David A. Spadaro

Sub-lethal effects of copper to benthic invertebrates explained by sediment properties and dietary exposure

The next generation of sediment quality guidelines (SQGs) requires better established causal links between the chronic exposure and effects of metals from both dissolved and dietary sources. The potential for dietary exposure from sediment metals to cause toxic effects to benthic invertebrates is strongly influenced by the metal-binding properties of the sediments. For relatively oxidized sediments, sublethal effects of copper to the epibenthic deposit-feeding amphipod, Melita plumulosa, and the benthic harpacticoid copepod, Nitocra spinipes, were investigated. Effects on reproduction were strongly influenced by the properties of the sediments and sediment-bound copper was found to be the major contribution to the toxicity. For sediments with the same total copper concentrations, effects were less for sediments with greater concentrations of fine particles (<63 μm sediment) or particulate organic carbon (OC). The OC-normalized copper concentration in the <63 μm sediment fraction provided a single effects threshold for all sediment types. For M. plumulosa and N. spinipes, the 10% effect concentrations (EC10s) were 5.2 and 4.8 mg <63 μm Cu g(-1) OC. These chronic EC10s indicate that a SQG of 3.5 mg <63 μm Cu g(-1) OC, that was previously proposed based on a species sensitivity distribution of acute no effects thresholds data for 12 benthic organisms, will be protective for these species. The study confirms the appropriateness of using SQGs that vary with sediment properties and that SQGs of this form provide adequate protection for metal exposure via both dissolved and dietary exposure pathways.

Performance and sensitivity of rapid sublethal sediment toxicity tests with the amphipod Melita plumulosa and copepod Nitocra spinipes

Sublethal whole-sediment toxicity tests are an important tool for assessing the potential effects of contaminated sediments. However, the longer duration required for evaluating potential chronic effects may increase endpoint variability and test costs compared to survival endpoints. In the present study we compare the performance and sensitivity to contaminants of 10-d sublethal sediment toxicity tests with the amphipod Melita plumulosa and harpacticoid copepod Nitocra spinipes. For both tests, sublethal effects were consistently observed when sediment contaminant concentrations exceeded sediment quality guideline (SQG) concentrations. The response of these bioassays in metal-contaminated sediments was shown to conform ideally with respect to the mean SQG quotient calculated on the basis of the Australian and New Zealand lower SQG trigger value, with toxicity being observed only in those sediments where the mean quotient exceeded one. Better predictions of nontoxicity were obtained when dilute acid-extractable rather than total metal concentrations were used. Using the upper SQG, toxicity frequently occurred at mean quotients below one. The effects were generally consistent with predictions from the acid-volatile sulfide and simultaneously extracted metal model. Effects on reproduction of M. plumulosa were detected for sediments that did not cause effects on survival and highlighted the environmental relevance and importance of using these sublethal endpoints. When using four replicates for M. plumulosa and five replicates for N. spinipes, the endpoint variability (standard error) was less than 10%. Variations in sediment particle size and organic carbon content did not affect endpoint variability. Both species are relatively easily cultured in the laboratory, and the estimated effort and cost of achieving the sublethal endpoints is 1.5 times that of the acute survival test endpoints.

Development and application of a rapid amphipod reproduction test for sediment-quality assessment.

Melita plumulosa is an epibenthic, detritivorous amphipod native to eastern Australia that has been adopted as a test organism for toxicity evaluations of contaminated estuarine sediments. In the present study, a 13-d amphipod reproduction test was developed that encompasses gametogenesis, fertilization, and embryo development before hatching. The primary endpoints for the test are fecundity (measured as the number of embryos per individual surviving female) and a fecundity index (fecundity multiplied by the stage of embryo development). This new test has been employed to scrutinize the sediments from a metal-contaminated coastal lagoon. Lake Macquarie (NSW, Australia) is a large, saltwater lagoon that has received metal pollution over many decades, leading to a concentration gradient of trace metals, including Pb, Zn, Cd, and Cu, in the sediments. Within one of the northern bays (Warners Bay), the concentrations of these metals either border on or exceed sediment quality guideline values prescribed by Australian and New Zealand Guidelines for Fresh and Marine Water Quality. In trials with the 13-d amphipod reproduction test, Warners Bay sediments significantly reduced fecundity in the test species. Subsequent tests with clean sediments spiked singly with Pb, Zn, or Cu indicated that no single metal was responsible for the observed toxicity in the field sediments. However, sediments spiked with various combinations of Pb, Zn, Cd, and Cu indicated that Zn in combination with one or more of the other metals was responsible for the reproductive toxicity observed in Warners Bay sediments. In all these tests, measured metal concentrations in overlying water and pore water were low, thus confirming that the observed effects on reproduction could be attributed to dietary exposure to metals.

Climate-driven mobilisation of acid and metals from acid sulfate soils

The recent drought in south-eastern Australia has exposed to air, large areas of acid sulfate soils within the River Murray system. Oxidation of these soils has the potential to release acidity, nutrients and metals. The present study investigated the mobilisation of these substances following the rewetting of dried soils with River Murray water. Trace metal concentrations were at background levels in most soils. During 24-h mobilisation tests, the water pH was effectively buffered to the pH of the soil. The release of nutrients was low. Metal release was rapid and the dissolved concentrations of many metals exceeded the Australian water quality guidelines (WQGs) in most tests. The concentrations of dissolved Al, Cu and Zn were often greater than 100× the WQGs and strong relationships existed between dissolved metal release and soil pH. Attenuation of dissolved metal concentrations through co-precipitation and adsorption to Al and Fe precipitates was an important process during mixing of acidic, metal-rich waters with River Murray water. The study demonstrated that the rewetting of dried acid sulfate soils may release significant quantities of metals and a high level of land and water management is required to counter the effects of such climate change events.

454 pyrosequencing-based analysis of gene expression profiles in the amphipod Melita plumulosa: transcriptome assembly and toxicant induced changes.

Next generation sequencing using Roches 454 pyrosequencing platform can be used to generate genomic information for non-model organisms, although there are bioinformatic challenges associated with these studies. These challenges are compounded by a lack of a standardized protocol to either assemble data or to evaluate the quality of a de novo transcriptome. This study presents an assembly of the control and toxicant responsive transcriptome of Melita plumulosa, an Australian amphipod commonly used in ecotoxicological studies. RNA was harvested from control amphipods, juvenile amphipods, and from amphipods exposed to either metal or diesel contaminated sediments. This RNA was used as the basis for a 454 based transcriptome sequencing effort. Sequencing generated 1.3 million reads from control, juvenile, metal-exposed and diesel-exposed amphipods. Different read filtering and assembly protocols were evaluated to generate an assembly that (i) had an optimal number of contigs; (ii) had long contigs; (iii) contained a suitable representation of conserved genes; and (iv) had long ortholog alignment lengths relative to the length of each contig. A final assembly, generated using fixed-length trimming based on the sequence quality scores, followed by assembly using the MIRA algorithm, produced the best results. The 26,625 contigs generated via this approach were annotated using Blast2GO, and the differential expression between treatments and control was determined by mapping with BWA followed by DESeq. Although the mapping generated low coverage, many differentially expressed contigs, including some with known developmental or toxicological function, were identified. This study demonstrated that 454 pyrosequencing is an effective means of generating reference transcriptome information for organisms, such as the amphipod M. plumulosa, that have no genomic information available in databases or in closely related sequenced species. It also demonstrated how optimization of read filtering protocols and assembly approaches changes the utility of results obtained from next generation sequencing studies, and establishes criteria to determine the quality of a de novo assembly in species lacking a reference genome. This new transcriptomic knowledge provides the genomic foundation for the creation of microarray and qPCR assays, serving as a reference transcriptome in future RNAseq studies, and allowing both the biology and ecotoxicology of this organism to be better understood. This approach will allow genomics-based methodology to be applied to a wider range of environmentally relevant species.

The impact of sediment bioturbation by secondary organisms on metal bioavailability, bioaccumulation and toxicity to target organisms in benthic bioassays: implications for sediment quality assessment

Bioturbation alters the properties of sediments and modifies contaminant bioavailability to benthic organisms. These naturally occurring disturbances are seldom considered during the assessment of sediment quality. We investigated how the presence (High bioturbation) and absence (Low bioturbation) of a strongly bioturbating amphipod within three different sediments influenced metal bioavailability, survival and bioaccumulation of metals to the bivalve Tellina deltoidalis. The concentrations of dissolved copper decreased and manganese increased with increased bioturbation. For copper a strong correlation was observed between increased bivalve survival (53-100%) and dissolved concentrations in the overlying water. Increased bioturbation intensity resulted in greater tissue concentrations for chromium and zinc in some test sediments. Overall, the results highlight the strong influence that the natural bioturbation activities from one organism may have on the risk contaminants pose to other organisms within the local environment. The characterisation of field-based exposure conditions concerning the biotic or abiotic resuspension of sediments and the rate of attenuation of released contaminants through dilution or readsorption may enable laboratory-based bioassay designs to be adapted to better match those of the assessed environment.

Incorporating bioavailability into management limits for copper in sediments contaminated by antifouling paint used in aquaculture

Although now well embedded within many risk-based sediment quality guideline (SQG) frameworks, contaminant bioavailability is still often overlooked in assessment and management of contaminated sediments. To optimise management limits for metal contaminated sediments, we assess the appropriateness of a range methods for modifying SQGs based on bioavailability considerations. The impairment of reproduction of the amphipod, Melita plumulosa, and harpacticoid copepod, Nitocra spinipes, was assessed for sediments contaminated with copper from antifouling paint, located below aquaculture cages. The measurement of dilute acid-extractable copper (AE-Cu) was found to provide the most useful means for monitoring the risks posed by sediment copper and setting management limits. Acid-volatile sulfide was found to be ineffective as a SQG-modifying factor as these organisms live mostly at the more oxidised sediment water interface. SQGs normalised to %-silt/organic carbon were effective, but the benefits gained were too small to justify this approach. The effectiveness of SQGs based on AE-Cu was attributed to a small portion of the total copper being present in potentially bioavailable forms (typically<10% of the total). Much of the non-bioavailable form of copper was likely present as paint flakes in the form of copper (I) oxide, the active ingredient of the antifoulant formulation. While the concentrations of paint-associated copper are very high in some sediments, as the transformation of this form of copper to AE-Cu appears slow, monitoring and management limits should assess the more bioavailable AE-Cu forms, and further efforts be made to limit the release of paint particles into the environment.

Bioavailability and Chronic Toxicity of Metal Sulfide Minerals to Benthic Marine Invertebrates: Implications for Deep Sea Exploration, Mining and Tailings Disposal.

The exploration and proposed mining of sulfide massive deposits in deep-sea environments and increased use deep-sea tailings placement (DSTP) in coastal zones has highlighted the need to better understand the fate and effects of mine-derived materials in marine environments. Metal sulfide ores contain high concentrations of metal(loid)s, of which a large portion exist in highly mineralized or sulfidised forms and are predicted to exhibit low bioavailability. In this study, sediments were spiked with a range of natural sulfide minerals (including chalcopyrite, chalcocite, galena, sphalerite) to assess the bioavailability and toxicity to benthic invertebrates (bivalve survival and amphipod survival and reproduction). The metal sulfide phases were considerably less bioavailable than metal contaminants introduced to sediment in dissolved forms, or in urban estuarine sediments contaminated with mixtures of metal(loid)s. Compared to total concentrations, the dilute-acid extractable metal(loid) (AEM) concentrations, which are intended to represent the more oxidized and labile forms, were more effective for predicting the toxicity of the sulfide mineral contaminated sediments. The study indicates that sediment quality guidelines based on AEM concentrations provide a useful tool for assessing and monitoring the risk posed by sediments impacted by mine-derived materials in marine environments.

The mismatch between bioaccumulation in field and laboratory environments: Interpreting the differences for metals in benthic bivalves

Laboratory-based bioaccumulation and toxicity bioassays are frequently used to predict the ecological risk of contaminated sediments in the field. This study investigates the bioassay conditions most relevant to achieving environmentally relevant field exposures. An identical series of metal-contaminated marine sediments were deployed in the field and laboratory over 31 days. Changes in metal concentrations and partitioning in both sediments and waters were used to interpret differences in metal exposure and bioaccumulation to the benthic bivalve Tellina deltoidalis. Loss of resuspended sediments and deposition of suspended particulate matter from the overlying water resulted in the concentrations of Cu, Pb and Zn (major contaminants) becoming lower in the 1-cm surface layer of field-deployed sediments. Lower exchange rates of overlying waters in the laboratory resulted in higher dissolved metal exposures. The prediction of metal bioaccumulation by the bivalves in field and laboratory was improved by considering the metal partitioning within the surface sediments.

Dissolved and Particulate Copper Exposure Induces Differing Gene Expression Profiles and Mechanisms of Toxicity in the Deposit Feeding Amphipod Melita plumulosa

Uptake of metals via ingestion is an important route of exposure for many invertebrates, and it has been suggested that the toxic response to metals accumulated via food differs from that of metals accumulated via the dissolved phase. To test this hypothesis, the deposit-feeding epibenthic amphipod Melita plumulosa was exposed to nontoxic or reproductively toxic concentrations of copper via the overlying water, via ingestion of sediment, or via a combination of the two. Rates of copper uptake from the two exposure routes were predicted using a biokinetic model. Gene expression profiles were measured via microarray analysis and confirmed via quantitative polymerase chain reaction. Differences in expression profiles were related to the exposure route more than to individual or combined rates of copper uptake. Chitinase and digestive protease transcript expression levels correlated to the copper uptake rate from sediment, rather than from the dissolved phase or combined total uptake rate. Overall, this study supports the hypothesis that metals accumulated via ingestion have a different mode of toxic action than metals taken up from water. Consequently, guidelines that only consider dissolved metal exposure, including equilibrium-partitioning-based guidelines, may underestimate the potential effects from deposited or resuspended metal-contaminated sediments.