Partitioning tissue metal concentration into subcellular compartments relecting 'toxicologically available'pools may provide good descriptors of the toxicological efects of metals on organisms. Here we investigatedthe relationships between internal compartmentalization of Cd, Pb and Zn and biomarker responses in a model soil organism: the earthworm. The aim of this study was to identify metal fractions reflecting the toxic pressure in an endogeic, naturally occurring earthworm species (Aporrectodea caliginosa) exposed torealistic field-contaminated soils.After a 21 days exposure experiment to 31 field contaminated soils, Cd, Pb and Zn concentrationsin earthworms and in three subcellular fractions (cytosol, debris and granules) were quantified. Differentbiomarkers were measured: the expression of a metallothionein gene (mt), the activity of catalase (CAT) andof glutathione-s-transferase (GST), and the protein, lipid and glycogen reserves. Biomarkers were furthercombined in an integrated biomarker index (IBR).The subcellular fractionation provided better predictors of biomarkers than the total internal contentshence supporting its use when assessing toxicological bioavailability of metals to earthworms. The mostsoluble internal pools of metals were not always the best predictors of biomarker responses. metallothioneinexpression responded to increasing concentrations of Cd in the insoluble fraction (debris + granules). Proteinand glycogen contents were also mainly related to Cd and Pb in the insoluble fraction. On the other hand,GST activity was better explained by Pb in the cytosolic fraction. CAT activity and lipid contents variationswere not related to metal subcellular distribution. The IBR was best explained by both soluble and insolublefractions of Cd and Pb.This study further extends the scope of mt expression as a robust and specific biomarker in an ecologicallyrepresentative earthworm species exposed to field-contaminated soils. The genetic lineage of the individuals,assessed by DNA barcoding with cytochrome c oxidase subunit I, did not inuence mt expression.

Partitioning tissue metal concentration into subcellular compartments relecting 'toxicologically available'pools may provide good descriptors of the toxicological efects of metals on organisms. Here we investigatedthe relationships between internal compartmentalization of Cd, Pb and Zn and biomarker responses in a model soil organism: the earthworm. The aim of this study was to identify metal fractions reflecting the toxic pressure in an endogeic, naturally occurring earthworm species (Aporrectodea caliginosa) exposed torealistic field-contaminated soils.After a 21 days exposure experiment to 31 field contaminated soils, Cd, Pb and Zn concentrationsin earthworms and in three subcellular fractions (cytosol, debris and granules) were quantified. Differentbiomarkers were measured: the expression of a metallothionein gene (mt), the activity of catalase (CAT) andof glutathione-s-transferase (GST), and the protein, lipid and glycogen reserves. Biomarkers were furthercombined in an integrated biomarker index (IBR).The subcellular fractionation provided better predictors of biomarkers than the total internal contentshence supporting its use when assessing toxicological bioavailability of metals to earthworms. The mostsoluble internal pools of metals were not always the best predictors of biomarker responses. metallothioneinexpression responded to increasing concentrations of Cd in the insoluble fraction (debris + granules). Proteinand glycogen contents were also mainly related to Cd and Pb in the insoluble fraction. On the other hand,GST activity was better explained by Pb in the cytosolic fraction. CAT activity and lipid contents variationswere not related to metal subcellular distribution. The IBR was best explained by both soluble and insolublefractions of Cd and Pb.This study further extends the scope of mt expression as a robust and specific biomarker in an ecologicallyrepresentative earthworm species exposed to field-contaminated soils. The genetic lineage of the individuals,assessed by DNA barcoding with cytochrome c oxidase subunit I, did not inuence mt expression.

The problematic of microplastics pollution in the marine environment is tightly linked to their colonization by a wide diversity of microorganisms, the so-called plastisphere. The composition of the plastisphere relies on a complex combination of multiple factors including the surrounding environment, the time of incubation along with the polymer type, making it difficult to understand how the biofilm evolves during the microplastic lifetime over the oceans. To better define bacterial community assembly processes on plastics, we performed a 5 months spatio-temporal survey of the plastisphere in an oyster farming area in the Bay of Brest (France). We deployed three types of plastic pellets in two positions in the foreshore and in the water column. Plastic-associated biofilm composition in all these conditions was monitored using 16 S rRNA metabarcoding and compared to free-living and attached bacterial members of seawater. We observed that bacterial families associated to plastic pellets were significantly distinct from the ones found in seawater, with a significant prevalence of filamentous Cyanobacteria on plastics. No convergence towards a unique plastisphere was detected between polymers exposed in the intertidal and subtidal area, emphasizing the central role of the surrounding environment on constantly shaping the plastisphere community diversity. However, we could define a bulk of early-colonizers of marine biofilms such as Alteromonas, Pseudoalteromonas or Vibrio. These early-colonizers could reach high abundances in floating microplastics collected in field-sampling studies, suggesting the plastic-associated biofilms could remain at early development stages across large oceanic scales. Our study raises the hypothesis that most members of the plastisphere, including putative pathogens, could result of opportunistic colonization processes and unlikely long-term transport.

Microorganisms able to form biofilms in marine ecosystems are selected depending on immersed surfaces and environmental conditions. Cell attachment directly on toxic surfaces like antifouling coatings suggests a selection of tolerant (or resistant) organisms with characteristics conferring adaptive advantages. We investigated if environment would drive metal resistance gene abundance in biofilms on artificial surfaces. Biofilms were sampled from three surfaces (a PVC reference and two antifouling coatings) deployed in three coastal waters with dissimilar characteristics: The Mediterranean Sea (Toulon) and Atlantic (Lorient) and Indian (Reunion) Oceans. The two coatings differed in metals composition, either Cu thiocyanate and Zn pyrithione (A3) or Cu2O (Hy). Metal resistance genes (MRG) specific to copper (cusA, copA, cueO) or other metals (czcA and pbrT) were monitored with qPCR in parallel to the microbial community using 16S rRNA gene metabarcoding. A lower α-diversity on A3 or Hy than on PVC was observed independent on the site. Weighted Unifrac suggested segregation of communities primarily by surface, with lower site effect. Metacoder log2 fold change ratio and LeFSe discrimination suggested Marinobacter to be specific of Hy and Altererythrobacter, Erythrobacter and Sphingorhabdus of A3. Likewise, the relative abundance of MRG (MRG/bacterial 16S rRNA) varied between surfaces and sites. A3 presented the greatest relative abundances for cusA, cueO and czcA. The latter could only be amplified from A3 communities, except at Toulon. Hy surface presented the highest relative abundance for copA, specifically at Lorient. These relative abundances were correlated with LeFSe discriminant taxa. Dasania correlated positively with all MRG except cueO. Marinobacter found in greater abundance in Hy biofilm communities correlated with the highest abundances of copA and Roseovarius with czcA. These results prove the selection of specific communities with abilities to tolerate metallic biocides forming biofilms over antifouling surfaces, and the secondary but significant influence of local environmental factors.

Nutrient loading is a major threat to estuaries and coastal environments worldwide, therefore, it is critical that we have good monitoring tools to detect early signs of degradation in these ecologically important and vulnerable ecosystems. Traditionally, bottom-dwelling macroinvertebrates have been used for ecological health assessment but recent advances in environmental genomics mean we can now characterize less visible forms of biodiversity, offering a more holistic view of the ecosystem and potentially providing early warning signals of disturbance. We carried out a manipulative nutrient enrichment experiment (0, 150 and 600 g N fertilizer m⁻²) in two estuaries in New Zealand to assess the effects of nutrient loading on benthic communities. After seven months of enrichment, environmental DNA (eDNA) metabarcoding was used to examine the response of eukaryotic (18S rRNA), diatom only (rbcL) and bacterial (16S rRNA) communities. Multivariate analyses demonstrated changes in eukaryotic, diatom and bacterial communities in response to nutrient enrichment at both sites, despite differing environmental conditions. These patterns aligned with changes in macrofaunal communities identified using traditional morphological techniques, confirming concordance between disturbance indicators detected by eDNA and current monitoring approaches. Clear shifts in eukaryotic and bacterial indicator taxa were seen in response to nutrient loading while changes in diatom only communities were more subtle. Community changes were discernible between 0 and 150 g N m⁻² treatments, suggesting that estuary health assessment tools could be developed to detect early signs of degradation. Increasing variation in community structure associated with nutrient loading could also be used as an indicator of stress or approaching tipping points. This work represents a first step towards the development of molecular-based estuary monitoring tools, which could provide a more holistic and standardized approach to ecosystem health assessment with faster turn-around times and lower costs.

Environmental pollution can disrupt the interactions between animals and their symbiotic bacteria, which can lead to adverse effects on the host even in the absence of direct chemical toxicity. It is therefore crucial to understand how environmental pollutants affect animal microbiomes, especially for those chemicals that are designed to target microbes. Here, we study the effects of two biocidal nanoparticles (NPs) (Ag and CuO) on the soil bacterial community and the resident gut microbiome of the earthworm Eisenia fetida over a 28-day period using metabarcoding techniques. Exposures to NPs were conducted following OECD test guidelines and effects on earthworm reproduction and juvenile biomass were additionally recorded in order to compare effects on the host to effects on microbiomes. By employing a full concentration series, we were able to link pollutants to microbiome effects in high resolution. Multivariate analysis, differential abundance analysis and species sensitivity distribution analysis showed that Ag-NPs are more toxic to soil bacteria than CuO-NPs. In contrast to the strong effects of CuO-NPs and Ag-NPs on the soil bacterial community, the earthworm gut microbiome is largely resilient to exposure to biocidal NPs. Despite this buffering effect, CuO-NPs did negatively affect the relative abundance of some earthworm symbionts, including ‘Candidatus Lumbricincola’. Changes in the soil bacterial community and the earthworm microbiome occur at total copper concentrations often found or modelled to occur in agricultural fields, demonstrating that soil bacterial communities and individual taxa in the earthworm microbiome may be at risk from environmental copper exposure including in nanomaterial form.

Oil spills offshore can cause long-term ecological effects on coastal marine ecosystems. Despite their important ecological roles in the cycling of energy and nutrients in food webs, effects on bacteria, protists or arthropods are often neglected. Environmental DNA (eDNA) metabarcoding was applied to characterize changes in the structure of micro- and macro-biota communities of surface sediments over a 7-year period since the occurrence of Hebei Spirit oil spill on December 7, 2007. Alterations in diversities and structures of micro- and macro-biota were observed in the contaminated area where concentrations of polycyclic aromatic hydrocarbons were greater. Successions of bacterial, protists and metazoan communities revealed long-term ecological effects of residual oil. Residual oil dominated the largest cluster of the community-environment association network. Presence of bacterial families (Aerococcaceae and Carnobacteriaceae) and the protozoan family (Platyophryidae) might have conferred sensitivity of communities to oil pollution. Hydrocarbon-degrading bacterial families (Anaerolinaceae, Desulfobacteraceae, Helicobacteraceae and Piscirickettsiaceae) and algal family (Araphid pennate) were resistant to adverse effects of spilt oil. The protistan family (Subulatomonas) and arthropod families (Folsomia, Sarcophagidae Opomyzoidea, and Anomura) appeared to be positively associated with residual oil pollution. eDNA metabarcoding can provide a powerful tool for assessing effects of anthropogenic pollution, such as oil spills on sediment communities and its long-term trends in coastal marine environments.

DNA metabarcoding can provide a high-throughput and rapid method for characterizing responses of communities to environmental stressors. However, within bulk samples, DNA metabarcoding hardly distinguishes live from the dead organisms. Here, both DNA and RNA metabarcoding were applied and compared in experimental freshwater mesocosms conducted for assessment of ecotoxicological responses of zooplankton communities to remediation treatment until 38 days post oil-spill. Furthermore, a novel indicator of normalized vitality (NV), sequence counts of RNA metabarcoding normalized by that of DNA metabarcoding, was developed for assessment of ecological responses. DNA and RNA metabarcoding detected similar taxa richness and rank of relative abundances. Both DNA and RNA metabarcoding demonstrated slight shifts in measured α-diversities in response to treatments. NV presented relatively greater magnitudes of differential responses of community compositions to treatments compared to DNA or RNA metabarcoding. NV declined from the start of the experiment (3 days pre-spill) to the end (38 days post-spill). NV also differed between Rotifer and Arthropoda, possibly due to differential life histories and sizes of organisms. NV could be a useful indicator for characterizing ecological responses to anthropogenic influence; however, the biology of target organisms and subsequent RNA production need to be considered.

Headwater streams are a hotspot of freshwater biodiversity, carrying indispensable resource pools of aquatic species. However, up to now, there remain many challenges to accurately and efficiently characterize the responses of this vulnerable ecosystem to human-induced changes. Here, we collected macroinvertebrate data from 12 different headwater streams in the Liao River of northeast China by DNA metabarcoding approach, to reveal biodiversity changes and ecological thresholds affected by human beings. Our data showed that the community composition and structure of headwater streams had unique and significant differences under human impacts, and 5-day biological oxygen demand (BOD₅) and ammonia nitrogen (NH₃–N) were the key variables explaining the variation in community structure. Although α diversity had a unimodal relationship with nutrients and organic loads, β diversity and its turnover component (species replacement) increased significantly. In addition, 22 and 33 indicative taxa were identified to have significant negative responses to BOD₅ and NH₃–N, respectively, and the change points derived from Threshold Indicator Taxa Analysis (TITAN) for the negative response of their frequency and abundance were BOD₅ >3.42 mg/L and NH₃–N >0.14 mg/L. Overall, this study reveals the biodiversity changes in headwater streams from the aspects of α and β diversity, and also determines the thresholds of BOD₅ and NH₃–N pollutants for one reach at one date from 12 headwater streams, suggesting the potential of DNA metabarcoding approach for threshold analyses in headwater streams.

Sertraline is a widely used antidepressant that becomes an aquatic pollutant through metabolic excretion and improper disposal. Determining the impact of sertraline on benthic microbial ecosystems is important for the transformation of river biogenic elements. However, the molecular initiating event induced by sertraline is more readily observed at higher levels, such as the individual or population level of larger organisms, and the effect is not pronounced in benthic organisms, which are directly involved in nitrogen transformation. Therefore, this study used DNA metabarcoding to analyze the effect of sertraline on the microbial ecosystem and material cycles in river sediment through the lens of a microbial food web. The presence of sertraline in the river sediment enhanced the mineralization capacity of nitrogen and increased the accumulation of nitrate in the sediment. Sertraline affected the structure of the microbial food web by stimulating different successions of bacteria and eukaryotes. A structural equation model revealed that sertraline affected the microbial food web model through top-down forces (predation) by reducing the trophic transfer efficiency from metazoans to protozoans. This effect resulted in decreases in the trophic transfer efficiency from protozoans to bacteria and increases in nitrogen mineralization capacity. This was followed by a gradual increase in the nitrification reaction under the action of nitrifying bacteria, increasing the threat to the ecological health of rivers. The results show that sertraline affects the material cycle of river ecosystems and emphasizes that the assessment of the ecological risks of sertraline needs to be considered from the perspective of the material cycle of ecosystems.