Assessing the impact of imidacloprid, glyphosate, and their mixtures on multiple biomarkers in Corbicula largillierti.

Pesticide mixtures are frequently utilized in agriculture, yet their cumulative effects on aquatic organisms remain poorly understood. Aquatic animals can be effective bioindicators and invasive bivalves, owing to their widespread distribution, provide an opportunity to assess these impacts. Glyphosate and imidacloprid, among the most prevalent pesticides globally, are frequently detected in freshwater systems in South America. This study aims to understand the cumulative effects of pesticide mixtures on aquatic organisms, using invasive Corbicula largillierti clams from a natural stream in northwestern Argentina. We conducted 48-hour exposure experiments using two concentrations of imidacloprid (20 and 200 µg L-1 a.i), two concentrations of glyphosate (0.3 and 3 mg L-1 a.i), and two combinations of these pesticides (both at low and high concentrations, respectively), simulating the direct contamination of both pesticides based on their agronomic recipe and observed values in Argentine aquatic environments. Clam metabolism was assessed through the examination of multiple oxidative stress parameters and measuring oxygen consumption rate as a proxy for standard metabolic rate (SMR). Our findings revealed that imidacloprid has a more pronounced effect compared to glyphosate. Imidacloprid significantly decreased clam SMR and cellular levels of reduced glutathione (GSH). However, when both pesticides were present, also cellular glycogen and thiobarbituric acid-reactive substances (TBARS) were affected. Proteins and glutathione S-Transferase (GST) activity were unaffected by either pesticide or their mixture at the assayed concentrations, highlighting the need to test several stress parameters to detect toxicological impacts. Our results indicated additive effects of imidacloprid and glyphosate across all measured parameters. The combination of multiple physiological and cytological biomarkers in invasive bivalves offers significant potential to enhance biomonitoring sensitivity and obtain insights into the origins and cellular mechanisms of chemical impacts. These studies can improve pollution regulatory policies and pesticide management.

Asian clam Corbicula fluminea as potential biomonitor of microplastics and metal(oid)s in a Patagonian River.

This study summarizes the concentration in dry weight (dw) of several metal(oid)s (As, Cd, Cr, Cu, Mn, Ni, Pb, and Zn), as well as the abundance and characteristics of microplastics (MP) in wet weight (ww) of the soft tissues of clam Corbicula fluminea from Chubut River (Patagonia, Argentina). The contents of essential elements were in the following decreasing order: Zn > Mn > Cu > Ni; meanwhile, non-essential elements Cd, Cr and Pb were below the detection limit (<0.5 µg/g dw). A high mean concentration of As (6.1 ± 0.3 µg/g dw) was found, surpassing the maximum allowable limit established by the Argentine Food Code for bivalve molluscs. The number of MP ranged from 0.07 to 1.27 items/ind. and from 0.2 to 2.9 items/g ww. Fibers were the most common shape, mainly transparent. The size of MP ranged from 42 to 1917 µm, accounting for 62 % of MP between 50 and 450 µm. The dominant polymer was PET based on the results of Raman spectroscopy. Based on the widespread distribution of MP in the environment and the wide range of effects on organisms, it is necessary to develop long-term monitoring programs for MP contamination in different environmental matrices. Understanding the bioaccumulation of MP in bivalves is crucial to assess the potential risk to human health through consumption and to the ecosystem. We propose that the widespread Asian clam could serve as a useful biomonitor for MP and As pollution in freshwater and estuarine environments such as the Chubut River.

What does the freshwater clam, Corbicula largillierti, have to tell us about chlorothalonil effects?

Chlorothalonil (CLT) is a broad spectrum, and non-systemic fungicide applied in foliar structures to prevent and treat pathogens. This compound reaches to aquatic environments and affects the biota. In this context, the main goal of this study was to assess the effects of CLT at biochemical, tissular, and individual levels of biological organization using the invasive bivalve Corbicula largillierti as a bioindicator species. Clams were exposed to different sublethal concentrations (0, 10, 20 and 50 µg. L-1 CLT) for 96 h. At biochemical level, the enzymatic activity (Glutathione-s-Transferase, Catalase, Acetyl-, Butiryl- and Carboxyl-esterases) and lipid peroxidation were measured in gills and the visceral mass. Also, the digestive gland morphometry through quantitative histological indexes was registered at the tissular level. Finally, filtering activity and burial behavior at the individual level were measured. At the highest CLT concentration, the most significant changes were observed in enzymatic activity (except for butyrylcholinesterase), lipid peroxidation and in digestive gland morphometry. It was also registered increases of the filtering activity and the latency time to burial. Most of the biomarkers assessed showed significant responses under CLT exposure. Therefore, taking into account that C. largillierti was affected by CLT, it can be expected that other species could be in a potential risk if this fungicide is present in freshwater systems.

Assessment of domestic landfill leachate toxicity to the Asian clam Corbicula fluminea via biomarkers.

In order to evaluate the effects of domestic landfill leachate to bivalves Corbicula fluminea, clams were exposed to different leachate concentrations (v/v): 2, 3, 6 and 10 percent, corresponding to dilutions observed along a stream that receives this effluent, or only to clean water for comparisons. After 5 and 15 days of exposure the activity of the biotransformation enzymes 7-ethoxyresorufin-O-deethylase (EROD) and glutathione S-transferase (GST), the multixenobiotic resistance mechanism (MXR) and lipid peroxidation (LPO) in gills and digestive gland and metallothionein (MT) content in gills were evaluated. Differences in biomarkers responses were observed between gills and digestive gland, except for MXR that decreased in both tissues of clams exposed to 6 percent for 5 days. EROD activity in gills was reduced in all leachate concentrations after 5 days and only in 2 percent after 15 days exposure, while an EROD increase was observed in digestive gland after 15 days exposure to 6 percent. GST activity increased only in the gills of clams exposed to 10 percent for 5 days. LPO varied between tissues and different conditions. A significant increase in LPO was observed in the gills, after 5 days exposure to 2 and 6 percent, and in digestive gland after 5 and 15 days exposure to 2 and 3 percent. MT content in the gills increased after 15 days exposure to 2 percent. In conclusion, different leachate concentrations tested here caused biochemical changes in C. fluminea, but due to the observed variability in biomarkers responses among leachate concentrations, it was difficult to determine patterns or thresholds concentrations.

Genotoxic and biochemical effects of atrazine and Roundup(®), alone and in combination, on the Asian clam Corbicula fluminea.

The present study aimed to evaluate biochemical and genotoxic effects of the herbicides atrazine (ATZ) and Roundup(®) (RD) separately, as well as their mixture, on the freshwater clam Corbicula fluminea after 96 h exposure. Animals were exposed to 2 and 10 ppb of ATZ (ATZ2 and ATZ10), 2 and 10 ppm of RD (RD2 and RD10) and the following mixtures: 2 ppb ATZ+2ppm RD (AR2) and 10 ppb ATZ+10 ppm RD (AR10). Activities of ethoxyresorufin-O-deethylase (EROD), glutathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR), as well as the multixenobiotic resistance mechanism (MXR), reduced glutathione concentrations (GSH) and lipid peroxidation (LPO) were measured in gills and digestive gland. DNA damage was determined in clams hemocytes through the comet assay. The gills were more susceptible to the action of the herbicides and the results showed that ATZ2 and ATZ10 caused a significant reduction in EROD and the mixture leads to a significant decrease in EROD and MXR. No significant change in the biotransformation parameters was observed in the digestive gland. Regarding the primary antioxidant defenses, SOD activity increased in the gills of clams exposed to ATZ10 and RD10 and in the digestive gland of animals exposed to RD2 and RD10, CAT activity was significantly reduced only in digestive gland of clams exposed RD10 while GPX increased in the gills after exposure to ATZ2 and RD10. The exposure to RD10 caused a significant increase in LPO in both gills and digestive gland. While the exposure to ATZ and RD separately did not increase DNA damage, the exposure to AR2 and AR10 caused a significant increase in the occurrence of DNA damage. In conclusion, this study showed that both herbicides applied alone caused effects on C. fluminea; ATZ interfered mostly in biotransformation while RD interfered mainly in antioxidant defenses leading to lipid peroxidation. The herbicides mixture showed antagonistic effects on the gills EROD and on lipid peroxidation in gills and digestive gland and synergistic effects on the gills MXR and on DNA damage in the hemocytes.

The influence of lead on different proteins in gill cells from the freshwater bivalve, Corbicula fluminea, from defense to repair biomarkers.

The objective of this study was to evaluate the influence of lead (Pb) on regulatory proteins linked to mechanisms of animal adaptation to polluted environments (using in vivo and in vitro tests) and to validate the in vitro assay as a tool for environmental assessment. Specimens of the bivalve Corbicula fluminea were exposed to nominal concentrations of Pb 5 mg l(-1) for 96 h. Isolated gill cells were exposed to three concentrations (1, 10, and 100 µM) for 5 h. Metal toxicity was evaluated by cell viability (trypan blue exclusion). We also analyzed Na+/K+ adenosine triphosphatase (ATPase) and carbonic anhydrase activity. Additionally, the multixenobiotic-resistance (MXR) phenotype was evaluated by the accumulation of rhodamine B (RB). Immunolabeling was used to quantify the expression of P-glycoproteins (C219) and proteins involved in ion transport, water movement, and cellular repair using antibodies against Na+/K+ ATPase, aquaporin 1, and heat-shock protein 70 (Hsp70). Pb was shown to be toxic in both in vivo and in vitro tests, in which cellular viability significantly decreased by approximately 25%. Cellular viability in the in vivo assays was determined by gill cell isolation after the entire animal was exposed to Pb. We observed that Na+/K+ ATPase activity was inhibited by 70%. Also, the expression of the MXR phenotype significantly increased in our in vivo tests. A statistically significant difference was observed in the expression of all proteins in the in vitro assays, whereas only Hsp70 increased in vivo. Employing these analyses, we could validate the sensitivity of the in vitro tests and can propose our in vitro model as a possible tool for environmental assessment.

Genetic damage in the bivalve mollusk Corbicula fluminea induced by the water-soluble fraction of gasoline.

Although gasoline is an important contaminant of aquatic ecosystems, information concerning the potential effects of this petroleum derivative on the DNA of aquatic biota is lacking. The present study aimed to evaluate the genotoxic and mutagenic effects of acute exposures (6, 24, and 96h) to gasoline water-soluble fraction (GWSF), diluted to 5%, on the Asian clam Corbicula fluminea. The comet assay and the micronucleus (MN) test were performed on hemocytes and gill cells of C. fluminea. For the three different times tested, the comet assay indicated DNA damage in hemocytes and gill cells of C. fluminea exposed to GWSF. The MN test detected significant damage in the genetic material of the hemocytes only after 96h of exposure to GWSF. The recovery capacity of organisms previously exposed for 6h to GWSF was also evaluated with the comet assay. The results revealed a great capacity of this species to repair DNA damage; following 6h of recovery, the comet score returned to that of the control groups. Overall, our findings indicate that GWSF has genotoxic and mutagenic effects on C. fluminea. In addition, the present results confirm the sensitivity of C. fluminea to damage caused by exposure to environmental contaminants, and, therefore, its suitability for use in environmental monitoring studies.

Detection of genotoxicity of water from an urbanized stream, in Corbicula fluminea (Mollusca) (in vivo) and CHO-K1 cells (in vitro) using comet assay.

The comet assay was utilized to investigate the quality of water from seven locations along the Cambé Stream, in vivo (Corbicula fluminea hemolymph), in vitro (CHO-K1 cells), in situ, and in laboratory studies. The Cambé Stream basin (Londrina, PR, Brazil) is almost completely urbanized and receives different forms of industrial and domestic runoff. The data indicated the occurrence of DNA damage in cells examined in vivo and in vitro, shown by the significant increase in frequencies of cells with DNA damage after exposure to water from all seven locations used in the study. Our results strongly suggest the presence of genotoxic agent(s) in water at all of the sampled locations, demonstrated by elevated numbers of cells with DNA damaged in field and laboratory tests. In all of the places sampled, domestic sewage influence appeared to be one important cause for the introduction of xenobiotics, environmental genotoxins, and pollutants into the water. Thus, the comet assay applied in these cell systems was able to detect adverse environmental conditions, proving to be a very adequate short-term test and should be included in batteries of tests utilized in the monitoring of aquatic environments.