Identification and quantification of Acanthamoeba spp. within seawater at four coastal lagoons on the east coast of Australia.

Acanthamoeba is an opportunistic free-living heterotrophic protist that is the most predominant amoeba in diverse ecological habitats. Acanthamoeba causes amoebic keratitis (AK), a painful and potentially blinding corneal infection. Major risk factors for AK have been linked to non-optimal contact lens hygiene practices and Acanthamoeba contamination of domestic and recreational water. This study investigated the incidence and seasonal variation of Acanthamoeba spp. within coastal lagoons located on the eastern coast of Australia and then examined the association between Acanthamoeba and water abiotic factors and bacterial species within the water. Water samples were collected from four intermittently closed and open lagoons (ICOLLs) (Wamberal, Terrigal, Avoca and Cockrone) every month between August 2019 to July 2020 except March and April. qPCR was used to target the Acanthamoeba 18S rRNA gene, validated by Sanger sequencing. Water abiotic factors were measured in situ using a multiprobe metre and 16S rRNA sequencing (V3-V4) was performed to characterise bacterial community composition. Network analysis was used to gauge putative associations between Acanthamoeba incidence and bacterial amplicon sequence variants (ASVs). Among 206 water samples analysed, 79 (38.3%) were Acanthamoeba positive and Acanthamoeba level was significantly higher in summer compared with winter, spring, or autumn (p = 0.008). More than 50% (23/45) water samples of Terrigal were positive for Acanthamoeba which is a highly urbanised area with extensive recreational activities while about 32% (16/49) samples were positive from Cockrone that is the least impacted lagoon by urban development. All sequenced strains belonged to the pathogenic genotype T4 clade except two which were of genotype clades T2 and T5. Water turbidity, temperature, intl1 gene concentration, and dissolved O2 were significantly associated with Acanthamoeba incidence (p < 0.05). The ASVs level of cyanobacteria, Pseudomonas spp., Candidatus spp., and marine bacteria of the Actinobacteria phylum and Acanthamoeba 18S rRNA genes were positively correlated (Pearson's r ≥ 0.14). The presence of Acanthamoeba spp. in all lagoons, except Wamberal, was associated with significant differences in the composition of bacterial communities (beta diversity). The results of this study suggest that coastal lagoons, particularly those in urbanised regions with extensive water recreational activities, may pose an elevated risk to human health due to the relatively high incidence of pathogenic Acanthamoeba in the summer. These findings underscore the importance of educating the public about the rare yet devastating impact of AK on vision and quality of life, highlighting the need for collaborative efforts between public health officials and educators to promote awareness and preventive measures, especially focusing lagoons residents and travellers.

Response of planktonic microbial assemblages to disturbance in an urban sub-tropical estuary.

Microbes are sensitive indicators of estuarine processes because they respond rapidly to dynamic disturbance events. As most of the world's population lives in urban areas and climate change-related disturbance events are becoming more frequent, estuaries bounded by cities are experiencing increasing stressors, at the same time that their ecosystem services are required more than ever. Here, using a multidisciplinary approach, we determined the response of planktonic microbial assemblages in response to seasonality and a rainfall disturbance in an urban estuary bounded by Australia's largest city, Sydney. We used molecular barcoding (16S, 18S V4 rRNA) and microscopy-based identification to compare microbial assemblages at locations with differing characteristics and urbanisation histories. Across 142 samples, we identified 8,496 unique free-living bacterial zOTUs, 8,175 unique particle associated bacterial zOTUs, and 1,920 unique microbial eukaryotic zOTUs. Using microscopy, we identified only the top <10% abundant, larger eukaryotic taxa (>10 µm), however quantification was possible. The site with the greater history of anthropogenic impact showed a more even community of associated bacteria and eukaryotes, and a significant increase in dissolved inorganic nitrogen following rainfall, when compared to the more buffered site. This coincided with a reduced proportional abundance of Actinomarina and Synechococcus spp., a change in SAR 11 clades, and an increase in the eukaryotic microbial groups Dinophyceae, Mediophyceae and Bathyoccocaceae, including a temporary dominance of the harmful algal bloom dinoflagellate Prorocentrum cordatum (syn. P. minimum). Finally, a validated hydrodynamic model of the estuary supported these results, showing that the more highly urbanised and upstream location consistently experienced a higher magnitude of salinity reduction in response to rainfall events during the study period. The best abiotic variables to explain community dissimilarities between locations were TDP, PN, modelled temperature and salinity (r = 0.73) for the free living bacteria, TP for the associated bacteria (r = 0.43), and modelled temperature (r = 0.28) for the microbial eukaryotic communities. Overall, these results show that a minor disturbance such as a brief rainfall event can significantly shift the microbial assemblage of an anthropogenically impacted area within an urban estuary to a greater degree than a seasonal change, but may result in a lesser response to the same disturbance at a buffered, more oceanic influenced location. Fine scale research into the factors driving the response of microbial communities in urban estuaries to climate related disturbances will be necessary to understand and implement changes to maintain future estuarine ecosystem services.

Rainfall leads to elevated levels of antibiotic resistance genes within seawater at an Australian beach.

Anthropogenic waste streams can be major sources of antibiotic resistant microbes within the environment, creating a potential risk to public health. We examined patterns in the occurrence of a suite of antibiotic resistance genes (ARGs) and their links to enteric bacteria at a popular swimming beach in Australia that experiences intermittent contamination by sewage, with potential points of input including stormwater drains and a coastal lagoon. Samples were collected throughout a significant rainfall event (40.8 mm over 3 days) and analysed using both qPCR and 16S rRNA amplicon sequencing. Before the rainfall event, low levels of faecal indicator bacteria and a microbial source tracking human faeces (sewage) marker (Lachno3) were observed. These levels increased over 10x following rainfall. Within lagoon, drain and seawater samples, levels of the ARGs sulI, dfrA1 and qnrS increased by between 1 and 2 orders of magnitude after 20.4 mm of rain, while levels of tetA increased by an order of magnitude after a total of 40.8 mm. After 40.8 mm of rain sulI, tetA and qnrS could be detected 300 m offshore with levels remaining high five days after the rain event. Highest levels of sewage markers and ARGs were observed adjacent to the lagoon (when opened) and in-front of the stormwater drains, pinpointing these as the points of ARG input. Significant positive correlations were observed between all ARGs, and a suite of Amplicon Sequence Variants that were identified as stormwater drain indicator taxa using 16S rRNA amplicon sequencing data. Of note, some stormwater drain indicator taxa, which exhibited correlations to ARG abundance, included the human pathogens Arcobacter butzleri and Bacteroides fragilis. Given that previous research has linked high levels of ARGs in recreationally used environments to antimicrobial resistant pathogen infections, the observed patterns indicate a potentially elevated human health risk at a popular swimming beach following significant rainfall events.

Molecular microbiological approaches reduce ambiguity about the sources of faecal pollution and identify microbial hazards within an urbanised coastal environment.

Urbanised beaches are regularly impacted by faecal pollution, but management actions to resolve the causes of contamination are often obfuscated by the inability of standard Faecal Indicator Bacteria (FIB) analyses to discriminate sources of faecal material or detect other microbial hazards, including antibiotic resistance genes (ARGs). We aimed to determine the causes, spatial extent, and point sources of faecal contamination within Rose Bay, a highly urbanised beach within Sydney, Australia's largest city, using molecular microbiological approaches. Sampling was performed across a network of transects originating at 9 stormwater drains located on Rose Bay beach over the course of a significant (67.5 mm) rainfall event, whereby samples were taken 6 days prior to any rain, on the day of initial rainfall (3.8 mm), three days later after 43 mm of rain and then four days after any rain. Quantitative PCR (qPCR) was used to target marker genes from bacteria (i.e., Lachnospiraceae and Bacteroides) that have been demonstrated to be specific to human faeces (sewage), along with gene sequences from Heliobacter and Bacteriodes that are specific to bird and dog faeces respectively, and ARGs (sulI, tetA, qnrS, dfrA1 and vanB). 16S rRNA gene amplicon sequencing was also used to discriminate microbial signatures of faecal contamination. Prior to the rain event, low FIB levels (mean: 2.4 CFU/100 ml) were accompanied by generally low levels of the human and animal faecal markers, with the exception of one transect, potentially indicative of a dry weather sewage leak. Following 43 mm of rain, levels of both human faecal markers increased significantly in stormwater drain and seawater samples, with highest levels of these markers pinpointing several stormwater drains as sources of sewage contamination. During this time, sewage contamination was observed up to 1000 m from shore and was significantly and positively correlated with often highly elevated levels of the ARGs dfrA1, qnrS, sulI and vanB. Significantly elevated levels of the dog faecal marker in stormwater drains at this time also indicated that rainfall led to increased input of dog faecal material from the surrounding catchment. Using 16S rRNA gene amplicon sequencing, several indicator taxa for stormwater contamination such as Arcobacter spp. and Comamonadaceae spp. were identified and the Bayesian SourceTracker tool was used to model the relative impact of specific stormwater drains on the surrounding environment, revealing a heterogeneous contribution of discrete stormwater drains during different periods of the rainfall event, with the microbial signature of one particular drain contributing up to 50% of bacterial community in the seawater directly adjacent. By applying a suite of molecular microbiological approaches, we have precisely pinpointed the causes and point-sources of faecal contamination and other associated microbiological hazards (e.g., ARGs) at an urbanised beach, which has helped to identify the most suitable locations for targeted management of water quality at the beach.

Legacy Metal Contaminants and Excess Nutrients in Low Flow Estuarine Embayments Alter Composition and Function of Benthic Bacterial Communities.

Coastal systems such as estuaries are threatened by multiple anthropogenic stressors worldwide. However, how these stressors and estuarine hydrology shape benthic bacterial communities and their functions remains poorly known. Here, we surveyed sediment bacterial communities in poorly flushed embayments and well flushed channels in Sydney Harbour, Australia, using 16S rRNA gene sequencing. Sediment samples were collected monthly during the Austral summer-autumn 2014 at increasing distance from a large storm drain in each channel and embayment. Bacterial communities differed significantly between sites that varied in proximity to storm drains, with a gradient of change apparent for sites within embayments. We explored this pattern for embayment sites with analysis of RNA-Seq gene expression patterns and found higher expression of multiple genes involved in bacterial stress response far from storm drains, suggesting that bacterial communities close to storm drains may be more tolerant of localised anthropogenic stressors. Several bacterial groups also differed close to and far from storm drains, suggesting their potential utility as bioindicators to monitor contaminants in estuarine sediments. Overall, our study provides useful insights into changes in the composition and functioning of benthic bacterial communities as a result of multiple anthropogenic stressors in differing hydrological conditions.

A weight-of-evidence approach for identifying potential sources of untreated sewage inputs into a complex urbanized catchment.

The Hawkesbury-Nepean River (HNR) is the largest catchment in the Sydney region and is undergoing unprecedented population growth. The HNR system receives a mix of anthropogenic inputs such as treated sewage, stormwater and agricultural runoff. Combined, these can diminish the ecological system health and pose potential concerns to human health. Of particular concern are inputs of untreated sewage, that can occur due to a range of different reasons including illegal point source discharges, failure of the sewerage network, and overloading of wastewater treatment plants during storm events. Here, we present findings of an intensive assessment across the HNR catchment where we used a weight-of-evidence (WOE) approach to identify untreated sewage contamination in surface waters against the background of treated effluent and diffuse inputs during post high flow conditions. Total nitrogen and phosphorus concentrations were used to assess treated effluent and diffuse inputs, and microbial analysis, including both culture-based traditional methods for E. coli and enterococci and qPCR analysis of Bacteroides and Lachnospiraceae, were used to assess raw sewage contamination. Despite a background of diffuse inputs from recent high flow events and the influence of treated wastewater, we found no gradient of faecal contamination along the HNR system or its tributaries. We observed two sites with evidence of untreated sewage contamination, where the human markers Bacteroides and Lachnospiraceae qPCR copy numbers were high. The biological and chemical approaches suggested these latter two hotspots originate from an industrial runoff source and possibly from a dry weather sewage leak. Our findings demonstrate the potential of a WOE approach in the assessment of human faecal signal in an urban river that can also pinpoint small sources of contamination as a strategy that can reshape the way monitoring is performed and the chemical end-points chosen to provide pertinent information on the potential risks to aquatic system health.

Climate change rapidly warms and acidifies Australian estuaries.

Climate change is impacting ecosystems worldwide. Estuaries are diverse and important aquatic ecosystems; and yet until now we have lacked information on the response of estuaries to climate change. Here we present data from a twelve-year monitoring program, involving 6200 observations of 166 estuaries along >1100 kilometres of the Australian coastline encompassing all estuary morphologies. Estuary temperatures increased by 2.16 °C on average over 12 years, at a rate of 0.2 °C year-1, with waters acidifying at a rate of 0.09 pH units and freshening at 0.086 PSU year-1. The response of estuaries to climate change is dependent on their morphology. Lagoons and rivers are warming and acidifying at the fastest rate because of shallow average depths and limited oceanic exchange. The changes measured are an order of magnitude faster than predicted by global ocean and atmospheric models, indicating that existing global models may not be useful to predict change in estuaries.

Sediment bacterial communities associated with environmental factors in Intermittently Closed and Open Lakes and Lagoons (ICOLLs).

Intermittently Closed and Open Lakes and Lagoons (ICOLLS) are important coastal systems that are periodically separated from the ocean by a sand barrier or a berm. In urban ICOLLs, continuous inputs of organic material and nutrients into coastal lagoons are contributing to eutrophic conditions that, together with natural environmental factors have implications for the resident sediment bacterial communities. We used molecular tools to investigate the ecological communities of four ICOLLs; Narrabeen, Dee Why, Curl Curl and Manly in Sydney, Australia, which have been subjected to increasing pressure from anthropogenic activities over the last century. We used targeted gene sequencing of the prokaryotic 16S ribosomal RNA gene to describe the bacterial diversity and community structure and discuss differences with respect to environmental factors at the ICOLL scale (e.g. size, shape, normalised N loading) and site scale (e.g. water and sediment quality) within each lagoon. Due to differences in hydrological patterns, we expected that sediment bacterial communities would be more heterogenous in displacement-dominated lagoons (Curl Curl and Manly) than the mixing-dominated lagoons (Narrabeen and Dee Why). Interestingly, we did not find strong relationships between the main bacterial groups and distance from the lagoon entrance (used as a proxy for salinity and silt) in the displacement-dominated lagoons. Moreover, we found that α diversity was highest in Narrabeen and Manly lagoons despite differences in lagoon size and shape. Furthermore, while bacterial community structure was similar in Curl Curl and Dee Why, communities in Manly and Narrabeen differed along temperature/TOC and salinity/silt gradients respectively. In Manly lagoon, we found relatively more anaerobic bacteria such as Epsilonbactereota which is involved in the oxidation and reduction of sulfur compounds. Moreover, we identified several bacterial taxa (including sulfur metabolising Chlorobiaceae) related to increasing TOC that could be investigated further as potential indicators of excess enrichment.

Using meta-omics of contaminated sediments to monitor changes in pathways relevant to climate regulation.

Microbially mediated biogeochemical processes are crucial for climate regulation and may be disrupted by anthropogenic contaminants. To better manage contaminants, we need tools that make real-time causal links between stressors and altered microbial functions, and the potential consequences for ecosystem services such as climate regulation. In a manipulative field experiment, we used metatranscriptomics to investigate the impact of excess organic enrichment and metal contamination on the gene expression of nitrogen and sulfur metabolisms in coastal sediments. Our gene expression data suggest that excess organic enrichment results in (i) higher transcript levels of genes involved in the production of toxic ammonia and hydrogen sulfide and (ii) lower transcript levels associated with the degradation of a greenhouse gas (nitrous oxide). However, metal contamination did not have any significant impact on gene expression. We reveal the genetic mechanisms that may lead to altered productivity and greenhouse gas production in coastal sediments due to anthropogenic contaminants. Our data highlight the applicability of metatranscriptomics as a management tool that provides an immense breadth of information and can identify potentially impacted process measurements that need further investigation.

Coastal acidification impacts on shell mineral structure of bivalve mollusks.

Ocean acidification is occurring globally through increasing CO 2 absorption into the oceans creating particular concern for calcifying species. In addition to ocean acidification, near shore marine habitats are exposed to the deleterious effects of runoff from acid sulfate soils which also decreases environmental pH. This coastal acidification is being exacerbated by climate change-driven sea-level rise and catchment-driven flooding. In response to reduction in habitat pH by ocean and coastal acidification, mollusks are predicted to produce thinner shells of lower structural integrity and reduced mechanical properties threatening mollusk aquaculture. Here, we present the first study to examine oyster biomineralization under acid sulfate soil acidification in a region where growth of commercial bivalve species has declined in recent decades. Examination of the crystallography of the shells of the Sydney rock oyster, Saccostrea glomerata, by electron back scatter diffraction analyses revealed that the signal of environmental acidification is evident in the structure of the biomineral. Saccostrea glomerata, shows phenotypic plasticity, as evident in the disruption of crystallographic control over biomineralization in populations living in coastal acidification sites. Our results indicate that reduced sizes of these oysters for commercial sale may be due to the limited capacity of oysters to biomineralize under acidification conditions. As the impact of this catchment source acidification will continue to be exacerbated by climate change with likely effects on coastal aquaculture in many places across the globe, management strategies will be required to maintain the sustainable culture of these key resources.

Interactive effects of multiple stressors revealed by sequencing total (DNA) and active (RNA) components of experimental sediment microbial communities.

Coastal waterways are increasingly exposed to multiple stressors, e.g. contaminants that can be delivered via pulse or press exposures. Therefore, it is crucial that ecological impacts can be differentiated among stressors to manage ecosystem threats. We investigated microbial community development in sediments exposed to press and pulse stressors. Press exposures were created with in situ mesocosm sediments containing a range of 'metal' concentrations (sediment contaminated with multiple metal(loid)s) and organic enrichment (fertiliser), while the pulse exposure was simulated by a single dose of organic fertiliser. All treatments and exposure concentrations were crossed in a fully factorial field experiment. We used amplicon sequencing to compare the sensitivity of the 1) total (DNA) and active (RNA) component of 2) bacterial (16S rRNA) and eukaryotic (18S rRNA) communities to contaminant exposures. Overall microbial community change was greater when exposed to press than pulse stressors, with the bacterial community responding more strongly than the eukaryotes. The total bacterial community represents a more time-integrated measure of change and proved to be more sensitive to multiple stressors than the active community. Metals and organic enrichment treatments interacted such that the effect of metals was weaker when the sediment was organically enriched. Taxa-level analyses revealed that press enrichment resulted in potential functional changes, mainly involving nitrogen cycling. Furthermore, enrichment generally reduced the abundance of active eukaryotes in the sediment. As well as demonstrating interactive impacts of metals and organic enrichment, this study highlights the sensitivity of next-generation sequencing for ecosystem biomonitoring of interacting stressors and identifies opportunities for more targeted application.

A database of chlorophyll a in Australian waters.

Chlorophyll a is the most commonly used indicator of phytoplankton biomass in the marine environment. It is relatively simple and cost effective to measure when compared to phytoplankton abundance and is thus routinely included in many surveys. Here we collate 173, 333 records of chlorophyll a collected since 1965 from Australian waters gathered from researchers on regular coastal monitoring surveys and ocean voyages into a single repository. This dataset includes the chlorophyll a values as measured from samples analysed using spectrophotometry, fluorometry and high performance liquid chromatography (HPLC). The Australian Chlorophyll a database is freely available through the Australian Ocean Data Network portal (https://portal.aodn.org.au/). These data can be used in isolation as an index of phytoplankton biomass or in combination with other data to provide insight into water quality, ecosystem state, and relationships with other trophic levels such as zooplankton or fish.

Seagrass ecosystem trajectory depends on the relative timescales of resistance, recovery and disturbance.

Seagrass ecosystems are inherently dynamic, responding to environmental change across a range of scales. Habitat requirements of seagrass are well defined, but less is known about their ability to resist disturbance. Specific means of recovery after loss are particularly difficult to quantify. Here we assess the resistance and recovery capacity of 12 seagrass genera. We document four classic trajectories of degradation and recovery for seagrass ecosystems, illustrated with examples from around the world. Recovery can be rapid once conditions improve, but seagrass absence at landscape scales may persist for many decades, perpetuated by feedbacks and/or lack of seed or plant propagules to initiate recovery. It can be difficult to distinguish between slow recovery, recalcitrant degradation, and the need for a window of opportunity to trigger recovery. We propose a framework synthesizing how the spatial and temporal scales of both disturbance and seagrass response affect ecosystem trajectory and hence resilience.

Scavenging rate ecoassay: a potential indicator of estuary condition.

Monitoring of estuary condition is essential due to the highly productive and often intensely impacted nature of these ecosystems. Assessment of the physico-chemical condition of estuaries is expensive and difficult due to naturally fluctuating water quality and biota. Assessing the vigour of ecosystem processes is an alternative method with potential to overcome much of the variability associated with physico-chemical measures. Indicators of estuary condition should have small spatial and temporal variability, have a predictable response to perturbation and be ecologically relevant. Here, we present tests of the first criterion, the spatio-temporal variability of a potential ecoassay measuring the rate of scavenging in estuaries. We hypothesised that the proposed scavenging ecoassay would not vary significantly among A) sites in an estuary, B) trips separated by weeks, or C) days in a trip. Because not all habitats are present in all estuaries, this test was undertaken in two habitats. When conducted over bare substrate there were occasional significant differences, but no discernible patterns, within levels of the experiment. When conducted over vegetated substrate, days within a trip did not vary significantly, but later trips experienced greater scavenging. This scavenging ecoassay shows potential as a tool for assessing the condition of estuarine ecosystems, and further exploration of this protocol is warranted by implementation in estuaries across a gradient of anthropogenic stress.

Unravelling complexity in seagrass systems for management: Australia as a microcosm.

Environmental decision-making applies transdisciplinary knowledge to deliver optimal outcomes. Here we synthesise various aspects of seagrass ecology to aid environmental decision-making, management and policy. Managers often mediate conflicting values and opinions held by different stakeholders. Critical to this role is understanding the drivers for change, effects of management actions and societal benefits. We use the diversity of seagrass habitats in Australia to demonstrate that knowledge from numerous fields is required to understand seagrass condition and resilience. Managers are often time poor and need access to synthesised assessments, commonly referred to as narratives. However, there is no single narrative for management of seagrass habitats in Australia, due to the diversity of seagrass meadows and dominant pressures. To assist the manager, we developed a classification structure based on attributes of seagrass life history, habitat and meadow form. Seagrass communities are formed from species whose life history strategies can be described as colonising, opportunistic or persistent. They occupy habitats defined by the range and variability of their abiotic environment. This results in seagrass meadows that are either transitory or enduring. Transitory meadows may come and go and able to re-establish from complete loss through sexual reproduction. Enduring meadows may fluctuate in biomass but maintain a presence by resisting pressures across multiple scales. This contrast reflects the interaction between the spatial and temporal aspects of species life history and habitat variability. Most management and monitoring strategies in place today favour enduring seagrasses. We adopt a functional classification of seagrass habitats based on modes of resilience to inform management for all seagrass communities. These concepts have world-wide relevance as the Australian case-studies have many analogues throughout the world. Additionally, the approach used to classify primary scientific knowledge into synthesised categories to aid management has value for many other disciplines interfacing with environmental decision-making.

Detrital diversity influences estuarine ecosystem performance.

Global losses of seagrasses and mangroves, eutrophication-driven increases in ephemeral algae, and macrophyte invasions have impacted estuarine detrital resources. To understand the implications of these changes on benthic ecosystem processes, we tested the hypotheses that detrital source richness, mix identity, and biomass influence benthic primary production, metabolism, and nutrient fluxes. On an estuarine muddy sandflat, we manipulated the availability of eight detrital sources, including mangrove, seagrass, and invasive and native algal species that have undergone substantial changes in distribution. Mixes of these detrital sources were randomly assigned to one of 12 treatments and dried detrital material was added to seventy-two 0.25 m(2) plots (n = 6 plots). The treatments included combinations of either two or four detrital sources and high (60 g) or low (40 g) levels of enrichments. After 2 months, the dark, light, and net uptake of NH4 (+) , dissolved inorganic nitrogen, and the dark efflux of dissolved organic nitrogen were each significantly influenced by the identity of detrital mixes, rather than detrital source richness or biomass. However, gross and net primary productivity, average oxygen flux, and net NOX and dissolved inorganic phosphorous fluxes were significantly greater in treatments with low than with high detrital source richness. These results demonstrate that changes in detrital source richness and mix identity may be important drivers of estuarine ecosystem performance. Continued impacts to estuarine macrophytes may, therefore, further alter detritus-fueled productivity and processes in estuaries. Specific tests that address predicted future changes to detrital resources are required to determine the consequences of this significant environmental problem.

The risk of harmful algal blooms (HABs) in the oyster-growing estuaries of New South Wales, Australia.

The spatial and temporal variability of potentially harmful phytoplankton was examined in the oyster-growing estuaries of New South Wales. Forty-five taxa from 31 estuaries were identified from 2005 to 2009. Harmful species richness was latitudinally graded for rivers, with increasing number of taxa southward. There were significant differences (within an estuary) in harmful species abundance and richness for 11 of 21 estuaries tested. Where differences were observed, these were predominately due to species belonging to the Pseudo-nitzschia delicatissima group, Dinophysis acuminata, Dictyocha octonaria and Prorocentrum cordatum with a consistent upstream versus downstream pattern emerging. Temporal (seasonal or interannual) patterns in harmful phytoplankton within and among estuaries were highly variable. Examination of harmful phytoplankton in relation to recognised estuary disturbance measures revealed species abundance correlated to estuary modification levels and flushing time, with modified, slow flushing estuaries having higher abundance. Harmful species richness correlated with bioregion, estuary modification levels and estuary class, with southern, unmodified lakes demonstrating greater species density. Predicting how these risk taxa and risk zones may change with further estuary disturbance and projected climate warming will require more focused, smaller scale studies aimed at a deeper understanding of species-specific ecology and bloom mechanisms. Coupled with this consideration, there is an imperative for further taxonomic, ecological and toxicological investigations into poorly understood taxa (e.g. Pseudo-nitzschia).