Effects of aminomethylphosphonic acid, the main breakdown product of glyphosate, on cellular and biochemical parameters of the mussel Mytilus galloprovincialis.

The effects of the breakdown products of herbicides on aquatic species are largely unknown. In a recent study, we evaluated the effects of glyphosate on the mussel Mytilus galloprovincialis. This study was designed to evaluate for the first time the impact of aminomethylphosphonic acid (AMPA) - the main breakdown product of glyphosate - on cellular and biochemical parameters of the mussel Mytilus galloprovincialis. Bivalves were exposed for 7, 14 and 21 days to 1, 10 and 100 µg/L of AMPA and various biomarkers were measured in haemolymph (total haemocyte counts, haemocyte diameter and volume, haemolymph pH, haemocyte proliferation, haemolymph lactate dehydrogenase activity, haemocyte lysate lysozyme and acid phosphatase activities), as well as in gills and digestive gland (superoxide dismutase, catalase, glutathione S-transferase and acetylcholinesterase activities). AMPA concentrations in seawater samples from the experimental tanks were also measured in order to correlate the biomarker responses of mussels with their exposure to the actual concentrations of AMPA. The MANOVA analysis demonstrated that the experimental variables considered (exposure dose, exposure duration, and their interaction) affected significantly biomarker responses. Nevertheless, the two-way ANOVA analysis revealed significant effects of AMPA on most of the biomarkers measured. The overall results of this study demonstrated that AMPA can affect cellular and biochemical parameters in mussels, similarly to glyphosate.

A multibiomarker approach in clams (Ruditapes philippinarum) for a toxicological evaluation of dredged sediments.

The Lagoon of Venice is often dredged for channel maintenance. To avoid harmful consequences to the ecosystem, a proper disposal of bottom sediments requires a preliminary evaluation of its potential toxicity before excavation. Here we evaluated the effects of polluted sediments on clams (Ruditapes philippinarum) using a multibiomarker approach. Bivalves were exposed for 3 and 14 days to five sediment samples collected along a navigation canal between Venice historical centre and the industrial area of Porto Marghera. Immunological, antioxidant, detoxification, and neurotoxicity biomarkers were analysed in haemolymph, gill, and digestive gland. As a control, sediment collected far from pollution sources was used. Two experiments were performed to assess potential seasonal/gametogenic influence in clam sensitivity. A different response of clam biomarkers was observed during the two experiments and among sampling sites. Clams' digestive gland resulted to be the most sensitive tissue analysed showing significant differences among sites in all biomarkers analysed. Greater differences were present due to seasonality rather than exposure. The concentrations of metals and organic pollutants increased from the city centre to the industrial area, highlighting the influence that industrial activities had on the lagoon ecosystem. However, bioaccumulation in clams did not follow the same clear pattern, suggesting low bioavailability of compounds due to relatively high organic matter content. Biomarkers modulation was mainly driven by metals, both present in sediments and bioaccumulated. In comparison, effects of organic pollutants on the biomarkers tested were negligible. Other sources of contamination not investigated (e.g. pesticides) were suggested by neurotoxicity biomarkers alteration.

New evidences in the complexity of contamination of the lagoon of Venice: polybrominated diphenyl ethers (PBDEs) pollution.

This study presents the first evaluation of the current pollution by polybrominated diphenyl ethers (PBDEs) of surface sediments from the Lagoon of Venice. We focused the research on tri-to hepta-BDEs, the main components of penta- and octa-mixtures, which are considered to be the most toxic for the biocoenosis. The results pointed out a quite homogeneous contamination of this keystone European transitional environment, with ∑ (13)PBDEs values ranging from 0.39 to 6.78 ng/g dry weight; these values reflect low to moderate pollution levels, which is in conformity to other coastal European ecosystems. The average PBDE profile of the lagoon sediments follows this decreasing trend of congeners: BDE-47>BDE-99> >BDE-190>BDE-28>BDE-153>BDE-154>BDE-138, BDE-183, and BDE-17, which is similar to the worldwide distribution pattern. BDE-47 and BDE-99 revealed a recent use of a penta-BDE mixture, while the presence of hepta-BDEs (BDE-183 and BDE-190) in all of the sites can indicate the actual use of a deca-BDE formulation, because these congeners are considered to be debrominated byproducts of BDE-209 degradation.

Effects of a mixture of glyphosate, 17α-ethynylestradiol and amyl salicylate on cellular and biochemical parameters of the mussel Mytilus galloprovincialis.

In this study the effects of a mixture of glyphosate (herbicide), 17a-ethinylestradiol (synthetic estrogen) and amyl salicylate (fragrance) to the mussel Mytilus galloprovincialis were evaluated. Mussels were exposed for 7 days to two realistic concentrations of the mixture (10 and 100 ng/L) and the effects on total haemocyte counts, haemocyte diameter and volume, haemocyte proliferation, haemolymph lactate dehydrogenase activity and haemocyte lysate lysozyme activity were measured. In addition, superoxide dismutase, catalase, acetylcholinesterase, glutathione-S-transferase and glutathione reductase activities were measured in gills and digestive gland. The survival-in-air test was also performed. Results demonstrated that the mixture affected both cellular and biochemical biomarkers, but not tolerance to aerial exposure of M. galloprovincialis. The negative effects recorded in this study suggested that more efforts should be done to assess the ecotoxicological risks posed by contaminant mixture to aquatic invertebrates.

New compounds, old problems. The case of C6O4 - a substitute of PFOA - and its effects to the clam Ruditapes philippinarum.

C6O4 (difluoro{[2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy}acetic acid) is a new surfactant and emulsifier used as a substitute of perfluorooctanoic acid (PFOA). Recently, C6O4 has been detected in aquatic environments, but, at present, no information concerning the effects of C6O4 on aquatic species, such as bivalves, are available in the literature. Therefore, in this study we evaluated for the first time the effects of C6O4 (0.1 and 1 µg/L) and PFOA (1 µg/L) to the clam Ruditapes philippinarum. Short-term (7 days) and long-term (21 days) exposures of clams to the two compounds were carried out and numerous biomarkers were measured in haemocytes/haemolymph, as well as in gills and digestive gland. The MANOVA analysis demonstrated statistically significant effects of the independent variables "treatment", "time" and "treatment-time interaction" on the whole dataset of biomarker responses. The two-way ANOVA analysis performed for each biomarker response indicated that the two compounds affected most of the cellular and tissue parameters measured. Despite preliminary, the results obtained suggested that C6O4 - similarly to PFOA - can affect both cellular and biochemical parameters of clams.

The new PFAS C6O4 and its effects on marine invertebrates: First evidence of transcriptional and microbiota changes in the Manila clam Ruditapes philippinarum.

There is growing concern for the wide use ofperfluorooctanoic acid (PFOA) because of its toxic effects on the environment and on human health. A new compound - the so called C6O4 (perfluoro ([5-methoxy-1,3-dioxolan-4-yl]oxy) acetic acid) - was recently introduced as one of the alternative to traditional PFOA, however this was done without any scientific evidence of the effects of C6O4 when dispersed into the environment. Recently, the Regional Agency for the Protection of the Environment of Veneto (Italy) detected high levels of C6O4 in groundwater and in the Po river, increasing the alarm for the potential effects of this chemical into the natural environment. The present study investigates for the first time the effects of C6O4 on the Manila clam Ruditapes philippinarum exposed to environmental realistic concentrations of C6O4 (0.1 µg/L and 1 µg/L) for 7 and 21 days. Furthermore, in order to better understand if C6O4 is a valid and less hazardous alternative to its substitute, microbial and transcriptomic alterations were also investigated in clams exposed to 1 µg/L ofPFOA. Results indicate that C6O4 may cause significant perturbations to the digestive gland microbiota, likely determining the impairment of host physiological homeostasis. Despite chemical analyses suggest a 5 times lower accumulation potential of C604 as compared to PFOA in clam soft tissues, transcriptional analyses reveal several alterations of gene expression profile. A large part of the altered pathways, including immune response, apoptosis regulation, nervous system development, lipid metabolism and cell membrane is the same in C6O4 and PFOA exposed clams. In addition, clams exposed to C6O4 showed dose-dependent responses as well as possible narcotic or neurotoxic effects and reduced activation of genes involved in xenobiotic metabolism. Overall, the present study suggests that the potential risks for marine organism following environmental contamination are not reduced by replacing PFOA with C6O4. In addition, the detection of both C6O4 and PFOA into tissues of clams inhabiting the Lagoon of Venice - where there are no point sources of either compounds - recommends a similar capacity to spread throughout the environment. These results prompt the urgent need to re-evaluate the use of C6O4 as it may represent not only an environmental hazard but also a potential risk for human health.

Fluoride toxicity and bioaccumulation in the invasive amphipod Dikerogammarus villosus (Sowinsky, 1894): a laboratory study.

The tolerance of the invasive amphipod Dikerogammarus villosus to fluoride (F⁻) toxicity was examined via laboratory experiments. 96-h LC50 and 240-h NOEC values were estimated to be 5.8 and 0.95 mg F⁻/L, respectively. Average whole-body fluoride content in control amphipods was 27.6 µg F⁻/g dry weight, whereas in exposed amphipods it ranged from 3,637 to 16,994 µg F⁻/g dry weight. All these results indicate that D. villosus is a very sensitive species to fluoride toxicity. Overall it is concluded that the potential risk of invasion for D. villosus in either natural or polluted freshwater ecosystems, exhibiting relatively high fluoride levels (at least ten-fold higher than the average freshwater background level of 0.15 mg F⁻/L), must be low.

Molecular and biochemical responses of vitellogenin in the mussel Mytilus galloprovincialis exposed to the glyphosate-based herbicide Roundup® Power 2.0.

Glyphosate-based herbicides (GBHs) occur in aquatic ecosystems at concentrations of hundreds of micrograms per liter. As formulation adjuvants are suspected to be endocrine-disrupting chemicals, we assessed the effects of the recent GBH formulation Roundup® Power 2.0 on vitellogenin (VTG) in Mytilus galloprovincialis. Mussels were exposed for 7, 14, and 21 days to two concentrations of the commercial formulation, corresponding to 100 and 1000 µg/L of glyphosate. The expression of the vtg gene in gonads of females and males, as well as the levels of alkali labile phosphates (ALP) in gonads and non-gonadal tissues from the two sexes were measured. No significant alterations were observed in vtg expression values during the exposure. Conversely, a significant reduction in gonadal ALP levels was observed in females exposed for 21 days and in males exposed for 7 days. In addition, ALP levels increased significantly in gonads from males exposed for 21 days to the two concentrations of Roundup®. As for non-gonadal tissues, ALP levels did not change significantly in females, whereas ALP levels decreased significantly in non-gonadal tissues from males exposed for 21 days to the lowest concentration tested. An overall statistically significant difference in ALP levels was found between females and males. Although preliminary, our study suggests that GBH can affect reproduction-related parameters in mussels.

Ecotoxicological hazard of a mixture of glyphosate and aminomethylphosphonic acid to the mussel Mytilus galloprovincialis (Lamarck 1819).

Assessment of the effects of chemical mixtures is a very important objective of the ecotoxicological risk assessment. This study was aimed at evaluating for the first time the effects of a mixture of glyphosate and its main breakdown product aminomethylphosphonic acid (AMPA) on various biomarkers in the mussel Mytilus galloprovincialis. Mussels were exposed for 7, 14 and 21 days to either 100 µg/L of glyphosate, 100 µg/L of AMPA or a mixture of both (100 + 100 µg/L). Various haemocyte parameters, such as total haemocyte counts, haemocyte diameter and volume, haemocyte proliferation, haemolymph lactate dehydrogenase activity and haemocyte lysate acid phosphatase activities were measured. In addition, the effects of exposure on the activity of antioxidant enzymes, acetylcholinesterase and glutathione-S-transferase were evaluated in gills and digestive gland from mussels. On the whole, this study demonstrated that the variables considered in the experimental plan, namely treatment, exposure time and their interaction, affect significantly biomarker responses in M. galloprovincialis. The effects of the mixture were comparable to those of the individual compounds, whereas their synergistic effects were occasionally observed, under the experimental conditions tested at least.

Ecotoxicological risk assessment for the herbicide glyphosate to non-target aquatic species: A case study with the mussel Mytilus galloprovincialis.

Glyphosate (GLY) is one of the most used herbicide worldwide. Considering that information concerning the impact of GLY on bivalves is scarce, in this study we evaluated for the first time the effects of environmentally realistic concentrations of GLY (10, 100 and 1000 µg/L) to the mussel Mytilus galloprovincialis. Mussels were exposed for 7, 14 and 21 days and several biomarkers were measured in haemocytes/haemolymph (total haemocyte counts, haemocyte diameter and volume, haemolymph pH, haemolymph lactate dehydrogenase activity, haemocyte lysate lysozyme and acid phosphatase activities), as well as in gills and digestive gland (antioxidant enzyme and acetylcholinesterase activities). The concentrations of GLY and its main metabolite aminomethylphosphonic acid in the experimental tanks were also measured. The MANOVA analysis demonstrated that the experimental variables considered (exposure concentration, exposure duration, and their interaction) affected significantly biomarker responses. In addition, the two-way ANOVA analysis indicated that GLY was able to affect most of the cellular parameters measured, whereas antioxidant enzyme activities resulted to be influenced moderately. Interestingly, exposure to GLY reduced significantly acetylcholinesterase activity in gills. Although preliminary, the results of this study demonstrated that GLY can affect both cellular and biochemical parameters in mussels, highlighting a potential risk for aquatic invertebrates.

Does the antibiotic amoxicillin affect haemocyte parameters in non-target aquatic invertebrates? The clam Ruditapes philippinarum and the mussel Mytilus galloprovincialis as model organisms.

Amoxicillin (AMX) is one of the most widely used antibiotics worldwide, and its levels in aquatic ecosystems are expected to be detectable. At present, information concerning the toxic effects of AMX on non-target aquatic organisms, such as bivalves, is scarce. Consequently, in this study, we investigated for the first time the effects of AMX on the haemocyte parameters of two bivalve species, the clam Ruditapes philippinarum and the mussel Mytilus galloprovincialis, which share the same habitat in the Lagoon of Venice, in order to compare the relative sensitivity of the two species. The bivalves were exposed to 100, 200 and 400 µg AMX/L for 1, 3 and 7 days, and the effects on the total haemocyte count (THC), the diameter and volume of the haemocytes, haemocyte proliferation, lactate dehydrogenase (LDH) activity in cell-free haemolymph, the haemolymph pH, and the formation of micronuclei were evaluated. The actual concentrations of AMX in the seawater samples from the experimental tanks were also measured. Overall, the obtained results demonstrated that AMX affected slightly the haemocyte parameters of bivalves. In addition, no clear differences in terms of sensitivity to AMX exposure were recorded between the two bivalve species.

Influence of temperature on fluoride toxicity and bioaccumulation in the nonindigenous freshwater mollusk Dreissena polymorpha Pallas, 1769.

Fluoride toxicity and bioaccumulation tests (short- and long-term) were performed on the nonindigenous freshwater mollusk Dreissena polymorpha at two different temperatures: 17 ± 0.5°C and 22 ± 0.5°C. Concentrations that did not result in toxicity in short-term experiments (96 h) induced effects over a longer period (17 weeks), especially at the warmest temperature, highlighting the role of this parameter. Fluoride bioaccumulation increased linearly with increasing concentration and temperature, reaching 4,202 µg F(-)/g dry weight in soft tissues only after 48 h of exposure at 22°C at a concentration of 640 mg F(-)/L. Comparing tolerance to fluoride and bioaccumulation values of this species with those of other freshwater invertebrates, D. polymorpha was much more resistant and revealed its capacity to accumulate a great quantity of this xenobiotic substance. The results of the present study demonstrated that fluoride accumulation in the soft tissue of this animal was much higher (up to 1,409.6 µg F(-)/g dry wt) than that in its shell (up to 706.4 µg F(-)/g dry wt). If we consider this datum and the fact that D. polymorpha is widespread in many aquatic ecosystems around the world, representing a food source for many birds and other vertebrates, we must acknowledge the possibility that it can represent a serious danger in view of fluoride biomagnification in the aquatic environment.