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Estuaries are critical habitats subject to a range of stressors requiring effective management. Microbes are gaining recognition as effective environmental indicators, however, the response of host associated communities to stressors remains poorly understood. We examined microbial communities from seawater, sediments and the estuarine fish Pelates sexlineatus, in Australia's largest urbanised estuary, and hypothesised that anthropogenic contamination would be reflected in the microbiology of these sample types. The human faecal markers Lachno3 and HF183 were not detected, indicating negligible influence of sewage, but a gradient in copy numbers of the class 1 integron (intI-1), which is often used as a marker for anthropogenic contamination, was observed in sediments and positively correlated with metal concentrations. While seawater communities were not strongly driven by metal contamination, shifts in the diversity and composition of the fish gut microbiome were observed, with statistical links to levels of metal contamination (F2, 21 = 1.536, p

An impact assessment of oceanic effluent releases from Belmont wastewater treatment works (WWTW) in Newcastle, Australia, was undertaken. Benthic infaunal assemblages in sandy sediments of ~25 m water depth were examined, at sites adjacent to the release point, and at increasing distances up to 2 km in both a NE and SW direction over five consecutive years (2016-2020). Localised impacts were evident for infaunal assemblages, with sites within 20 m of the outfall ("Impact" site types) exhibiting lower taxa richness and Shannon diversity, higher abundances of polychaetes and/or nematodes, higher polychaete ratios, and shifts in assemblage composition in comparison to sites at greater distances during some years. Taxa with increased localised abundances at the outfall were identified as indicators for monitoring impacts, including deposit-feeding polychaetes (Families Polygordiidae, Paraonidae and Dorvilleidae) and Phylum Nematoda. Future infaunal monitoring could include molecular tools and paired sediment analyses.

Very little is currently known regarding the effects of estrogenic endocrine disrupting chemicals on embryonic and larval development in molluscs, nor the potential effects of parental (F0) exposure on resultant F1 offspring. In this study, we assessed the embryotoxic impacts of exposure to environmentally relevant concentrations of the synthetic estrogen, 17α-ethinylestradiol (EE2), to male and female parents (50 ng/L) and their offspring (5 and 50 ng/L) in the native Australian Sydney rock oyster, Saccostrea glomerata. There were no detectable effects of parental exposure on fertilisation success, proportions of early larval (F1) morphs and unfertilised eggs. Offspring impacts were evidenced in terms of developmental delays, with decreased percentages of D-veligers retained by 45 μm mesh, along with a reduction of swimming capabilities of larvae at 2 days post-fertilisation (dpf) when both parents had been exposed to 50 ng/L EE2. Although no significant parental effects were found on the survival of F1 larvae at 9 dpf, retardation of shell growth was observed on F1 larvae in treatments where both parents had been exposed to 50 ng/L EE2. Subsequent larval exposure from 2 to 9 dpf caused declines in survival and reduction of shell length in F1 larvae at both 5 and 50 ng/L EE2 across all parental exposure treatments. Collectively, parental EE2 imparts effects on offspring in terms of retardation of larval development, and subsequent offspring exposure to EE2 further exacerbates impacts to development. Future research should aim to understand the potential mechanisms of EE2 induced toxicity and its transmission resulting in altered phenotypes of the F1 generation.

This study aimed to reveal possible alterations to lipidomic profiles in Sydney rock oysters, Saccostrea glomerata, exposed to estrogenic mixtures (i.e., estrone, E1; 17β-estradiol, E2; estriol, E3; 17α-ethinylestradiol, EE2; bisphenol A, BPA; 4-t-octylphenol, 4-t-OP; and 4-nonylphenol, 4-NP) at “low” and “high” concentrations, typical of those detected in Australian and global receiving waters. A seven-day acute exposure window exhibited significantly lower abundances of many non-polar metabolites in digestive gland, gills, and gonads. Overall, there was a strong effect of the carrier solvent ethanol (despite a low exposure of 0.0002%), with all solvent containing treatments exhibiting lower abundances of lipidic metabolites, especially in the gill and digestive gland. No significant changes of the lipidome were exhibited in the male gonad by estrogenic exposure. However, in the female gonad, significant reductions of phospholipids and phosphatidylcholine were associated with exposure to high estrogenic mixtures. We hypothesise that the decreases in these phospholipids in the female gonad may be attributable to 1) lower algal consumption and thus lower uptake of lipidic building blocks; 2) a reduction of available substrates for phospholipid and phosphatidylcholine synthesis; and/or 3) induction of reactive oxygen species via estrogen metabolism, which may cause lipid peroxidation and lower abundance of phospholipids.

The current study investigated the effect of environmentally relevant mixtures of estrogens at levels representative of receiving waters on the metabolome of the Sydney rock oyster, Saccostrea glomerata. Oysters were exposed to a "low" and a "high" mixture of (xeno) estrogens (representative of Australian and global receiving waters respectively) for 7 days and digestive gland, gill, and gonad tissue were sampled for quantification of polar metabolites by 1H NMR spectroscopy. Exposure to both mixtures lowered body mass and altered the metabolite profile in the digestive glands. Comparatively, gills, and ovaries demonstrated lesser sensitivity to the mixtures, with significant metabolomic alterations observed only for the high mixture. The male gonad did not respond to either estrogenic exposure. In the responsive tissues, major metabolites including amino acids, carbohydrates, intermediates of the tricarboxylic acid cycle and ATP were all down-regulated and exhibited tissue-specific patterns of down-regulation with the greatest proportion of metabolites down-regulated due to estrogenic exposure in the digestive gland. Exposure to (xeno) estrogen mixtures representative of concentrations reported in receiving waters in Australia and globally can impact the metabolome and associated energy metabolism, especially in the digestive gland, translating to lower pools of available ATP energy for potential cellular homeostasis, somatic maintenance and growth, reproduction and fitness.

Soil in urban areas contains the residues of past land-uses and practices. Urban farming (keeping chickens, vegetable gardening) requires soil disturbance and can increase exposure of residents to these contaminants. We measured the level of lead, arsenic, cadmium, copper and zinc contaminants in soil and eggs from 26 backyard chicken coops across the Lower Hunter, NSW Australia. We compared the levels of metals in soil to Health Investigation Levels and metals in home-grown eggs to the levels in commercial eggs tested in this study or published by Food Standards Australia New Zealand. The levels of arsenic, cadmium, copper and zinc were low, both in soil and in home-grown eggs and were comparable to commercial eggs tested in this study. The Health Investigation Level for lead in soil (300mglead/kg soil) was exceeded at 7 of the 26 sites. The level of lead in home-grown eggs was generally higher than in commercial eggs. The reference health standard for meat (including chicken), fruit and vegetables of 0.1mglead/kg produce was exceeded in home-grown eggs from 7 of the 26 sites. There was a significant relationship between the lead level in eggs and the lead level in soil accessible to chickens. As soil lead increased, concentrations of lead in eggs tended to increase. No relationship was detected between the lead level in feed and in eggs. We recommend strategies to reduce ingestion of soil by chickens thereby reducing metal contamination in home-grown eggs.

Same-sex genital interactions (SSGIs) occur across the order primates, yet explaining their maintenance in evolutionary terms appears problematic; as such interactions seem to counteract reproductive goals. We hypothesised that in more promiscuous species, where sexual motivation, mating effort, and non-conceptive heterosexual behaviour are greater, SSGIs may also occur at greater frequencies without necessarily impeding reproduction. We found that the expression of both male and female SSGIs were greater in multimale systems than in unimale ones. Both male and female SSGIs were positively correlated with the degree of promiscuity (relative testes mass). As mating system confers biases in the sex ratio that may influence the expression of SSGIs, we controlled for availability of members of the same-sex. When employing this control, results were largely congruent. For males, SSGIs were expressed more frequently in multimale systems. For both sexes, SSGIs were expressed more frequently with greater relative testes mass. We suggest SSGIs in primates may be a neutral by-product of selection for increases in promiscuous sexual activity, and that in certain instances these interactions may be co-opted to facilitate adaptive social functions.

The metal-binding protein metallothionein (MT) is widely used as a biomarker of metal contamination. In this study, we cloned a MT gene (sgMT) from the Sydney rock oyster Saccostrea glomerata. The gene encodes a MT-I protein with a classical αβ domain structure and is expressed as two transcripts resulting from alternative polyadenylation. The gene promoter contains two putative metal-responsive elements (MREs) which are known to be required for metal-inducible transcription. A specific and efficient qPCR assay was developed to quantify sgMT mRNA expression. Further, we assessed whether prior metal exposure history influences sgMT mRNA expression upon subsequent metal exposure. Oysters with varying prior metal exposure histories (contaminated and reference) were exposed to Cu, Cd and Zn. Expression of sgMT generally increased with metal dose, and oysters with an elevated past metal exposure history exhibited higher sgMT expression under Cd and Zn stress, representing a potential acclimatory response to prior metal exposure.

We aimed to determine if offspring of oysters from contaminated locations were more tolerant to metals, and whether this tolerance could be attributed to acclimation. Oysters from 10 estuaries were sampled, representing a gradient in metal contamination. Tolerance to metals of the F1 offspring from adults residing in these estuaries was assessed. Then, adults from these estuaries were translocated to a single estuary and their offspring tolerance reassessed. No linear relationship was found between the Cu concentrations of adults and their offspring's tolerance to Cu. A positive linear relationship was found between the Zn concentration of adults and the Zn EC50's of their offspring. Zn tolerance was lost after translocation. Zn EC50 values of offspring from transplanted adults bore no relation to the Zn EC50's of their location of origin. Thus the initial tolerance observed could be attributed to acclimation transferred to the F1 generation.

In vertebrates, estrogens and estrogen mimicking chemicals modulate gene expression mainly through a genomic pathway mediated by the estrogen receptors (ERs). Although the existence of an ER orthologue in the mollusc genome has been known for some time, its role in estrogen signalling has yet to be deciphered. This is largely due to its constitutive (ligand-independent) activation and a limited mechanistic understanding of its regulation. To fill this knowledge gap, we cloned and characterised an ER cDNA (sgER) and the 5'-flanking region of the gene from the Sydney rock oyster Saccostrea glomerata. The sgER cDNA is predicted to encode a 477-amino acid protein that contains a DNA-binding domain (DBD) and a ligand-binding domain (LBD) typically conserved among both vertebrate and invertebrate ERs. A comparison of the sgER LBD sequence with those of other ligand-dependent ERs revealed that the sgER LBD is variable at several conserved residues known to be critical for ligand binding and receptor activation. Ligand binding assays using fluorescent-labelled E2 and purified sgER protein confirmed that sgER is devoid of estrogen binding. In silico analysis of the sgER 5'-flanking sequence indicated the presence of three putative estrogen responsive element (ERE) half-sites and several putative sites for ER-interacting transcription factors, suggesting that the sgER promoter may be autoregulated by its own gene product. sgER mRNA is ubiquitously expressed in adult oyster tissues, with the highest expression found in the ovary. Ovarian expression of sgER mRNA was significantly upregulated following in vitro and in vivo exposure to 17β-estradiol (E2). Notably, the activation of sgER expression by E2 in vitro was abolished by the specific ER antagonist ICI 182, 780. To determine whether sgER expression is epigenetically regulated, the in vivo DNA methylation status of the putative proximal promoter in ovarian tissues was assessed using bisulfite genomic sequencing. The results showed that the promoter is predominantly hypomethylated (with 0-3.3% methylcytosines) regardless of sgER mRNA levels. Overall, our investigations suggest that the estrogen responsiveness of sgER is regulated by a novel ligand-dependent receptor, presumably via a non-genomic pathway(s) of estrogen signalling.

The Sydney rock oyster, Saccostrea glomerata, has been demonstrated as a useful biomonitor of estrogenic compounds following laboratory exposures, yet its utility in the assessment of estrogenic exposure and effects under field conditions requires investigation. To achieve this aim, S. glomerata were deployed in Newcastle, Australia in the effluent receiving marine waters of Burwood Beach WWTP (Burwood Beach "near"

Although mounting evidence suggests exposure to estrogenic contaminants increases vitellogenin production in molluscs, demonstration of dose-response relationships and knowledge of the temporal nature of the vitellogenin response with continual exposure is currently lacking for biomarker utility. To address this knowledge gap, adult Sydney rock oysters, Saccostrea glomerata, were exposed to a range of environmentally relevant concentrations of 17α-ethynylestradiol (EE2) (0, 6.25, 12.5, 25 or 50 ng/l) in seawater under laboratory conditions. Vitellogenin induction and gonadal development was assessed following 4, 21 and 49 days exposure to EE2. Vitellogenin was found to increase in a dose dependent manner with EE2 exposure for females (4 and 49 days) and males (4 and 21 days). Histological examination of gonads revealed a number of individuals exhibited intersex (ovotestis) in 50 ng/l EE2 (after 21 days) and in 6.25 and 12.5 ng/l EE2 (after 49 days). Furthermore, a significant shift towards females was observed following 49 days exposure at 50 ng/l EE2 suggesting estrogenic exposure is capable of facilitating a progression for protandric males from male-intersex-female gametal status. Increases in female vitellogenin (4 days) were predictive of later increases in female developmental stages at 21 days and increases in oocyte area following 49 days. Male vitellogenin (4 days) was predictive of decreased male percentages and lower male developmental stages at 49 days. Vitellogenin in S. glomerata is a predictive biomarker of estrogenic exposure and effect if sampled soon after exposure and at the commencement of a gonadal development cycle.

The current study represents the first investigation of the suitability of marsupial and eutherian mammalian hair as indicator tissue for metal exposure and accumulation within contaminated Australian terrestrial ecosystems. A soil metal contamination gradient was established across 22 sites at increasing distances from a decommissioned Lead/Zinc smelter in NSW, Australia. Within each site, soil and small mammal populations were sampled. An Australian native marsupial, the insectivorous Brown Antechinus, Antechinus stuartii: Dasyuridae, and introduced rodents, the omnivorous Brown or Norway Rat, Rattus norvegicus: Muridae and the Black Rat, Rattus rattus: Muridae were assessed for hair concentrations of Cadmium (Cd), Copper (Cu), Lead (Pb) and Zinc (Zn). Metals in soil were most elevated at sites within close proximity to the smelter, with soil metal concentrations decreasing with distance from the smelter. The non-essential metals Pb and Cd were accumulated in hair, both metals exhibiting positive linear relationships with environmental exposure (soil metal concentrations). When the variables of weight and snout-vent length were considered, no further contribution in terms of explaining the variability in hair Cd or Pb was observed for all species examined. The essential metals Cu and Zn were regulated in hair, remaining similar across the metal contamination gradient. A significant negative correlation between snout-vent length and hair Cu concentration was found for the Brown Rat; greater hair Cu concentrations were found in smaller individuals of this species. Accumulation of Pb to hair was similar among species while concentrations of Cd in Brown Rat hair were higher than both Black Rat and Brown Antechinus hair. As each of the three aforementioned species exhibit similar bioaccumulation relationships for Pb, we suggest that sampling hair from introduced rodents (pest species) may provide a suitable proxy for the assessment of Pb bioavailability for a range of small mammals within Australian urban remnants.

Adult Saccostrea glomerata were exposed to environmentally relevant concentrations of 4-nonylphenol (1microg/L and 100microg/L) and 17alpha-ethynylestradiol (5ng/L and 50ng/L) in seawater over 8 weeks. Exposures were performed to assess effects on vitellogenin induction and gonadal development during reproductive conditioning. Chronic direct estrogenicity within gonadal tissue was assessed via an estrogen receptor-mediated, chemical-activated luciferase reporter gene-expression assay (ER-CALUX). Estradiol equivalents (EEQ) were greatest in the 100microg/L 4-nonylphenol exposure (28.7+/-2.3ng/g tissue EEQ) while 17alpha-ethynylestradiol at concentrations of 50ng/L were 2.2+/-1.5ng/g tissue EEQ. Results suggest 4-nonylphenol may be accumulated in tissue and is partly resistant to biotransformation; maintaining its potential for chronic estrogenic action, while 17alpha-ethynylestradiol, although exhibiting greater estrogenic potency on biological endpoints possibly exerts its estrogenic action before being rapidly metabolised and/or excreted. A novel methodology was developed to assess vitellogenin using high-performance liquid chromatography (HPLC). Exposure to both 17alpha-ethynylestradiol (50ng/L) and 4-nonylphenol (100microg/L) produced increases in vitellogenin for females, whereas males exhibited increases in vitellogenin when exposed to 50ng/L 17alpha-ethynylestradiol only. Females exhibited greater vitellogenin responses than males at 50ng/L 17alpha-ethynylestradiol only. Histological examination of gonads revealed a number of individuals exhibiting intersex (ovotestis) in 50ng/L 17alpha-ethynylestradiol exposures. Male individuals in 1microg/L and 100microg/L 4-nonylphenol exposures and 5ng/L 17alpha-ethynylestradiol were at earlier stages of spermatogenic development than corresponding controls.

The ability of animals to act in a bioremediative capacity is not widely known. Animals are rarely considered for bioremediation initiatives owing to ethical or human health concerns. Nonetheless, specific examples in the literature reveal that some animal species are effective remediators of heavy metals, microbial contaminants, hydrocarbons, nutrients and persistent organic pollutants, particularly in an aquatic environment. Recent examples include deploying pearl oysters to remove metals and nutrients from aquatic ecosystems and the harvest of fish to remove polychlorinated biphenyls (PCBs) from the Baltic. It is probable that many animal taxa will possess attributes amenable to bioremediation. We introduce zoological equivalents of the definitions used in phytoremediation literature (zooextraction, zootransformation, zoostabilization and animal hyperaccumulation), to serve as useful benchmarks in the evaluation of candidate animal species for zooremediation initiatives, and propose that recognition of the concept of zooremediation would act to stimulate discussion and future research in this area.

Variation in glutathione antioxidant biochemistry in response to metal contamination and accumulation under field conditions was examined in the brachyurid grapsid, Parasesarma erythodactyla. Significant relationships suggesting accumulation were found between sediment metals and metals in crab tissue for Pb, Cu, Cr, Zn, and Se in males and Cd, Pb, Cr Zn, As, and Se in females. Higher pH and lower organic content were associated with greater uptake of selected metals in males and females. Higher salinity was related to increased metal uptake for Cu and Zn in males and lower salinities to increased Se uptake for males and females. When examining metals, which were elevated in crabs, patterns of site discrimination were similar to sediment metal site discrimination for both males and females. In terms of biochemical responses, glutathione levels remained constant while glutathione peroxidase activity was elevated in individuals where metals were elevated. Only females with the highest levels of accumulated metals exhibited increases in lipid peroxidation products. Glutathione peroxidase activity may be a sensitive biomarker of metal exposure and biological effect and lipid peroxides as a secondary marker when accumulated metals are high.

The Akoya pearl oyster (Pinctada imbricata) was experimentally exposed to (a) constant levels of lead (Pb) at 180 microg L(-1) for nine weeks, or (b) two short term (pulse) exposures of Pb at 180 microg L(-1) (three weeks each) with an intervening depuration period (three weeks), to assess its utility as an (i) accumulative monitor of Pb contamination and an (ii) archival monitor for discriminating constant versus pulsed Pb exposure events. P. imbricata showed similar reductions in growth (based on shell morphology and wet weight) and Pb accumulation patterns for whole tissue and shell in response to both Pb exposure regimes. Thus the whole oyster was deemed an inappropriate accumulative monitor for assessing short-term temporal variation of Pb exposure and effect. However, using secondary ion mass spectrometry, Pb was shown to accumulate in the successively deposited nacreous layers of the shell of P. imbricata, documenting the exposure history of constant versus pulsed Pb events. Patterns of Pb deposition not only reflected the frequency of Pb exposure events but also their relative durations. Thus, the shell of P. imbricata may be employed as a suitable biological archive of Pb exposure.

The use of pearl oysters has recently been proposed as an environmental remediation tool in coastal ecosystems. This study quantified the nitrogen, phosphorus and heavy metal content of the tissue and shell of pearl oysters harvested from a small pearl oyster farm at Port Stephens, Australia. Each tonne of pearl oyster material harvested resulted in approximately 703 g metals, 7452 g nitrogen, and 545 g phosphorus being removed from the waters of Port Stephens. Increasing current farm production of 9.8 tyr(-1) to 499 tyr(-1) would balance current nitrogen loads entering Port Stephens from a small Sewage Treatment Plant (STP) located on its southern shores. Furthermore, manipulation of harvest dates to coincide with oyster condition would likely remove substantially greater quantities of nutrients. This study demonstrates that pearl aquaculture may be used to assist in the removal of pollutants from coastal waters while producing a commercially profitable commodity.

Surveys conducted after a crude oil spill indicated that the intertidal gastropod mollusc Austrocochlea porcata may be highly sensitive to the pollutant, and therefore also valuable as a biomonitoring organism. Toxicity tests conducted in the laboratory and field established cause-effect for A. porcata mortalities on exposure to environmentally relevant concentrations of crude oil constituents. Glutathione antioxidant system components (glutathione and glutathione peroxidase, GPx) and oxidative damage (lipid peroxidation) in A. porcata were measured to determine whether any of these biochemical parameters showed potential as biomarkers of sublethal oil exposure. GPx was the most promising candidate for field-based biomarker studies after showing a dose-dependent induction to a crude oil water accommodated fraction (WAF) in laboratory assays. However, subsequent manipulative field experimentation indicated that the GPx response was not sufficiently sensitive and not necessarily predictive of population level effects when measured in situ.