 (at 5 mg CaCO3 L−1, pH 8.3) to 184 μg Zn L−1 (at 92 mg CaCO3 L−1, pH 6.7). Increases in hardness from 93 to 402 mg CaCO3 L−1 generally resulted in no significant (p > 0.05) reduction in zinc toxicity. DGT-labile zinc measurements did not correspond with the observed changes in zinc toxicity as hardness was varied within a pH treatment. This suggests that cationic competition from increased hardness is decreasing zinc toxicity, rather than changes in metal lability. This study highlighted that current hardness algorithms used in water quality guidelines may not be sufficiently protective of sensitive species, such as Chlorella sp., in high hardness waters.

Fish bacterial communities provide functions critical for their host’s survival in contrasting environments. These communities are sensitive to environmental-specific factors (i.e., physicochemical parameters, bacterioplankton), and host-specific factors (i.e., host genetic background). The relative contribution of these factors shaping Amazonian fish bacterial communities is largely unknown. Here, we investigated this topic by analyzing the gill bacterial communities of 240 wild flag cichlids (Mesonauta festivus) from 4 different populations (genetic clusters) distributed across 12 sites in 2 contrasting water types (ion-poor/ acidic black water and ion-rich/circumneutral white water). Transcriptionally active gill bacterial communities were characterized by a 16S rRNA metabarcoding approach carried on RNA extractions. They were analyzed using comprehensive data sets from the hosts genetic background (Genotyping-By-Sequencing), the bacterioplankton (16S rRNA) and a set of 34 environmental parameters. Results show that the taxonomic structure of 16S rRNA gene transcripts libraries were significantly different between the 4 genetic clusters and also between the 2 water types. However, results suggest that the contribution of the host’s genetic background was relatively weak in comparison to the environment-related factors in structuring the relative abundance of different active gill bacteria species. This finding was also confirmed by a mixed-effects modeling analysis, which indicated that the dissimilarity between the taxonomic structure of bacterioplanktonic communities possessed the best explicative power regarding the dissimilarity between gill bacterial communities’ structure, while pairwise fixation indexes (FST) from the hosts’ genetic data only had a weak explicative power. We discuss these results in terms of bacterial community assembly processes and flag cichlid fish ecology.

Increased focus on the development and application of bioavailability-based metal water quality guideline values requires increased understanding of the influence of water chemistry on metal bioavailability and toxicity. Development of empirical models, such as multiple linear regression models, requires the assessment of the influence of individual water quality parameters as toxicity-modifying factors. The present study investigated the effect of pH on the lability and toxicity of zinc (Zn) to a tropical green microalga (Chlorella sp.). Zinc speciation and lability were explored using the Windermere Humic Aqueous Model (WHAM7), ultrafiltration, and diffusive gradients in thin films (DGT). Zinc toxicity increased significantly with increasing pH from 6.7 to 8.3, with 50% growth inhibition effect concentrations decreasing from 185 to 53 µg l-1 across the pH range. Linear relationships between DGT-labile Zn and dissolved Zn did not vary across the tested pH range, nor did the linear relationship between dissolved (

Amazonian blackwaters are extremely biodiverse systems containing some of the most naturally acidic, dissolved organic carbon-rich and ion-poor waters on Earth. Physiological adaptations of fish facing these ionoregulatory challenges are unresolved but could involve microbially-mediated processes. Here, we characterize the physiological response of 964 fish-microbe systems from four blackwater Teleost species along a natural hydrochemical gradient, using dual RNA-Seq and 16S rRNA of gill samples. We find that responses to blackwaters are host-species-specific, but occasionally include the overexpression of Toll-receptors and integrins associated to interkingdom communication. Blackwater gill microbiomes are characterized by a transcriptionally-active betaproteobacterial cluster potentially interfering with epithelial permeability. We explore further blackwater fish-microbe interactions by analyzing transcriptomes of 320 axenic zebrafish larvae exposed to sterile, non-sterile and inverted (non-native bacterioplankton) blackwater. We find that axenic zebrafish do not survive well when exposed to sterile/inverted blackwater, suggesting an essential role of endogenous symbionts in blackwater fish physiology.

Environmental context Having appropriate and robust models used for developing water quality guidelines is critical for sound environmental management. Methods used to validate models have only been demonstrated appropriate for a small portion of data types used in these models. This study has found that models using certain data types would be more appropriately validated using alternative evaluation criteria. This study serves as an important reference for developing and evaluating robust models. Rationale Bioavailability-based toxicity models for metals often have performance assessed by whether it can predict toxicity data within a factor of 2 of their paired observed toxicity data. This method has only been verified for median effect values (EC50) for acute fish and daphnia data, however toxicity models have been developed for a much broader range of effect levels (i.e. EC10/EC20) and species (e.g. microalga). This study tested whether the factor-of-2 rule is appropriate for a wider range of organisms and effect concentrations than previously studied. Methodology Toxicity estimate data from repeated tests conducted under the same conditions were collated to assess variation in results and compare this variation to a range of 4 (a factor of 2 above and below the mean) and a range of 9 (a factor of 3 above and below the mean) to assess if a factor-of-3 rule may be more appropriate for some species and effect levels. Results and discussion Overall, the factor-of-2 rule is broadly applicable for metal toxicity to a range of species for EC50 data. The EC10 datasets highlighted that larger variability exists in low effect levels and supported the use of a factor-of-3 rule, while the either the factor-of-2 or factor-of-3 rule could be applied to microalgae. The level of performance evaluation chosen may depend on the application of the bioavailability model. This study also found that while repeated toxicity test data is routinely generated, it is rarely published. Publication of such data would enable expansion of the present study to include inter-laboratory comparisons, an important consideration as most bioavailability models are based on data pooled from multiple sources.

Associations between host genotype and the microbiome of holobionts have been shown in a variety of animal clades, but studies on teleosts mostly show weak associations. Our study aimed to explore these relationships in four sympatric Serrasalmidae (i.e. piranha) teleosts from an Amazonian lake, using datasets from the hosts genomes (SNPs from GBS), skin and gut microbiomes (16S rRNA metataxonomics), and diets (COI metabarcoding) from the same fish individuals. Firstly, we investigated whether there were significant covariations of microbiome and fish genotypes at the inter and intraspecific scales. We also assessed the extent of co-variation between Serrasalmidae diet and microbiome, to isolate genotypic differences from dietary effects on community structure. We observed a significant covariation of skin microbiomes and host genotypes at interspecific (R2=24.4%) and intraspecific (R2=6.2%) scales, whereas gut microbiomes correlated poorly with host genotypes. Serrasalmidae diet composition was significantly correlated to fish genotype only at the interspecific scale (R2=5.4%), but did not covary with gut microbiome composition (Mantel R=-0.04; only 6 microbiome taxa involved). Secondly, we tested whether microbial taxa represent reliable host traits to complement host genotypic variations in these species. By using an NMDS ordination-based approach, we observed that subsets of the skin and gut microbiomes selected by a machine-learning Random Forest algorithm can complement host genotypic variations by increasing significantly the average interspecific differentiation. The complementarity of genome and microbiome variations suggests that combining both markers could potentially benefit our understanding of the evolution of Serrasalmidae in future studies.

Fish microbiomes provide functions critical for their host’s survival in contrasting environments. These communities are sensitive to a range of environmental-specific factors (i.e. physicochemical parameters, free-living bacterioplankton) and host-specific factors (i.e. host genetic background). The relative contribution of these genomic and environmental factors shaping Amazonian fish microbiomes is still unknown. Here, we investigated this topic by analyzing the gill microbiomes of 240 wild flag cichlids (Mesonauta festivus) from four different populations (genetic clusters) distributed in 12 sites of two contrasting water types (ion-poor/acidic black water and ion-rich/circumneutral white water). The transcriptionally active gill microbiomes were characterized by a 16S rRNA metabarcoding approach carried on RNA extractions. They were analyzed in light of comprehensive datasets from the hosts genetic background (Genotyping-By-Sequencing), the bacterioplanktonic pool of bacteria (16S rRNA) and a set of 34 environmental parameters. Results show that the transcriptional activity of gill microbiome samples was significantly different between the genetic clusters and between water types. However, they suggest that the contribution of the host’s genetic background was relatively weak in comparison to the environment-related factors in structuring the relative abundance of different gill microbiome transcripts. This result was also confirmed by a mixed-effects modeling analysis, which suggested that the dissimilarity between the transcriptional activity of bacterioplanktonic communities possessed the best explicative power regarding the dissimilarity between gill microbiomes transcripts, while pairwise fixation indexes (FST) from the hosts’ genetic data only had a weak explicative power. We discuss these results in terms of microbiome assembly processes and flag cichlid fish ecology.ImportanceHost-associated microbial communities respond to a range of factors specific to the host physiology, genetic backgrounds and life history. However, these communities also show different degrees of sensitivity to environment-dependant factors such as abiotic physico-chemical parameters and ecological interactions. The relative importance of host-versus environment-associated factors in shaping teleost microbiomes is still understudied and is paramount for their conservation and aquaculture. Here, we studied the relative importance of host- and environment-associated factors structuring teleost microbiomes using gill samples from a wild Amazonian teleost model (Mesonauta festivus) sampled in contrasting habitats along a 1500 km section of the Amazonian basin, thus ensuring high genetic diversity. Results showed that the contribution of the host’s genetic background was weak compared to environment-related bacterioplanktonic communities in shaping gill microbiomes, thereby suggesting that our understanding of teleost microbiome assembly could benefit from further studies focused on the ecological interplay between host-associated and free-living communities.

Amazonia is characterized by very heterogeneous riverscapes dominated by two drastically divergent water types: black (ion-poor, dissolved organic carbonate rich and acidic) and white (nutrient rich and turbid) waters. Recent phylogeographic and genomic studies have associated the ecotone formed by these environments to ecologically driven speciation in fishes. With the objective of better understanding the evolutionary forces behind the Amazonian Teleostean diversification, we sampled 240 Mesonauta festivus from 12 sites on a wide area of the Amazonian basin. These sites included three confluences of black and white water environments to seek for repeated evidences of ecological speciation at these ecotones. Results obtained through our genetic assessment based on 41,268 SNPs contrast with previous findings and supports a low structuring power of water types. Conversely, we detected a strong pattern of isolation by unidirectional downstream water current and evidence of past events of vicariance potentially linked to the Amazon River formation. Using a combination of population genetic, phylogeographic analysis and environmental association models, we decomposed the spatial variance from the environmental genetic variance specifically to assess which evolutive forces have shaped inter-population differences in M. festivus’ genome. Our sampling design comprising four major Amazonian rivers and three confluences of black and white water rivers supports the possibility that past studies potentially confounded ecological speciation with a site effect unrepresentative of the full Amazonian watershed. While ecological speciation admittedly played a role in Amazonian fish species diversification, we argue that neutral evolutionary processes explain most of the divergence between M. festivus populations.

Zinc is a contaminant of concern in aquatic environments and is a known toxicant to many aquatic organisms. Dissolved organic matter (DOM) is a toxicity modifying factor for zinc and is an important water chemistry parameter. This study investigated the influence of DOM concentration, source, and water pH on the chronic toxicity of zinc to a freshwater microalga, Chlorella sp. The influence of DOM on zinc toxicity was dependent on both concentration and source. In the absence of DOM, the 72-h EC50 was 112 μg Zn.L

Amazonia is characterized by very heterogeneous riverscapes dominated by two drastically divergent water types: black (ion-poor, dissolved organic carbonate rich and acidic) and white (nutrient rich and turbid) waters. Recent phylogeographic and genomic studies have associated the ecotone formed by these environments to ecologically driven speciation in fish species. With the objective of better understanding the evolutionary forces behind the Amazonian Teleostean diversification, we sampled 240 Mesonauta festivus from 12 sites on a wide area of the Amazonian basin. These sites included three confluences of black and white water environments to seek for repeated evidences of ecological speciation at these ecotones. Our genetic dataset of 41,268 SNPs is contrasting with previous results and supports a low structuring power of water types. Conversely, we detected a strong pattern of isolation by unidirectional downstream water current and evidence of past events of vicariance potentially linked to the Amazon River formation and salt-water incursions that occurred 2.5 Mya. Using a combination of population genetic, phylogeographic analysis and environmental association models, we decomposed the spatial variance from the environmental genetic variance specifically to assess which evolutive forces have shaped inter-population differences in M. festivus’ genome. Our sampling design comprising four major Amazonian rivers and three confluences of black and white water rivers supports the possibility that past studies potentially confounded ecological speciation with a site effect unrepresentative of the full Amazonian watershed. While ecological speciation admittedly played a role in Amazonian fish species diversification, we argue that neutral evolutionary processes explain most of the divergence between M. festivus populations.

Contamination of freshwaters is increasing globally, with microalgae considered one of the most sensitive taxa to metal pollution. Here, we used 72 h bioassays to explore the biochemical effects of copper (Cu) on the amino acid (AA) profile and proteome of Chlorella sp. and advance our understanding of the molecular changes that occur in algal cells during exposure to environmentally realistic Cu concentrations. The Cu concentrations required to inhibit algal growth rate by 10% (EC10) and 50% (EC50) were 1.0 (0.7–1.2) µg L−1 and 2.0 (1.9–2.4) µg L−1, respectively. The AA profile of Chlorella sp. showed increases in glycine and decreases in isoleucine, leucine, valine, and arginine, with increasing Cu. Proteomic analysis revealed the modulation of several proteins involved in energy production pathways, including: photosynthesis, carbon fixation, glycolysis, and oxidative phosphorylation, which likely assists in meeting increased energy demands under Cu-stressed conditions. Copper exposure also caused up-regulation of cellular processes and signalling proteins, and the down-regulation of proteins related to ribosomal structure and protein translation. These changes in biomolecular pathways have direct effects on the AA profile and total protein content and provide an explanation for the observed changes in amino acid profile, cell growth and morphology. This study shows the complex mode of action of Cu on Chlorella under environmentally realistic Cu concentrations and highlights several potential biomarkers for future investigations.

Stormwater runoff typically contains significant quantities of metal contaminants that enter urban waterways over short durations and represent a potential risk to water quality. The origin of metals within the catchment and processes that occur over the storm can control the partitioning of metals between a range of different forms. Understanding the fraction of metals present in a form that is potentially bioavailable to aquatic organisms is useful for environmental risk assessment. To help provide this information, the forms and dynamics of metal contaminants in an urban system were assessed across a storm. Temporal patterns in the concentration of metals in dissolved and particulate (total suspended solids; TSS) forms were assessed from water samples, and diffusive gradients in thin-films (DGTs) were deployed to measure the DGT-labile time-integrated metal concentration. Results indicate that the concentrations of dissolved and TSS-associated metals increased during the storm, with the metals Al, Cd, Co, Cu, Pb and Zn representing the greatest concern relative to water quality guideline values (GVs). The portion of labile metal as measured by DGT devices indicated that during the storm a substantial fraction (∼98%) of metals were complexed and pose a lower risk of acute toxicity to aquatic organisms. Comparison of DGT results to GVs indicate that current GVs are likely quite conservative when assessing stormwater pollution risks with regards to metal contaminants. This study provides valuable insight into the forms and dynamics of metals in an urban system receiving stormwater inputs and assists with the development of improved approaches for the assessment of short-term, intermittent discharge events.

The Amazon River basin sustains dramatic hydrochemical gradients defined by three water types: white, clear and black waters. Black waters contain important loads of allochthonous humic dissolved organic carbon (DOC), mostly coming from bacteria-mediated lignin degradation, a process that remains understudied. Here, we identified the main bacterial taxa and functions associated with contrasting Amazonian water types, and shed light on their potential implication in the lignin degradation process. We performed an extensive field bacterioplankton sampling campaign from the three Amazonian water types, and combined our observations to a meta-analysis of 90 Amazonian basin shotgun metagenomes used to build a tailored functional inference database. We showed that the overall quality of DOC is a major driver of bacterioplankton structure, transcriptional activity and functional repertory. We also showed that among the taxa mostly associated to differences between water types, Polynucleobacter sinensis particularly stood out, as its abundance and transcriptional activity was strongly correlated to black water environments, and specially to humic DOC concentration. Screening the reference genome of this bacteria, we found genes coding for enzymes implicated in all the main lignin degradation steps, suggesting that this bacteria may play key roles in the carbon cycle processes within the Amazon basin.

Macroinvertebrates play a key role in aquatic food webs, with amino acids (AA) playing an important role in determining their nutritional value to higher consumers. This study aimed to determine whether AA profile varies among four macroinvertebrate taxa, spatially and seasonally (summer/winter). The freshwater prawn, Macrobrachium australiense; freshwater shrimp, Paratya australiensis; freshwater snail, Physa sp. and caddisfly, Triplectides sp. were collected from seven sites along the Murray–Darling Basin, Australia. Sampling was conducted during summer 2015 (October–December), winter 2016 (May–June) and summer 2016 (October–December). AA profiles were found to be significantly different among the four taxa, with the highest total amino acid content found in decapods (M. australiense and P. australiensis). Based on the total essential AA content from our study, decapods had higher nutritional value compared to Physa. Seasonal variations in AA profiles of decapods were observed, with an increased proportion of the non-essential amino acid glycine and decreased proportion of different essential AAs found in individuals collected during winter compared to summer. No seasonal variation in the AA profiles of Physa sp. or Triplectides sp. was shown. Spatial variation in the AA profile of macroinvertebrates was only recorded in P. australiense during winter 2016. These findings expand the current understanding of the AA profile of freshwater macroinvertebrates showing that AA profiles are taxa specific and vary seasonally and spatially depending on taxa.

There have been limited studies on the effects of toxicity-modifying factors, such as dissolved organic matter (DOM), on the toxicity of metal mixtures to aquatic biota. The present study investigated the effects of DOM concentration (low, 2.8 ± 0.1 mg C/L; high, 11 ± 1.0 mg C/L) and DOM source (predominantly terrestrial or microbial) on the chronic toxicity of copper (Cu) and nickel (Ni) binary mixtures to the green freshwater microalga Chlorella sp. This was assessed by using a full factorial design of 72-h growth inhibition bioassays. Measured algal growth rate was compared with growth predicted by the concentration addition and independent action reference models. Model predictions were based on concentrations of dissolved metals, labile metals (measured by diffusive gradients in thin films [DGT]), and calculated free metal ions (determined by the Windermere Humic Aqueous Model). Copper/Ni mixture toxicity was synergistic to Chlorella sp. in the absence of added DOM, with evidence of metal concentration-dependent toxicity at low effect concentrations. As DOM concentration increased, the mixture interaction changed from synergism to noninteraction or antagonism depending on the metal speciation method used. The DOM source had no significant effect on mixture interaction when based on dissolved and free metal ion concentrations but was significantly different when based on DGT-labile metal concentrations. Ratio-dependent mixture interaction was observed in all treatments, with increased deviation from the reference model predictions as the mixture changed from Ni- to Cu-dominated. The present study demonstrated that both DOM concentration and source can significantly change metal mixture toxicity interactions and that these interactions can be interpreted differently depending on the metal speciation method used. Environ Toxicol Chem 2021;40:1908-1918. © 2021 SETAC.

Cladocerans are important filter-feeders transferring energy up the food web to different invertebrate and vertebrate predators. Along the flood period, cladocerans are one of the primary food sources for juvenile fish in floodplain. Resting egg banks allow cladoceran populations to overcome the environmental stress, related to several limnological changes, including complete drying of temporary wetlands. After drought, resting egg banks influence cladoceran community attributes during the cyclic and successional processes driven by episodic flood events. In this study we compared the taxonomic richness of active (from the water column) and dormant (from the sediment) Cladocera assemblages and analyzed the structure of resting egg banks, comparing the diversity, abundance and apparent viability/unviability of the eggs, between six temporary and six permanent wetlands, located along the Ovens River Floodplain, Victoria, Australia. The qualitative analysis shows higher taxonomic richness in active assemblages from temporary (24 taxa) than permanent (13 taxa) wetlands compared to dormant assemblages present in resting egg banks (9 taxa) from temporary and permanent wetlands. However, richness was influenced by taxonomic level of identification, with the majority of resting eggs only being identified to the taxonomic level of family (i.e. Chydoridae). Total taxa richness within egg banks was similar between wetland types, however, on average higher Shannon’s diversity of resting eggs was found within permanent (1.53) than temporary (0.82) wetlands. This is likely to be due to more stable wetlands not providing appropriate cues to trigger dormancy induction or breakage for specific populations, leading to higher values of evenness in permanent than temporary wetlands. Comparing permanent and temporary wetlands, higher abundance of resting eggs (more than four times) consisting of higher abundance of unviable eggs and similar viable egg abundance to permanent wetlands, was found within temporary wetlands, suggesting that the increased resting egg abundance in temporary wetlands is balanced by the losses due to factors such as predation, parasitism or other physical damage, during the terrestrial phase. Despite resistant outer shell structure, this study highlights that the damage to egg integrity is intensified in wetlands that undergo dry phases. Cladoceran resting egg banks represent the potential assemblage to recover after disturbance events such as drying, and information about these is important to ensure appropriate management and conservation of floodplain biodiversity.

Nickel is often a metal of interest in regulatory settings given its increasing prevalence in disturbed freshwaters and as a known toxicant to fish and algae. Dissolved organic matter (DOM) is a toxicity modifying factor for nickel and a ubiquitous water physicochemical parameter. This study investigated the effect of DOM concentration and source on the chronic toxicity of nickel to Chlorella sp. using three DOM at two concentrations (3.1 ± 1.8 and 12 ± 1.3 mg C/L). Nickel toxicity to Chlorella sp. was not strongly influenced by DOM concentration. In the absence of DOM, the 72-h EC50 for Chlorella sp. was 120 μg Ni/L. In the low DOM treatment, nickel toxicity was either unchanged or slightly increased (87–140 μg Ni/L) and unchanged or slightly decreased in the high DOM treatment (130–240 μg Ni/L). DOM source also had little effect on nickel toxicity, the largest differences in nickel toxicity occurring in the high DOM treatment. Labile nickel (measured by diffusive gradients in thin-films, DGT) followed strong linear relationships with dissolved nickel (R2 > 0.97). DOM concentration and source had limited effect on DGT-labile nickel. DGT-labile nickel decreased with increasing DOM concentration for only one of the three DOM. Modelled labile nickel concentrations (expressed as maximum dynamic concentrations, cdynmax) largely agreed with DGT-labile nickel and suggested that toxicity is explained by free Ni2+ concentrations. This study confirms that nickel toxicity is largely unaffected by DOM concentration or source and that both measured (DGT) and modelled (cdynmax and free Ni2+) nickel concentrations can explain nickel toxicity.

Teleost fish represent an invaluable repertoire of host species to study the factors shaping animal-associated microbiomes. Several studies have shown that the phylogenetic structure of the fish gut microbiome is driven by species-specific (e.g., host ancestry, genotype, or diet) and habitat-specific (e.g., hydrochemical parameters and bacterioplankton composition) factors. However, our understanding of other host-associated microbial niches, such as the skin mucus microbiome, remains limited. The goal of our study was to explore simultaneously the phylogenetic structure of the fish skin mucus and gut microbiome and compare the effect of species- and habitat-specific drivers on the structure of microbial communities in both tissues. We sampled 114 wild fish from 6 populations of 3 ecologically and phylogenetically contrasting Amazonian teleost species. Water samples were collected at each site, and 10 physicochemical parameters were characterized. The skin mucus, gut, and water microbial communities were characterized using a metabarcoding approach targeting the V3-V4 regions of the 16S rRNA. Our results showed a significant distinction between the phylogenetic profile and diversity of the microbiome from each microbial niche. Skin mucus and bacterioplankton communities were significantly closer in composition than gut and free-living communities. Species-specific factors mostly modulated gut bacterial communities, while the skin mucus microbiome was predominantly associated with environmental physicochemistry and bacterioplankton community structure. These results suggest that the variable skin mucus community is a relevant target for the development of microbial biomarkers of environmental status, while the more conserved gut microbiome is better suited to study long-term host-microbe interactions over evolutionary time scales. IMPORTANCE Whether host-associated microbiomes are mostly shaped by species-specific or environmental factors is still unresolved. In particular, it is unknown to what extent microbial communities from two different host tissues from the same host respond to these factors. Our study is one of the first to focus on the microbiome of teleost fish to shed a light on this topic as we investigate how the phylogenetic structure of microbial communities from two distinct fish tissues are shaped by species- and habitat-specific factors. Our study showed that in contrast to the teleost gut microbiome, skin mucus communities are highly environment dependent. This result has various implications: (i) the skin mucus microbiome should be used, rather than the gut, to investigate bacterial biomarkers of ecosystem perturbance in the wild, and (ii) the gut microbiome is better suited for studies of the drivers of phylosymbiosis, or the coevolution of fish and their symbionts.

Dissolved organic matter (DOM) plays important roles in both abiotic and biotic processes within aquatic ecosystems, and these in turn depend on the quality of the DOM. We collected and characterized chromophoric DOM (CDOM) from different Australian freshwater types (circumneutral, naturally acidic and groundwater-fed waterways), climatic regions and seasons. CDOM quality was characterized using absorbance and fluorescence spectroscopy. Excitation emission scans followed by parallel factor (PARAFAC) analysis showed that CDOM was characterized by three main components: protein-like, fulvic-like and humic-like components commonly associated with various waters globally in the Openfluor database. Principal component analysis showed that CDOM quality varied between naturally acidic, circumneutral and groundwater-fed waters, with unique CDOM quality signatures shown for each freshwater type. CDOM quality also differed significantly within some sites between seasons. Clear differences in dominant CDOM components were shown between freshwater types. Naturally acidic waters were dominated by highly aromatic (as indicated by the specific absorbance co-efficient (SAC340) and the specific UV absorbance (SUVA254) values which ranged between 31 and 50 cm2 mg−1 and 3.9–5.7 mg C−1 m−1 respectively), humic-like CDOM of high molecular weight (as indicated by abs254/365 which ranged from 3.8 to 4.3). In contrast, circumneutral waters were dominated by fulvic-like CDOM of lower aromaticity (SAC340: 7–21 cm2 mg−1 and SUVA254: 1.5–3.0 mg C−1 m−1) and lower molecular weight (abs254/365 5.1–9.3). The groundwater-fed site had a higher abundance of protein-like CDOM, which was the least aromatic (SAC340: 2–5 cm2 mg−1 and SUVA254: 0.58–1.1 mg C−1 m−1). CDOM was generally less aromatic, of a lower molecular weight and more autochthonous in nature during the summer/autumn sampling compared to winter/spring. Significant relationships were shown between various CDOM quality parameters and pH. This is the first study to show that different freshwater types (circumneutral, naturally acidic and groundwater-fed) contain distinct CDOM quality signatures in Australia, a continent with unique flora and geology.

Acid mine drainage (AMD) is one of the major environmental problems impacting aquatic ecosystems globally. We studied changes in the community composition of macroinvertebrates and amino acid (AA) profiles of dominant taxa along an AMD contamination gradient within the Dee River, Queensland, Australia to understand how AMD can affect the biomolecular composition of macroinvertebrates. Taxa richness and community composition of macroinvertebrates changed widely along the AMD gradient with significantly lower taxa richness recorded at the polluted sites compared to upstream and downstream sites. The Dipteran families: Chironomidae and Ceratopogonidae, the Odonata family Gomphidae, and the Coleoptera family Dytiscidae were the only families found at all sampling sites and were used here for AA analysis. There were significant variations in the AA profiles among the studied taxa. The AA profile of each taxon also varied among upstream, polluted and downstream sites suggesting that contamination of a river system with acid mine drainage not only alters the overall macroinvertebrate community composition but also significantly influences the AA profile of organisms that are tolerant to AMD. This study highlights the potential of using AA profiling to study the response of aquatic organisms to contamination gradients such as those associated with AMD.

MMCP Collaboration (MMCP) is a project supported by the Joint State Governments and the Murray-Darling Basin Authority to generate and adopt freshwater ecological knowledge through collaboration, to maintain research capability and contribute supporting science to underpin the Basin-Wide Watering Strategy.This project, is one of five research themes that make up MMCP. This research theme investigated the biofilm succession patterns and ecosystem dynamics in the Edward-Wakool River system.

MMCP Collaboration (MMCP) is a project supported by the Joint State Governments and the Murray-Darling Basin Authority to generate and adopt freshwater ecological knowledge through collaboration, to maintain research capability and contribute supporting science to underpin the Basin-Wide Watering Strategy.The biofilm succession theme is one of five themes that make up MMCP. This study examined the successional patterns of biofilms through time, under relatively constant flow conditions.

Fertilizers containing phosphate (PO4 3- ) are commonly used within the agricultural industry and are known to increase the bioavailability and mobility of metalloids like arsenic (As). This may increase plant uptake of As and hence pose a risk to human health. Arsenic and antimony (Sb) often co-occur in contaminated soils; however, little is known about the interactions between As and Sb with PO4 3- on their bioavailability, accumulation, and toxicity in plants. The present study investigated individual and combined As and Sb-contaminated soils across 2 soil PO4 3- concentrations using a commonly consumed leafy vegetable, choy sum (Brassica chinensis var. parachinensis). Increased soil PO4 3- had no clear influence on the bioavailability of As or Sb (derived from a sequential extraction procedure). At high PO4 3- concentration, B. chinensis accumulated higher amounts of As in the shoots and roots in both individual and co-contaminated soil, whereas Sb accumulation increased only when Sb was the only contaminant. When As was the only contaminant, the translocation of As from roots to shoots decreased as soil PO4 3- increased. Increased soil PO4 3- had no influence on Sb translocation from root to shoot. Although As was toxic (impaired growth) at low PO4 3- soil concentration, no toxicity was observed in the high-PO4 3- soil. No toxicity was observed for Sb in either low- or high-PO4 3- soils. Increased soil PO4 3- concentration ameliorated or masked As toxicity to plant growth and led to higher As concentration in the plant's edible parts. The addition of high soil PO4 3- concentrations ameliorated or masked As toxicity to plant growth in both individually and As + Sb co-contaminated soil; however, the plant's edible parts accumulated higher As and Sb concentrations. Environ Toxicol Chem 2020;39:1233-1243. © 2020 SETAC.

Antimony (Sb) is an emerging contaminant and until recently it was assumed to behave in a similar way to arsenic (As). Arsenic and Sb often co-occur in contaminated sites, yet most investigations consider their toxicity to plants singly. More research is needed to understand the interactions between As and Sb in soils and plants. This study investigated the interactive effect of As and Sb in terms of soil bioavailability, plant toxicity and bioaccumulation on the commercially important agricultural plant, water spinach (Ipomoea aquatica) using a pot experiment. Plants were exposed to As and Sb individually (As (individual), Sb (individual)) and as a mixture (As + Sb (combined)) at different concentrations. Plant growth was measured using shoot and root dry mass, length and chlorophyll a content of leaves. At the end of the bioassay, bioavailable metalloids were extracted from the soil as per a sequential extraction procedure (SEP) and plant tissue was analysed for metalloid content. For As, there were no differences observed between the bioavailability of As in the As + Sb (combined) and As (individual) treatments. For Sb, no increase in bioavailability was observed with co-contamination compared to single-Sb exposures for most concentrations except at 1250 mg/kg. Single-Sb was not toxic to I. aquatica shoot dry mass and length, but there was greater shoot Sb accumulation in the As + Sb (combined) than the Sb (individual) treatment. In contrast, single-As was toxic to I. aquatica growth. When As and Sb were present together in the soil, there was a synergistic toxicity to shoot dry mass (EC50 Toxic Unit (TU) was less than 1) and additive toxicity (EC50 equal to 1 TU) to shoot length. This work shows that the co-occurrence of As and Sb in soil increases Sb bioavailability and can cause synergistic toxicity to an important agricultural crop.

International audience; The dinoflagellate Alexandrium minutum produces toxic compounds, including paralytic shellfish toxins, but also some unknown extracellular toxins. Although copper (Cu) is an essential element, it can impair microalgal physiology and increase their toxic potency. This study investigated the effect of different concentrations of dissolved Cu (7 nM, 79 nM and 164 nM) on A. minutum allelochemical potency, here defined as negative effects of a protist on competing protists through the release of chemicals. This was studied in relation to its physiology. The effects of Cu were assessed on A. minutum growth, reactive oxygen species level, photosynthesis proxies, lipid metabolism, exudation of dissolved organic compounds, allelochemical potency and on the associate free bacterial community of A. minutum. Only the highest Cu exposure (164 nM) inhibited and delayed the growth of A. minutum, and only in this treatment did the allelochemical potency significantly increase, when the dissolved Cu concentration was still toxic. Within the first 7 days of the high Cu treatment, the physiology of A. minutum was severely impaired with decreased growth and photosynthesis, and increased stress responses and free bacterial density per algal cell. After 15 days, A. minutum partially recovered from Cu stress as highlighted by the growth rate, reactive oxygen species level and photosystem II yields. This recovery could be attributed to the apparent decrease in background dissolved Cu concentration to a non-toxic level, suggesting that the release of exudates may have partially decreased the bioavailable Cu fraction. Overall, A. minutum appeared quite tolerant to Cu, and this work suggests that the modifications in the physiology and in the exudates help the algae to cope with Cu exposure. Moreover, this study shows the complex interplay between abiotic and biotic factors that can influence the dynamic of A. minutum blooms. Modulation in allelochemical potency of A. minutum by Cu may have ecological implications with an increased competitiveness of this species in environments contaminated with Cu.

The acidification of waterways through anthropogenic means is a global problem. Treatment options for anthropogenically acidified waterways are currently limited. This review proposes the use of humic substance (HS) to treat waterways acidified via anthropogenic means such as those affected by acid rain, acid mine drainage and acid sulphate soils. In laboratory settings, HS have been shown to successfully remove metals from solutions. HS also have the added benefit of being present in all natural waters and have been shown to positively influence aquatic organism’s survival to acidification, metals and the combined stressors of low pH and metal mixtures commonly associated with anthropogenically acidified waters. Comparative food webs of waterways affected by natural and anthropogenic acidification are provided along with a third conceptual food web showing the potential benefits of adding HS to an affected system. HS may represent a sustainable remediation option as it may be locally sourced from environmentally friendly options such as green waste. However, if such options are to be fully developed and utilised, the specific requirements for treating anthropogenically acidified waters with different HS sources (e.g. treatment volumes and efficacy) will require further study.

Australian tropical freshwaters can experience extreme seasonal variability in rainfall and run off, particularly due to pulse events such as storms and cyclones. This study investigated how seasonal variability in dissolved organic matter (DOM) quality impacted the chronic toxicity of copper to a tropical green alga (Chlorella sp.) in the presence of two concentrations of DOM (low: ∼2 mg C/L; high: ∼10 mg C/L) collected from three tropical waters. Copper speciation and lability were explored using diffusive gradients in thin-films (DGT) and modelled maximum dynamic concentrations (cdynmax) using data derived from the Windermere Humic Aqueous Model (WHAM VII). Relationships between copper lability and copper toxicity were assessed as potential tools for predicting toxicity. Copper toxicity varied significantly with DOM concentration, source and season. Copper toxicity decreased with increasing concentrations of DOM, with 50% growth inhibition effect concentrations (EC50) increasing from 1.9 μg Cu/L in synthetic test waters with no added DOM (0.34 mg C/L) up to 63 μg Cu/L at DOM concentrations of 9.9 mg C/L. Copper toxicity varied by up to 2-fold between the three DOM sources and EC50 values were generally lower in the presence of wet season DOM compared to dry season DOM. Linear relationships between DGT-labile copper and dissolved copper were significantly different between DOM source, but not concentration or season. Modelled cdynmax consistently under-predicted labile copper in high DOM treatments compared to DGT measurements but performed better in low DOM treatments, indicating that this method is DOM-concentration dependent. Neither speciation method was a good surrogate for copper toxicity in the presence of different sources of natural DOM. Our findings show that DOM source and season, not just DOM concentration, affect copper toxicity to freshwater biota. Therefore, DOM quality should be considered as a toxicity-modifying factor for future derivation of bioavailability-based site-specific water quality guideline values.

This study adds further critical information to the limited body of knowledge on the ameliorative ability of Australian dissolved organic carbon (DOC), reinforcing the importance of DOC source and concentration as significant factors controlling the risk copper poses to organisms in freshwater systems. The ameliorative ability of five unstudied DOCs on the chronic toxicity of copper to the tropical alga Chlorella sp. was compared. Sensitivity to copper varied dramatically; effect concentrations at the 50 percent effect level (EC50) increased by up to 22-fold in the high DOC treatment compared to controls and more than 2-fold between DOCs at the same concentration. The analytical techniques, diffusive gradients in thin films (DGT) and Chelex column, were used to understand whether differences in copper toxicity could be explained by copper lability. Labile copper mirrored the trends seen in the toxicity tests; lability decreased with increasing DOC concentration and varied between DOCs at the same concentration. The equilibrium model, WHAM VII, was also used to better understand the role of the free copper ion on the toxicity observed. Disagreement between EC50 values derived using the WHAM-predicted free Cu2+ concentrations and agreement between DGT-labile and the maximum dynamic concentration ( cmaxdyn) suggest free copper is not the sole contributor to toxicity and that the source of the specific DOCs also plays a role.

Dissolved organic matter (DOM) within freshwaters is essential for broad ecosystem function. The concentration and type of DOM within rivers depends on the relative contributions of allochthonous sources and the production and consumption of DOM by microbes. In this work we have examined the temporal patterns in DOM quality and productivity in three lowland rivers in dryland Australia using fluorescence excitation emission scans. We assessed the production and consumption of DOM within light and dark bottle assays to quantify the relative contribution of bacteria and algae to the DOM pool and simultaneously assessed whether the systems were autotrophic or heterotrophic. DOM varied temporally within the three river systems over the course of the study period. Characterisation of DOM within light and dark bottles following a 6-hour incubation revealed microbial consumption of a humic-like component and production of protein-like components similar in nature to the amino acids tryptophan and tyrosine. The lack of a significant difference in DOM quality between the light and dark bottles indicated that the protein-like DOM is likely derived from bacterial activity. Respiration was shown to be higher than gross primary production in both whole river and bottle assays, yielding negative net production values and demonstrating that these rivers were predominately heterotrophic. Our work suggests that bacterial metabolism of DOM may be a significant contributor to the production of protein-like components within heterotrophic freshwater systems.

The recent deployment of fluorescent dissolved organic matter (fDOM) probes in dam catchments and drinking water treatment plants (DWTP) for water quality monitoring purposes has resulted in the production of a large amount of data that requires scientific evaluation. This study introduces a comprehensive, transferable methodological framework for scientists and water professionals to model fluorescence site-specific quenching on fDOM probe readings caused by temperature, suspended particles, and the inner filter effect (IFE) and applies it to an Australian subtropical reservoir. The findings revealed that quenching due to turbidity and IFE effects were best predicted by threshold autoregressive models. Raw fDOM probe measurements were validated as being more reliable if they were systematically compensated using the proposed procedure. The developed fDOM compensation procedure must consider the instrument features (i.e., wavelength broadband and responsiveness) and site-specific conditions (i.e., DOM characteristics and suspended particles). A finding of particular interest was that the compensated normalized fDOM readings had a high correlation with the low (&lt, 500 Da) molecular weight fraction of the DOM, which is more recalcitrant to removal by coagulation. As a consequence, there is potential to use compensated fDOM probe readings to provide real-time, in situ information on DOM properties in freshwater systems, which will enable water treatment plant operators to optimize the coagulation process.

The use of field data to derive guideline water quality trigger values is likely to be more environmentally relevant than laboratory estimates. In the present study, macroinvertebrate responses to conductivity (specific conductance at 25 °C) within 5 bioregions in Victoria, Australia, were derived from 19 yr of macroinvertebrate field data. Varying response to electrical conductivity (EC) occurred among taxa. Ninety-five percent extirpation concentrations (XC95) for EC were calculated for each genus and species and ranged from 25 to 23 600 µS/cm. Hazardous concentration 5th percentiles (HC05) were calculated for each bioregion from species sensitivity distributions developed using genus and species XC95 values. Genus HC05 values varied substantially between bioregions: bioregion 1 (29 µS/cm), 2 (78 µS/cm), 3 (143 µS/cm), 4 (1068 µS/cm), and 5 (2226 µS/cm). No substantial differences in HC05 values were shown between genus- and species-level calculations in bioregions 1 to 3 and 5; however, a decrease of approximately 300 µS/cm was shown for bioregion 4. The substantial differences in HC05 values between bioregions supports the need for region-specific determination of effects of EC. We explore the use of HC05 values as water quality guidelines across a bioregion gradient and provide a comprehensive analysis of macroinvertebrate responses to changes in EC, with important implications for waterway management. Environ Toxicol Chem 2019;38:1334-1342. © 2019 SETAC.

Acid mine drainage (AMD) is a global problem leading to the acidification of freshwaters, as well as contamination by heavy metals. The ability of humic substances (HS) such as humic acid (HA) to decrease toxicity of heavy metals is widely known, whereas limited studies have examined the ability of HS to decrease toxicity linked with multiple stressors such as those associated with AMD. This study investigated the ability of HA to decrease acute toxicity defined as morbidity and ventilation frequency (measured via the time elapsed for ten operculum movements) in eastern rainbowfish ( Melanotaenia splendida splendida ) exposed to the multiple stressors of AMD-driven heavy metal concentrations, together with low pH. Water from the Mount Morgan open pit (a now closed gold and copper mine site), located at Mount Morgan, Central Queensland, Australia, was used as the AMD source. Fish were exposed to zero per cent (pH 7.3), two per cent (pH 6.7), three per cent (pH 5.7) and four per cent (pH 4.6) AMD in the presence of 0, 10 and 20 mg/L Aldrich Humic Acid (AHA) over 96 h. HA was shown to significantly decrease the acute toxicity of AMD and its adverse effects on ventilation frequency. These results are important in showing that HA can influence toxicity of metal mixtures and low pH, thus indicating a potential role for HA in decreasing toxicity of multiple environmental stressors more widely, and possible value as a rehabilitation aid.

Acidification of freshwaters via anthropogenic means is a global problem. Decreases in pH in these systems have devastating effects on macroinvertebrate fauna especially Ephemeropterans. However, fauna in naturally acidic waters with high concentrations of humic substances (HS) appear to be less affected by acidification. This study investigated the ability of HS to decrease toxicity of low pH to mayflies (Atalophlebia spp.) in both hard and soft water environments. Laboratory trialling with both hard and soft water was conducted to represent water chemistries commonly associated with anthropogenic and naturally acidic freshwaters. Mayflies were exposed to decreases in pH between 7 (control) and 3.5 in the presence of 0, 10 and 20 mg/L HS (Aldrich humic acid). HS increased mayfly survival by up to 45% in soft water and up to 39% in hard water. HS were also shown to increase 96 h LC50 values in both soft (4.29, 0 mg/L; 3.99, 10 mg/L and 3.97, 20 mg/L) and hard water (4.58, 0 mg/L; 4.22, 10 mg/L and 4.07, 20 mg/L). The results of this study are important in showing that HS can influence the toxicity of low pH environments; thus providing insight into why contrasting effects on biota are recorded from naturally acidic compared with anthropogenically-acidified environments.

Differences relating to the ability of various types of humic substances (HS) to influence toxicity of pollutants have been reported in the literature, but there still remains a gap in understanding whether various HS will have the same influence on the toxicity of acid mine drainage (AMD). This study investigated differences in the ability of Aldrich humic acid (AHA), Suwannee River humic acid and Suwannee River fulvic acid to decrease toxicity of AMD to the freshwater shrimp (Caridina sp. D). Toxicity tests were conducted over 96 h and used Mount Morgan open pit water as source of AMD and Dee River water as control/diluents. Concentrations of 0-4 % AMD at 0 mg/L HS, 10 mg/L AHA, 10 mg/L Suwannee River humic acid and 10 mg/L Suwannee River fulvic acid were used. Significantly higher survival of shrimp was recorded in the HS treatments compared with the treatment containing no HS. No significant differences were found among HS type. HS considerably increased LC50 values irrespective of type, from 1.29 (0 mg/L HS) to 2.12 % (AHA); 2.19 (Suwannee River humic acid) and 2.22 % (Suwannee River fulvic acid). These results support previous work that HS decrease the toxicity of AMD to freshwater organisms, but with the novel finding that this ability occurs irrespective of HS type. These results increase the stock of knowledge regarding HS and may contribute to a possible remediation option for AMD environments.

Humic substances (HS) have been shown to decrease the toxicity of environmental stressors, but knowledge of their ability to influence the toxicity of multiple stressors such as metal mixtures and low pH associated with acid mine drainage (AMD) is still limited. The present study investigated the ability of HS to decrease toxicity of AMD to mayflies (Atalophlebia spp.). The AMD was collected from the Mount Morgan (Mount Morgan, Queensland, Australia) open pit. Mayflies were exposed to concentrations of AMD at 0%, 1%, 2%, 3%, and 4% in the presence of 0 mg/L, 10 mg/L, and 20 mg/L Aldrich humic acid (AHA). A U-shaped response was noted in all AHA treatments, with higher rates of mortality recorded in the 2% and 3% dilutions compared with 4%. This result was linked with increased precipitates in the lower concentrations. A follow-up trial showed significantly higher concentrations of precipitates in the 2% and 3% AMD dilutions in the 0 mg/L AHA treatment and higher precipitates in the 2% AMD, 10 mg/L and 20 mg/L AHA, treatments. Humic substances were shown to significantly increase survival of mayflies exposed to AMD by up to 50% in the 20 mg/L AHA treatment. Humic substances may have led to increased survival after AMD exposure through its ability to influence animal physiology and complex heavy metals. These results are valuable in understanding the ability of HS to influence the toxicity of multiple stressors. Environ Toxicol Chem 2014;33:567–572. © 2013 SETAC

High amounts of humic substances (HS) are commonly found in natural acidic waterways and have been suggested to offer some protection against low pH. This study investigated the ability of HS to decrease respiratory stress in eastern rainbowfish (Melanotaenia splendida splendida) exposed to decreases in pH (range of 7-3.5) in soft and hard water. Repeated measures ANOVA revealed a significant difference in respiration (time taken for ten operculum movements) between pH and HS treatments, with a significant interaction between pH and HS present in 5/6 trials. Respiratory stress was shown to increase with increasing acidity, but significantly decreased in treatments with HS (10 and 20 mg/L) compared to those without. The fish exposed to pH treatments without HS also displayed increased hyperactivity, larger operculum movements and increased mucous production. Increased morbidity was shown in HS treatments at pH 3.5 (soft water) and at pH 4 (hard water) compared to treatment without HS. This indicates that HS is helpful in ameliorating the effects of decreased pH on respiration at sublethal pH levels; however, as pH decreases further, it seems that HS increases the toxicity (morbidity) of the low pH.

To date, most research on freshwater cyanotoxin(s) has focused on understanding the dynamics of toxin production and decomposition, as well as evaluating the environmental conditions that trigger toxin production, all with the objective of informing management strategies and options for risk reduction. Comparatively few research studies have considered how this information can be used to understand the broader ecological role of cyanotoxin(s), and the possible applications of this knowledge to the management of toxic blooms. This paper explores the ecological, toxicological, and genetic evidence for cyanotoxin production in natural environments. The possible evolutionary advantages of toxin production are grouped into two main themes: That of "competitive advantage" or "physiological aide". The first grouping illustrates how compounds produced by cyanobacteria may have originated from the need for a cellular defence mechanism, in response to grazing pressure and/or resource competition. The second grouping considers the contribution that secondary metabolites make to improved cellular physiology, through benefits to homeostasis, photosynthetic efficiencies, and accelerated growth rates. The discussion also includes other factors in the debate about possible evolutionary roles for toxins, such as different modes of exposures and effects on non-target (i.e., non-competitive) species. The paper demonstrates that complex and multiple factors are at play in driving evolutionary processes in aquatic environments. This information may provide a fresh perspective on managing toxic blooms, including the need to use a "systems approach" to understand how physico-chemical conditions, as well biological stressors, interact to trigger toxin production.