In vitro effects of glyphosate-based herbicides and related adjuvants on primary culture of hemocytes from Haliotis tuberculata.

Glyphosate-based herbicides are among the most produced and widely-used herbicides. Studies have shown that commercial formulations and adjuvants may be more toxic to non-target organisms than the active ingredients alone, but the mechanisms of action of these chemicals remain unclear. The aim of this study was to investigate the in vitro effects of glyphosate, a commercial formulation and adjuvant alone using primary culture of hemocytes from the European abalone Haliotis tuberculata, a commonly farmed shellfish. Glyphosate was found to have negligible effects on viability, phagocytic activities and lysosome stability even with very high doses (i.e. 100 mg L-1). By contrast, greater effects on viability were observed for the commercial formulation and adjuvant alone, with EC50 values of 41.42 mg L-1 and 1.85 mg L-1, respectively. These results demonstrate that the toxic sublethal effects (i.e. phagocytic activity and destabilization of lysosomal membranes) of formulated glyphosate came from adjuvants and suggest they may be related to cell and organelle membrane destabilization.

An integrated approach to determine interactive genotoxic and global gene expression effects of multiwalled carbon nanotubes (MWCNTs) and benzo[a]pyrene (BaP) on marine mussels: evidence of reverse 'Trojan Horse' effects.

The interactions between carbon-based engineered nanoparticles (ENPs) and organic pollutants might enhance the uptake of contaminants into biota. The present integrated study aimed to assess this potential 'Trojan Horse', probing the interactive effects of purpose-made multi-walled carbon nanotubes (MWCNTs), a representative ENP, and benzo[a]pyrene (BaP), a ubiquitous polycyclic aromatic hydrocarbon (PAH) pollutant, on the marine mussel Mytilus galloprovincialis. Mussels were exposed to MWCNTs and BaP either alone or in various combinations. The co-exposure of BaP with MWCNTs revealed that the presence of MWCNTs enhanced the aqueous concentrations of BaP, thereby reducing the uptake of this pollutant by mussels as evidenced by lowering BaP concentrations in the tissues. Determination of DNA damage (comet assay) showed a concentration-dependent response for BaP alone which was absent when MWCNTs were present. Global gene expression using microarray analyses indicated that BaP and MWCNTs, in combination, differentially activated those genes which are involved in DNA metabolism compared to the exposures of BaP or MWCNTs alone, and the gene expression response was tissue-specific. Mechanisms to explain these results are discussed and relate primarily to the adsorption of BaP on MWCNTs, mediated potentially by van der Waals interactions. The use of a novel approach based on gold-labeled MWCNTs to track their uptake in tissues improved the traceability of nanotubes in biological samples. Overall, our results did not indicate the 'Trojan Horse' effects following co-exposure to the contaminants and clearly showed that the adsorption of BaP to MWCNTs modified the uptake of the pollutant in marine mussels.

Mixtures of tritiated water, zinc and dissolved organic carbon: Assessing interactive bioaccumulation and genotoxic effects in marine mussels, Mytilus galloprovincialis.

Release of tritium (3H) in the marine environment is of concern with respect to its potential bioaccumulation and detrimental impact on the biota. Previous studies have investigated the uptake and toxicity of this radionuclide in marine mussels, and the interaction of 3H with dissolved organic ligands and elevated temperature. However, despite the well-established view that toxicity is partly governed by chemical speciation, and that toxic effects of mixture of contaminants are not always additive, there have been no studies linking the prevailing chemistry of exposure waters with observed biological effects and tissue specific accumulation of 3H in combination with other constituents commonly found in natural waters. This study exposed the marine mussel Mytilus galloprovincialis for 14 days to mixtures of 3H (as tritiated water, HTO) and zinc (Zn) at 5 Mbq L-1, and 383, 1913 and 3825 nM Zn, respectively, to investigate (a) 3H and Zn partitioning in soft tissues of mussels, and (b) DNA damage in haemocytes, determined using the single cell gel electrophoresis or the comet assay. Additionally, the extent of association of 3H with dissolved organic carbon (DOC, added as humic acid) over the exposure period was investigated in order to aid the interpretation of biological uptake and effects. Results concluded a clear antagonistic effect of Zn on 3H-induced DNA damage at all Zn concentrations used, likely explained by the importance of Zn in DNA repair enzymes. The interaction of DOC with 3H was variable, with strong 3H-DOC associations observed in the first 3 d of the experiment. The secretion of 3H-binding ligands by the mussels is suggested as a possible mechanism for early biological control of 3H toxicity. The results suggest risk assessments for radionuclides in the environment require consideration of potential mixture effects.

An integrated approach to assess the impacts of zinc pyrithione at different levels of biological organization in marine mussels.

The mechanisms of sublethal toxicity of the antifouling biocide, zinc pyrithione (ZnPT), have not been well-studied. This investigation demonstrates that 14-d sublethal exposure to ZnPT (0.2 or 2 µM, alongside inorganic Zn and sea water controls) is genotoxic to mussel haemocytes but suggests that this is not caused by oxidative DNA damage as no significant induction of oxidised purines was detected by Fpg-modified comet assay. More ecologically relevant endpoints, including decreased clearance rate (CR), cessation of attachment and decreased tolerance of stress on stress (SoS), also showed significant response to ZnPT exposure. Our integrated approach was underpinned by molecular analyses (qRT-PCR of stress-related genes, 2D gel electrophoresis of proteins) that indicated ZnPT causes a decrease in phosphoenolpyruvate carboxykinase (PEPCK) expression in mussel digestive glands, and that metallothionein genes are upregulated; PEPCK downregulation suggests that altered energy metabolism may also be related to the effects of ZnPT. Significant relationships were found between % tail DNA (comet assay) and all higher level responses (CR, attachment, SoS) in addition to PEPCK expression. Principal component analyses suggested that expression of selected genes described more variability within groups whereas % tail DNA reflected different ZnPT concentrations.

Assessing the impact of Benzo[a]pyrene on Marine Mussels: Application of a novel targeted low density microarray complementing classical biomarker responses.

Despite the increasing use of mussels in environmental monitoring and ecotoxicological studies, their genomes and gene functions have not been thoroughly explored. Several cDNA microarrays were recently proposed for Mytilus spp., but putatively identified partial transcripts have rendered the generation of robust transcriptional responses difficult in terms of pathway identification. We developed a new low density oligonucleotide microarray with 465 probes covering the same number of genes. Target genes were selected to cover most of the well-known biological processes in the stress response documented over the last decade in bivalve species at the cellular and tissue levels. Our new 'STressREsponse Microarray' (STREM) platform consists of eight sub-arrays with three replicates for each target in each sub-array. To assess the potential use of the new array, we tested the effect of the ubiquitous environmental pollutant benzo[a]pyrene (B[a]P) at 5, 50, and 100 µg/L on two target tissues, the gills and digestive gland, of Mytilus galloprovincialis exposed invivo for three days. Bioaccumulation of B[a]P was also determined demonstrating exposure in both tissues. In addition to the well-known effects of B[a]P on DNA metabolism and oxidative stress, the new array data provided clues about the implication of other biological processes, such as cytoskeleton, immune response, adhesion to substrate, and mitochondrial activities. Transcriptional data were confirmed using qRT-PCR. We further investigated cellular functions and possible alterations related to biological processes highlighted by the microarray data using oxidative stress biomarkers (Lipofuscin content) and the assessment of genotoxicity. DNA damage, as measured by the alkaline comet assay, increased as a function of dose.DNA adducts measurements using 32P-postlabeling method also showed the presence of bulky DNA adducts (i.e. dG-N2-BPDE). Lipofiscin content increased significantly in B[a]P exposed mussels. Immunohistochemical analysis of tubulin and actin showed changes in cytoskeleton organisation. Our results adopting an integrated approach confirmed that the combination of newly developed transcriptomic approcah, classical biomarkers along with chemical analysis of water and tissue samples should be considered for environmental bioimonitoring and ecotoxicological studies to obtain holistic information to assess the impact of contaminants on the biota.

Exposure to tritiated water at an elevated temperature: Genotoxic and transcriptomic effects in marine mussels (M. galloprovincialis).

Temperature is an abiotic factor of particular concern for assessing the potential impacts of radionuclides on marine species. This is particularly true for tritium, which is discharged as tritiated water (HTO) in the process of cooling nuclear institutions. Additionally, with sea surface temperatures forecast to rise 0.5-3.5 °C in the next 30-100 years, determining the interaction of elevated temperature with radiological exposure has never been more relevant. We assessed the tissue-specific accumulation, transcriptional expression of key genes, and genotoxicity of tritiated water to marine mussels at either 15 or 25 °C, over a 7 day time course with sampling after 1 h, 12 h, 3 d and 7d. The activity concentration used (15 MBq L-1) resulted in tritium accumulation that varied with both time and temperature, but consistently produced dose rates (calculated using the ERICA tool) of <20 Gy h-1, i.e. considerably below the recommended guidelines of the IAEA and EURATOM. Despite this, there was significant induction of DNA strand breaks (as measured by the comet assay), which also showed a temperature-dependent time shift. At 15 °C, DNA damage was only significantly elevated after 7 d, in contrast to 25 °C where a similar response was observed after only 3 d. The transcription profiles of two isoforms of hsp70, hsp90, mt20, p53 and rad51 indicated potential mechanisms behind this temperature-induced acceleration of genotoxicity, which may be the result of compromised defence. Specifically, genes involved in protein folding, DNA double strand break repair and cell cycle checkpoint control were upregulated after 3 d HTO exposure at 15 °C, but significantly downregulated when the same exposure occurred at 25 °C. This study is the first to investigate temperature effects on radiation-induced genotoxicity in an ecologically relevant marine invertebrate, Mytilus galloprovincialis. From an ecological perspective, our study suggests that mussels (or similar marine species) exposed to increased temperature and HTO may have a compromised ability to defend against genotoxic stress.

Radiation dose estimation for marine mussels following exposure to tritium: Best practice for use of the ERICA tool in ecotoxicological studies.

Accurate dosimetry is critically important for ecotoxicological and radioecological studies on the potential effects of environmentally relevant radionuclides, such as tritium ((3)H). Previous studies have used basic dosimetric equations to estimate dose from (3)H exposure in ecologically important organisms, such as marine mussels. This study compares four different methods of estimating dose to adult mussels exposed to 1 or 15 MBq L(-1) tritiated water (HTO) under laboratory conditions. These methods were (1) an equation converting seawater activity concentrations to dose rate with fixed parameters; (2) input into the ERICA tool of seawater activity concentrations only; (3) input into the ERICA tool of estimated whole organism concentrations (woTACs), comprising dry activity plus estimated tissue free water tritium (TFWT) activity (TFWT volume × seawater activity concentration); and (4) input into the ERICA tool of measured whole organism activity concentrations, comprising dry activity plus measured TFWT activity (TFWT volume × TFWT activity concentration). Methods 3 and 4 are recommended for future ecotoxicological experiments as they produce values for individual animals and are not reliant on transfer predictions (estimation of concentration ratio). Method 1 may be suitable if measured whole organism concentrations are not available, as it produced results between 3 and 4. As there are technical complications to accurately measuring TFWT, we recommend that future radiotoxicological studies on mussels or other aquatic invertebrates measure whole organism activity in non-dried tissues (i.e. incorporating TFWT and dry activity as one, rather than as separate fractions) and input this data into the ERICA tool.

Assessment of growth, genotoxic responses and expression of stress related genes in the Pacific oyster Crassostrea gigas following chronic exposure to ionizing radiation.

Marine organisms are exposed to low doses of anthropogenic contaminants during their entire life. Authorized amounts of radionuclides are discharged in the Channel by nuclear facilities. The Pacific oyster was used to investigate the potential impact of chronic exposure to ionizing radiation. Though we exposed larvae and spat for two weeks to much higher concentrations than those encountered near nuclear facilities, oyster growth and expression of 9 selected stress genes were not significantly changed. To determine potential DNA damage, 2year old oysters were exposed for two weeks to tritiated water. The comet assay was used to evaluate the level of DNA strand breaks in haemocytes, whilst the 'clearance rate' was used as a measure of physiological effects. Whilst other parameters did not alter, DNA damage significantly increased. Our results highlight the significance of the observed DNA damage and their potential consequences at higher levels of biological organization.

Applications of biological tools or biomarkers in aquatic biota: A case study of the Tamar estuary, South West England.

Biological systems are the ultimate recipients of pollutant-induced damage. Consequently, our traditional reliance on analytical tools is not enough to assess ecosystem health. Biological responses or biomarkers are therefore also considered to be important tools for environmental hazard and risk assessments. Due to historical mining, other anthropogenic activities, and its conservational importance (e.g. NATURA sites, SACs), the Tamar estuary in South West England is an ideal environment in which to examine applications of such biological tools. This review presents a thorough and critical evaluation of the different biological tools used in the Tamar estuary thus far, while also discussing future perspectives for biomarker studies from a global perspective. In particular, we focus on the challenges which hinder applications of biological tools from being more readily incorporated into regulatory frameworks, with the aim of enabling both policymakers and primary stakeholders to maximise the environmental relevance and regulatory usefulness of such tools.

Physiological perturbations in juvenile cuttlefish Sepia officinalis induced by subchronic exposure to dissolved zinc.

Although cephalopod early life stage development often occurs in coastal areas where contamination is real and continuous, the physiological perturbations induced by contaminants have been rarely investigated. This study focused on the Zn as it is one of the trace metals the most concentrated in coastal waters, worldwide. As Zn-tolerance limits were unknown in juvenile Sepia officinalis, the aim of this study was to estimate the threshold inducing mortality during the 2-first weeks post-hatching, and to determine its sensitivity using digestive and immune enzymatic assays, as well as growth and behavior follow-up during the first 5weeks post-hatching. Our study highlighted a Zn-mortality threshold lying between 185 and 230µgl(-1), and growth reductions occurring after 5-week at 108µgl(-1) and above, associated with enzymatic perturbations. These results underline a relatively important sensitivity of juvenile cuttlefish to Zn, pointed out by a wide diversity of biomarkers.

Enhanced toxicity of 'bulk' titanium dioxide compared to 'fresh' and 'aged' nano-TiO2 in marine mussels (Mytilus galloprovincialis).

Marine bivalves (Mytilus galloprovincialis) were exposed to titanium dioxide (10 mg L(-1)) either as engineered nanoparticles (nTiO2; fresh, or aged under simulated sunlight for 7 days) or the bulk equivalent. Inductively coupled plasma-optical emission spectrometry analyses of mussel tissues showed higher Ti accumulation (>10-fold) in the digestive gland compared to gills. Nano-sized TiO2 showed greater accumulation than bulk, irrespective of ageing, particularly in digestive gland (>sixfold higher). Despite this, transcriptional expression of metallothionein genes, histology and histochemical analysis suggested that the bulk material was more toxic. Haemocytes showed significantly enhanced DNA damage, determined by the modified comet assay, for all treatments compared to the control, but no significant differences between the treatments. Our integrated study suggests that for this ecologically relevant organism photocatalytic ageing of nTiO2 does not significantly alter toxicity, and that bulk TiO2 may be less ecotoxicologically inert than previously assumed.

Oxidative DNA damage may not mediate Ni-induced genotoxicity in marine mussels: assessment of genotoxic biomarkers and transcriptional responses of key stress genes.

Nickel (Ni) is a known carcinogenic and mutagenic compound and an important contaminant of aquatic environments. Ni toxicity and its potential impact on aquatic organisms are, however, not well understood. This study used an integrated approach to evaluate genotoxic effects, tissue-specific accumulation and transcriptional profiles of key genes in mussels, Mytilus galloprovincialis, exposed to a range of concentrations of Ni. The genotoxic effects assessed were total and oxidative DNA damage (DNA strand breaks measured using the enzyme modified comet assay), and induction of micronuclei (MN; clastogenic and/or aneugenic effects) using haemocytes as the target cells. Six genes (pgp, mt10, mt20, sod, hsp70 and gst) were selected for transcriptional analysis in the gills based on their key role in the stress response. Following exposure to sublethal concentrations of Ni (0-3600µgL(-1)) for 5 days, mussel haemocytes showed significant genotoxicity at >1800µgL(-1) (4-fold increase for DNA strand breaks and 3-fold increase for MN induction). There was no significant difference between buffer (control) and enzyme treatments which target oxidised DNA bases (formamidopyrimidine glycosylase or endonuclease IIII). This suggested that, in haemocytes, oxidative DNA damage is not a major mechanism for Ni-induced genotoxicity. The expression of mt20 and gst genes in gill was up-regulated at genotoxic concentrations, whilst pgp expression was markedly up-regulated, particularly at 18µgL(-1) Ni (19-fold increase). Pearson's correlation analysis revealed significant associations between % tail DNA and MN induction in haemocytes (r=0.88, p<0.05), and between Ni accumulation in foot (r=0.47, p<0.05) and digestive gland (r=0.41, p<0.05) and induction of MN in the haemocytes. Our results are the first to suggest that Ni-induced genotoxicity in mussel haemocytes may not be a result of oxidative DNA damage, and that multixenobiotic resistance (MXR) may play an important role in Ni detoxification in this species.

Relative sensitivity of two marine bivalves for detection of genotoxic and cytotoxic effects: a field assessment in the Tamar Estuary, South West England.

The input of anthropogenic contaminants to the aquatic environment is a major concern for scientists, regulators and the public. This is especially relevant in areas such as the Tamar valley in SW England, which has a legacy of contamination from industrial activity in the nineteenth and twentieth centuries. Following on from previous laboratory validation studies, this study aimed to assess the relationship between genotoxic and cytotoxic responses and heavy metal concentrations in two bivalve species sampled from locations along the Tamar estuary. Adult cockles, Cerastoderma edule, and blue mussels, Mytilus edulis, were sampled from five locations in the Tamar and one reference location on the south Devon coast. Bivalve haemocytes were processed for comet and neutral red retention (NRR) assays to determine potential genotoxic and cytotoxic effects, respectively. Sediment and soft tissue samples were analysed for metal content by inductively coupled plasma mass spectrometry. Sediment concentrations were consistent with the physico-chemical nature of the Tamar estuary. A significant correlation (P = 0.05) was found between total metal concentration in sediment and C. edule soft tissues, but no such correlation was found for M. edulis samples. DNA damage was elevated at the site with highest Cr concentrations for M. edulis and at the site with highest Ni and Pb concentrations for C. edule. Analysis of NRR revealed a slight increase in retention time at one site, in contrast to comet data. We conclude that the comet assay is a reliable indicator of genotoxic damage in the field for both M. edulis and C. edule and discuss reasons for the apparent discrepancy with NRR.

Assessing the impact of ionizing radiation on aquatic invertebrates: a critical review.

There is growing scientific, regulatory and public concern over anthropogenic input of radionuclides to the aquatic environment, especially given the issues surrounding existing nuclear waste, future energy demand and past or potential nuclear accidents. A change in the approach to how we protect the environment from ionizing radiation has also underlined the importance of assessing its impact on nonhuman biota. This review presents a thorough and critical examination of the available information on the effects of ionizing radiation on aquatic invertebrates, which constitute approximately 90% of extant life on the planet and play vital roles in ecosystem functioning. The aim of the review was to assess the progress made so far, addressing any concerns and identifying the knowledge gaps in the field. The critical analysis of the available information included determining yearly publications in the field, qualities of radiation used, group(s) of animals studied, and levels of biological organization at which effects were examined. The overwhelming conclusion from analysis of the available information is that more data are needed in almost every area. However, in light of the current priorities in human and environmental health, and considering regulatory developments, the following are areas of particular interest for future research on the effects of ionizing radiation on nonhuman biota in general and aquatic invertebrates in particular: (1) studies that use end points across multiple levels of biological organization, including an ecosystem level approach where appropriate, (2) multiple species studies that produce comparable data across phylogenetic groups, and (3) determination of the modifying (i.e. antagonistic, additive or synergistic) effects of biotic and abiotic factors on the impact of ionizing radiation. It is essential that all of these issues are examined in the context of well-defined radiation exposure and total doses received and consider the life stages and life span of the species studied. The review also provides future directions for studies in this stimulating area of research to protect human and environmental health.