Exposure to environmental pharmaceuticals affects the macromolecular composition of mussels digestive glands.

Human pharmaceuticals represent a major challenge in natural environment. A better knowledge on their mechanisms of action and adverse effects on cellular pathways is fundamental to predict long-term consequences for marine wildlife. The FTIRI Imaging (FTIRI) spectroscopy represents a vibrational technique allowing to map specific areas of non-homogeneous biological samples, providing a unique biochemical and ultrastructural fingerprint of the tissue. In this study, FTIRI technique has been applied, for the first time, to characterize (i) the chemical building blocks of digestive glands of Mytilus galloprovincialis, (ii) alterations and (iii) resilience of macromolecular composition, after a 14-days exposure to 0.5 µg/L of carbamazepine (CBZ), valsartan (VAL) and their mixture, followed by a 14-days recovery period. Spectral features of mussels digestive glands provided insights on composition and topographical distribution of main groups of biological macromolecules, such as proteins, lipids, and glycosylated compounds. Pharmaceuticals caused an increase in the total amount of protein and a significant decrease of lipids levels. Changes in macromolecular features reflected the modulation of specific molecular and biochemical pathways thus supporting our knowledge on mechanisms of action of such emerging pollutants. Overall, the applied approach could represent an added value within integrated strategies for the effects-based evaluation of environmental contaminants.

Evaluation of Chronic Effects of Potassium Chloride and Nickel on Survival, Growth, and Reproduction of a Unionid Mussel (Lampsilis siliquoidea).

The ASTM International standard test method for freshwater mussels (E2455-13) recommends 4-week toxicity testing with juveniles to evaluate chronic effects on survival and growth. However, concerns remain that the method may not adequately address the sensitivity of mussels to longer term exposures (>4 weeks), particularly in relation to potential reproductive impairments. No standard method directly evaluates toxicant effects on mussel reproduction. The objectives of the present study were to (1) evaluate toxicity endpoints related to reproduction in fatmucket (Lampsilis siliquoidea) using two common reference toxicants, potassium chloride (KCl) and nickel (Ni); (2) evaluate the survival and growth of juvenile fatmucket in standard 4-week and longer term (12-week) KCl and Ni tests following a method refined from the standard method; and (3) compare the sensitivity of the reproductive endpoints with the endpoints obtained from the juvenile mussel tests. Reproductive toxicity tests were conducted by first exposing female fatmucket brooding mature larvae (glochidia) to five test concentrations of KCl and Ni for 6 weeks. Subsamples of the glochidia were then removed from the adults to determine three reproductive endpoints: (1) the viability of brooded glochidia; (2) the viability of free glochidia in a 24-h exposure to the same toxicant concentrations as their mother; and (3) the success of glochidia parasitism on host fish. Mean viability of brooded glochidia was significantly reduced in the high KCl concentration (26 mg K/L) relative to the control, with a 20% effect concentration (EC20) of 14 mg K/L, but there were no significant differences between the control and any Ni treatment (EC20 > 95 µg Ni/L). The EC20s for viability of free glochidia after the additional 24-h exposure and parasitism success were similar to the EC20s of brooded glochidia. The EC20s based on the most sensitive biomass endpoint in the 4-week juvenile tests were 15 mg K/L and 91 µg Ni/L, similar to or greater than the EC20s from the reproductive KCl and Ni tests, respectively. When exposure duration in the juvenile tests was extended from 4 to 12 weeks, the EC20s decreased by more than 50% in the KCl test but by only 8% in the Ni test. Overall, these results indicate that a standard 4-week test with juvenile mussels can prove effective for estimating effects in chronic exposures with different life stages although a longer term 12-week exposure with juvenile mussels may reveal higher sensitivity of mussels to some toxicants, such as KCl. Environ Toxicol Chem 2024;43:1097-1111. © 2024 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Reduced Freshwater Mussel Juvenile Production as a Result of Agricultural and Urban Contaminant Mixture Exposures.

Freshwater mussels provide invaluable ecological services but are threatened by habitat alteration, poor water quality, invasive species, climate change, and contaminants, including contaminants of emerging concern (CECs). Contaminants of emerging concerns are well documented in aquatic environments, including the Great Lakes Basin, but limited information is available on how environmentally relevant mixtures affect freshwater mussel biology throughout their varied life stages. Our main goal was to assess mussels' reproductive output in response to exposure to agricultural and urban CEC mixtures during glochidial development through juvenile transformation and excystment focusing on how exposure duration and treatment affect: (1) the number of glochidia prematurely released by brooding females, (2) glochidial transformation through host-fish excystment, and (3) the number of fully metamorphosed juveniles able to continue the lifecycle. Mussels and host fish were exposed to either a control water (CW), control ethanol (CE), agriculture CEC mixture (AM), or urban CEC mixture (UM) for 40 and 100 days. We found no effect from treatment or exposure duration on the number of glochidia prematurely released. Fewer partially and fully metamorphosed AM juveniles were observed during the 100-day exposure, compared with the 40-day. During the 40-day exposure, CW produced more fully metamorphosed individuals compared with CE and UM, but during the 100-day exposure AM produced more fully metamorphosed individuals compared with the CW. There was reduction in fully metamorphosed juveniles compared with partially metamorphosed for CE and UM during the 40-day exposure, as well as in the CW during the 100-day exposure. These results will be important for understanding how mussel populations are affected by CEC exposure. The experiments also yielded many insights for laboratory toxicology exposure studies. Environ Toxicol Chem 2024;43:1112-1125. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Self-Enhanced Antibacterial and Antifouling Behavior of Three-Dimensional Porous Cu2O Nanoparticles Functionalized by an Organic-Inorganic Hybrid Matrix.

Cu2O is currently an important protective material for domestic engineering and equipment used to exploit marine resources. Cu+ is considered to have more effective antibacterial and antifouling activities than Cu2+. However, disproportionation of Cu+ in the natural environment leads to its reduced bioavailability and weakened reactivity. Novel and functionalized Cu2O composites could enable efficient and environmentally friendly applications of Cu+. To this end, a series of three-dimensional porous Cu2O nanoparticles (3DNP-Cu2O) functionalized by organic (redox gel, R-Gel)-inorganic (reduced graphene oxide, rGO) hybrids─3DNP-Cu2O/rGOx@R-Gel─at room temperature by immobilization-reduction method was prepared and applied for protection against marine biofouling. 3DNP-Cu2O/rGO1.76@R-Gel includes rGO and R-Gel shape 3D porous Cu2O nanoparticles with diameters ∼177 nm and strong dispersion and antioxidant stability. Compared with commercial Cu2O (Cu2O-0), 3DNP-Cu2O/rGO1.76@R-Gel exhibited an ∼50% higher bactericidal rate, ∼96.22% higher water content, and ∼75% lower adhesion of mussels and barnacles. Moreover, 3DNP-Cu2O/rGOx@R-Gel maintains the same excellent, stable, and long-lasting bactericidal performance as Cu2O-0@R-Gel while reducing the average copper ion release concentration by ∼56 to 76%. This was also confirmed by X-ray diffraction, X-ray photoelectric spectroscopy (XPS), atomic absorption spectroscopy, and antifouling tests. In addition, XPS tests of rGO-Cu2+ and R-Gel-Cu2+, photocurrent tests of 3DNP-Cu2O/rGO1.76@R-Gel, and energy-dispersive spectrometry pictures of bacteria confirm that R-Gel and rGO act as electron donors and transfer substrates driving the reduction of Cu2+ (Cu2+ → Cu+) and the diffusion of Cu+. Thus, a self-growing antibacterial and antifouling system of 3DNP-Cu2O/rGO1.76@R-Gel was achieved. The mechanism of accelerated bacterial inactivation and resistance to mussel and barnacle adhesion by 3DNP-Cu2O/rGO1.76@R-Gel was interpreted. It is shown that rGO and R-Gel are important players in the antibacterial and antifouling system of 3DNP-Cu2O/rGO1.76@R-Gel.

Characterization of oxidative damage induced by nanoparticles via mechanism-driven machine learning approaches.

The wide application of TiO2-based engineered nanoparticles (nTiO2) inevitably led to release into aquatic ecosystems. Importantly, increasing studies have emphasized the high risks of nTiO2 to coastal environments. Bivalves, the representative benthic filter feeders in coastal zones, acted as important roles to assess and monitor the toxic effects of nanoparticles. Oxidative damage was one of the main toxic mechanisms of nTiO2 on bivalves, but the experimental variables/nanomaterial characteristics were diverse and the toxicity mechanism was complex. Therefore, it was very necessary to develop machine learning model to characterize and predict the potential toxicity. In this study, thirty-six machine learning models were built by nanodescriptors combined with six machine learning algorithms. Among them, random forest (RF) - catalase (CAT), k-neighbors classifier (KNN) - glutathione peroxidase (GPx), neural networks - multilayer perceptron (ANN) - glutathione s-transferase (GST), random forest (RF) - malondialdehyde (MDA), random forest (RF) - reactive oxygen species (ROS), and extreme gradient boosting decision tree (XGB) - superoxide dismutase (SOD) models performed good with high accuracy and balanced accuracy for both training sets and external validation sets. Furthermore, the best model revealed the predominant factors (exposure concentration, exposure periods, and exposure matrix) influencing the oxidative stress induced by nTiO2. These results showed that high exposure concentrations and short exposure-intervals tended to cause oxidative damage to bivalves. In addition, gills and digestive glands could be vulnerable to nTiO2-induced oxidative damage as tissues/organs differences were the important factors controlling MDA activity. This study provided insights into important nano-features responsible for the different indicators of oxidative stress and thereby extended the application of machine learning approaches in toxicological assessment for nanoparticles.

Biogenic metallic nanoparticles (Ag, TiO2, Fe) as potential fungicides for agriculture: are they safe for the freshwater mussel Anodontites trapesialis?

Silver (Ag), titanium dioxide (TiO2), and iron (Fe) nanoparticles (NPs) synthesized using the fungus Trichoderma harzianum are effective against the agriculture pathogen Sclerotinia sclerotiorum. However, their effects should be evaluated in aquatic organisms, as agriculture practices can contaminate the aquatic environment. Thus, this work evaluated sublethal effects of acute exposure (24 h) to AgNP, TiO2NP and FeNP, synthesized with T. harzianum, on the Neotropical freshwater bivalve Anodontites trapesialis, considering the hypothesis that suspension-feeding bivalves are susceptible to NPs toxicity. Individuals of A. trapesialis were divided into four groups (n = 8/group): a control group, kept in water only; a group exposed to AgNP; a group exposed to TiO2NP; and a group exposed to FeNP. The bioaccumulation of Ag, Ti, and Fe was evaluated in the gills, hemolymph, mantle, digestive gland, and muscle (foot). Lipoperoxidation, activities of the glutathione S-transferase, catalase, and superoxide dismutase, and glycogen concentration were quantified in the gills, mantle, and digestive gland. Ions (Na+, K+, Cl-, Ca2+, and Mg+2) and glucose concentrations were quantified in the hemolymph. Na+/K+-ATPase, H+-ATPase, Ca2+-ATPase, and carbonic anhydrase activities were assessed in the gills and mantle. Acetylcholinesterase activity was determined in the foot and adductor muscle. The mussels exposed to AgNP accumulated Ag in the gills, hemolymph, and foot, and showed a decrease in hemolymph concentrations of Na+ and Cl-, which was associated with the action of Ag ion (Ag+). The exposures to TiO2NP and FeNP led to the accumulation of Ti and Fe in the hemolymph, respectively, but did not promote additional effects. Accordingly, A. trapesialis showed bioaccumulation potential and susceptibility to AgNP, but was not susceptible to TiO2NP and FeNP. Thus, the preferential agricultural use of TiO2NP and FeNP over AgNP is highlighted.

A novel peptide identified from visceral ganglia induces oocyte maturation, spermatozoa active motility, and spawning in the pen shell Atrinapectinata.

The identification of novel peptides that regulate reproduction is essential for studying reproductive physiology in bivalves. Therefore, we aimed to identify peptides that affect the reproductive physiology of bivalves. We identified an oocyte maturation-, sperm motility-, and spawning-inducing peptide from the visceral ganglia of the pen shell, Atrina pectinata. The peptide consisted of 26 amino acid residues (GFDSINFPGTIDGFKDYSSNKKERLL). This peptide induced oocyte maturation and sperm motility activation at less than 1 nM upon the treatment of gonad fragments and induced spawning at 1 nmol when injected into mature individuals. Mature eggs and sperms artificially spawned by peptide administration were fertilized, and we confirmed that the development proceeded normally to veliger (D-shape) larvae. These results indicated that GFDSINFPGTIDGFKDYSSNKKERLL stimulated the gonads of pen shells and induced oocyte maturation, sperm motility activation, and spawning.

Sol-gel-derived hard coatings from tetraethoxysilane and organoalkoxysilanes bearing zwitterionic and isothiazolinone groups and their antifouling behaviors.

Current environmentally friendly marine antifouling (AF) coatings are mainly polymeric with a relatively low hardness. Hard sol-gel-derived AF coatings for underwater robot-cleaning are seldom used. In this work, two new organoalkoxysilanes, i.e., (N-methoxyacylethyl)-3-aminopropyltriethoxysilane and 2-(2-hydroxy-3-(3-(trimethoxysilyl)propoxy)propyl)benzo[d]isothiazol-3(2H)-one, were synthesized by a facile method. These two precursors were used with tetraethoxysilane (TEOS) to produce three series of hybrid AF coatings with zwitterionic group (Z-χ), antibacterial group (1,2-benzisothiazolin-3-one) (A-χ) and zwitterionic and antibacterial groups (S-χ) by a sol-gel process. The hardness of the coatings was measured using a pencil hardness tester and the AF behaviors of the coatings were examined by laboratory and field assays. A pencil hardness up to 5 H was achieved and slight deterioration was observed after 9 months of immersion in artificial seawater for the A-χ and S-χ coatings at a sufficiently high TEOS content. A synergistic effect between the zwitterion and antimicrobial agents existed but was not obvious. A higher TEOS content led to a higher hardness and better AF performance regardless of the type of AF group. Even with the same biofilm formation after field assay, coatings with a higher TEOS content exhibited a better resistance to mussel settlement.

Bioaccumulation and function analysis of glutathione S-transferase isoforms in Manila clam Ruditapes philippinarum exposed to different kinds of PAHs.

This study analyzed the function of different glutathione S-transferase (GST) isoforms and detoxification metabolism responses in Manila clam, Ruditapes philippinarum, exposed to 4 kinds of polycyclic aromatic hydrocarbons (PAHs) single, and their mixtures for 15 days under laboratory conditions. 13 kinds of GSTs in R. philippinarum were classified, and the results of tissue distribution indicated that 12 kinds of GSTs (except GST sigma 3) expressed most in digestive glands. We detected the mRNA expression levels of aryl hydrocarbon receptor signaling pathway, and detoxification system in digestive glands of clams exposed to benzo[a]pyrene (BaP), chrysene (CHR), benzo[a]anthracene (BaA), benzo[b]fluoranthene (BbF), and BaP + CHR + BaA + BbF, respectively. Among these genes, we selected GST-sigma, GST-omega and GST-pi as potential indicators to BaP; GST-sigma, GST-A and GST-rho to CHR; GST-pi, GST-sigma, GST-A, GST-rho and GST-microsomal to BaA; GST-theta and GST-mu to BbF; while GST-pi and GST-mu to the mixture of BaP, CHR, BaA and BbF. Additionally, the bioaccumulation of PAHs in tissues increased remarkably over time, and showed an obvious dose-effect. Under the same concentration, the bioaccumulation in single exposure group was higher than that in mixture group, and the bioaccumulation of PAHs in tissues with different concentrations of stress was irregular. The results revealed the metabolic differences and bioaccumulation rules in clams exposed to four kinds of PAHs, and provided more valuable information for the PAHs risk assessment.

Microplastics hamper the fertilization success of a broadcast spawning bivalve through reducing gamete collision and gamete fusion efficiency.

By employing external fertilization (broadcast spawning) as a mating strategy, the gametes and subsequent fertilization of various marine invertebrates are directly subjected to pollution. Although microplastics (MPs) are ubiquitous in marine environments, their potential effects on the fertilization of broadcast spawners remain largely unknown. Therefore in this study, the impacts of polystyrene MPs on the fertilization success of broadcast spawning bivalve (Tegillarca granosa) were investigated. In order to reveal the underlying mechanisms affecting fertilization, the sperm swimming performance, sperm ATP status, sperm viability, DNA integrity, gamete collision probability, gamete fusion efficiency, enzymatic antioxidants, and key ion transport enzyme activities were analyzed. The results showed that MPs weakened the sperm swimming performance through reducing ATP production and cell viability, thus leading to the decreased probability of gamete collision. Furthermore, MPs affected ion transport in the gametes by inducing oxidative stress, which resulted in gamete fusion failure. In conclusion, this study demonstrates that MPs could significantly decrease the fertilization success of T. granosa through reducing gamete collision and lowering gamete fusion efficiency.

Microplastics influence physiological processes, growth and reproduction in the Manila clam, Ruditapes philippinarum.

Microplastics (<5 mm) are widely distributed in marine environments and pose a serious threat to bivalves. Here, the ingestion and accumulation of polystyrene microplastics (PS microplastics, diameters 5 and 10 µm) by the Manila clam, Ruditapes philippinarum, and their impacts on physiological processes, growth and reproduction were studied. The results showed that both PS microplastics were ingested by the Manila clam and accumulated in their gills, hepatopancreases and intestines. Furthermore, the accumulation of 5 and 10 µm PS microplastics significantly increased the rates of respiration and excretion while significantly decreasing feeding and absorption efficiency (AE), leading to a dramatically reduced amount of energy available for growth (SfG) and ultimately led to slower growth. The dynamic energy budget (DEB) model predicts that PS microplastic exposure for 200 days would cause lower shell/flesh growth rates and reproductive potentiality. Transcriptomic profiles support these results, as carbon and protein metabolism and oxytocin and insulin-related signaling pathways were significantly altered in clams in response to PS microplastics. This study provides evidence that microplastics strongly affect the physiological activities, energy allocation, growth and reproduction of filter-feeding bivalves.

How can environmental conditions influence dicofol genotoxicity on the edible Asiatic clam, Meretrix meretrix?

Genotoxic effects of dicofol on the edible clam Meretrix meretrix were investigated through a mesocosm experiment. Individuals of M. meretrix, were exposed to environmental concentration (D1 = 50 ng/L) and supra-environmental concentration (D2 = 500 ng/L) of dicofol for 15 days, followed by the same depuration period. DNA damage (i.e., strand breaks and alkali-labile sites) was evaluated at day 1, 7 and 15, during uptake and depuration, using Comet assay (alkaline version) and nuclear abnormalities (NAs) as genotoxicity biomarkers. The protective effects of dicofol against DNA damage induced by ex vivo hydrogen peroxide (H2O2) exposure were also assessed. Comet assay results revealed no significant DNA damages under dicofol exposure, indicating 1) apparent lack of genotoxicity of dicofol to the tested conditions and/or 2) resistance of the animals due to optimal adaptation to stress conditions. Moreover, ex vivo H2O2 exposure showed an increase in the DNA damage in all the treatments without significant differences between them. However, considering only the DNA damage induced by H2O2 during uptake phase, D1 animals had significantly lower DNA damage than those from other treatments, revealing higher protection against a second stressor. NAs data showed a decrease in the % of cells with polymorphic, kidney shape, notched or lobbed nucleus, along the experiment. The combination of these results supports the idea that the clams used in the experiment were probably collected from a stressful environment (in this case Pearl River Delta region) which could have triggered some degree of adaptation to those environmental conditions, explaining the lack of DNA damages and highlighting the importance of organisms' origin and the conditions that they were exposed during their lives.

Gene expression and functional analysis of different heat shock protein (HSPs) in Ruditapes philippinarum under BaP stress.

Heat shock proteins (HSPs) are a class of highly conserved proteins which can protect cells against various types of stress. However, little information on the mechanism involved in the organic contaminants stress response of HSPs is available, especially in marine invertebrates. The present study was conducted to evaluate the responses of HSPs in clams (Ruditapes philippinarum) under Benzo[a] pyrene (BaP) exposure. The clams were exposed to BaP (concentrations: 0, 0.1, 1, 10 µg/L) for 15 days. 6 HSPs mRNA were classified, and the results of tissue distribution indicated that 4 HSPs gene expressed most in the digestive glands. The transcription level of 6 HSPs (HSP22-1, HSP22-2, HSP40A, HSP60, HSP70, HSP90) genes and the aryl hydrocarbon receptor signaling pathway-related genes, and detoxification system-related enzymes activities were analyzed at 0, 1, 3, 6, 10 and 15 days. The activities of phase II detoxification metabolic enzymes and signaling pathway related genes in clams were severely affected by BaP stress and presented significant difference. Our result suggested that HSPs were produced in the presence of BaP and participated in the process of detoxification metabolism to a certain extent. Additionally, the transcription of HSP40A gene may be used as a potential biomarker of BaP exposure due to its evident concentration- and time-dependent expression pattern. Overall, the study investigated the classification of HSPs in R. philippinarum, provided information about the expression profiles of various HSPs after BaP exposure and broadened the understanding mechanism of HSPs in detoxification defense system under PAHs stress in mollusks.

Ecotoxicological impacts of oil sand mining activity to endemic caged mussels Pyganodon grandis.

The intense mining extraction of oil sand (OS) has increased over the last few decades, raising concerns about the release of OS contaminants and toxicity in resident aquatic organisms in the Athabasca River (Alberta, Canada). To address this, endemic Pyganodon grandis mussels were caged for 6 weeks at various upstream and downstream sites of industrial OS mining activities. Post-exposure mussels were then analyzed for light/medium/heavy polyaromatic hydrocarbons (PAHs) in tissues, general health (weight to length ratio, growth rate, air survival time), biotransformation (cytochrome P4501A and 3A and glutathione S-transferase activities), oxidative stress/inflammation (lipid peroxidation-LPO and arachidonate cyclooxygenase-COX), genotoxicity (DNA strand breaks), and gonad status (triglycerides, GSI and vitellogenin-like proteins). The following effects significantly differed between OS mining area and natural/background sites: health condition, growth rate, air survival time, COX (immune/inflammation) activity, P4501A/GST activity, LPO and DNA breaks in the digestive gland and vitellogenin-like proteins in the gonad. Correlation analysis revealed that the biochemical responses were scaled to at least one of the following impacts at the individual level: air survival time, weight to length ratio, growth rate and vitellogenin-like proteins. These indices were therefore identified as key adverse outcome pathways of mussels impacted by OS mining activities. Based on the relative levels of light/medium/heavy PAHs in tissues, the observed effects appears to be associated rather to the disturbance of OS in this area than contamination from OS tailing ponds leaching into the aquatic environment.

Natural Benzo/Acetophenones as Leads for New Synthetic Acetophenone Hybrids Containing a 1,2,3-Triazole Ring as Potential Antifouling Agents.

Marine biofouling is a natural process that represents major economic, environmental, and health concerns. Some booster biocides have been used in biofouling control, however, they were found to accumulate in environmental compartments, showing negative effects on marine organisms. Therefore, it is urgent to develop new eco-friendly alternatives. Phenyl ketones, such as benzophenones and acetophenones, have been described as modulators of several biological activities, including antifouling activity (AF). In this work, acetophenones were combined with other chemical substrates through a 1,2,3-triazole ring, a strategy commonly used in Medicinal Chemistry. In our approach, a library of 14 new acetophenone-triazole hybrids was obtained through the copper(I)-catalyzed alkyne-azide cycloaddition "click" reaction. All of the synthesized compounds were evaluated against the settlement of a representative macrofouling species, Mytilus galloprovincialis, as well as on biofilm-forming marine microorganisms, including bacteria and fungi. The growth of the microalgae Navicula sp. was also evaluated after exposure to the most promising compounds. While compounds 6a, 7a, and 9a caused significant inhibition of the settlement of mussel larvae, compounds 3b, 4b, and 7b were able to inhibit Roseobacter litoralis bacterial biofilm growth. Interestingly, acetophenone 7a displayed activity against both mussel larvae and the microalgae Navicula sp., suggesting a complementary action of this compound against macro- and microfouling species. The most potent compounds (6a, 7a, and 9a) also showed to be less toxic to the non-target species Artemia salina than the biocide Econea®. Regarding both AF potency and ecotoxicity activity evaluation, acetophenones 7a and 9a were put forward in this work as promising eco-friendly AF agents.

From Natural Xanthones to Synthetic C-1 Aminated 3,4-Dioxygenated Xanthones as Optimized Antifouling Agents.

Biofouling, which occurs when certain marine species attach and accumulate in artificial submerged structures, represents a serious economic and environmental issue worldwide. The discovery of new non-toxic and eco-friendly antifouling systems to control or prevent biofouling is, therefore, a practical and urgent need. In this work, the antifouling activity of a series of 24 xanthones, with chemical similarities to natural products, was exploited. Nine (1, 2, 4, 6, 8, 16, 19, 21, and 23) of the tested xanthones presented highly significant anti-settlement responses at 50 µM against the settlement of mussel Mytilus galloprovincialis larvae and low toxicity to this macrofouling species. Xanthones 21 and 23 emerged as the most effective larval settlement inhibitors (EC50 = 7.28 and 3.57 µM, respectively). Additionally, xanthone 23 exhibited a therapeutic ratio (LC50/EC50) > 15, as required by the US Navy program attesting its suitability as natural antifouling agents. From the nine tested xanthones, none of the compounds were found to significantly inhibit the growth of the marine biofilm-forming bacterial strains tested. Xanthones 4, 6, 8, 16, 19, 21, and 23 were found to be non-toxic to the marine non-target species Artemia salina (<10% mortality at 50 µM). Insights on the antifouling mode of action of the hit xanthones 21 and 23 suggest that these two compounds affected similar molecular targets and cellular processes in mussel larvae, including that related to mussel adhesion capacity. This work exposes for the first time the relevance of C-1 aminated xanthones with a 3,4-dioxygenated pattern of substitution as new non-toxic products to prevent marine biofouling.

Xenobiotics Result in Hormonal and Enzymatic Dysregulations in the Red Mussel Mytilus galloprovincialis (Lamarck, 1819) (Bivalvia, Mytilidae).

<b>Background and Objective:</b> The contaminants in a marine ecosystem like mercury and synthetic hormones can disrupt the regulation of natural endocrine and reproductive systems of most organisms. This study aims to study the effect of organic and inorganic mercury on the viscera of <i>Mytilus galloprovincialis</i> after intracoelomic injection of 17α-ethinylestradiol, 17ß-estradiol and Dichlorodiphenyltrichloroethane (DDT) and check the histological changes in the gonads. <b>Materials and Methods:</b> Mussels are collected during June-August, 2018 from Ras el tin beach of the Mediterranean Sea of Alexandria, Egypt. This study aims to: test the effect of 17α-ethinylestradiol, 17ß-estradiol and DDT on vitellogenin (VTG) synthesis, enzymes dysfunction through intracoelomic injection of methyl mercury in a 0.75 µg/0.1 mL and mercury chloride to a 75 µg/0.1 mL. Gonads are studied histologically in control and treated mussels. Water-administered E2 and EE2 at 120 µL dose induced VTG expression in males 14 days exposure. <b>Results:</b> The relative concentration of VTG in the induced groups increases significantly as compared to the control. Alterations in the gonadal tissues and the maturation stages of the mussels are observed. The imposex mussels are characterized by concomitant secondary male sexual characteristics and the female gonad shows testicular structure. Superoxide Dismutase (SOD) activity in mussel digestive glands differed significantly (p = 0.002) after 72 hrs of MeHg exposure. <b>Conclusion:</b> Significant correlation can be observed between the activities of Glutathione S-Transferases (GST) and Glutathione Reductase (GR) in the digestive glands of mussels treated with MeHg, the enzyme activities of digestive glands treated with HgCl₂ and between Superoxide Dismutase<i>-</i>Catalase (SOD-CAT), SOD-GR and GST-GR.

Acute benzo[a]pyrene exposure induced oxidative stress, neurotoxicity and epigenetic change in blood clam Tegillarca granosa.

The blood clam (Tegillarca granosa) is being developed into a model bivalve mollusc for assessing and monitoring marine pollution on the offshore seabed. However, the information on the response of blood clam to PAHs, an organic pollutant usually deposited in submarine sediment, remains limited. Herein, we employed multiple biomarkers, including histological changes, oxidative stress, neurotoxicity and global DNA methylation, to investigate the effects of 10 and 100 µg/L Bap exposure on the blood clams under laboratory conditions, as well as the potential mechanisms. Acute Bap exposure can induce significant morphological abnormalities in gills as shown through hematoxylin-eosin (H.E) staining, providing an intuitive understanding on the effects of Bap on the structural organization of the blood clams. Meanwhile, the oxidative stress was significantly elevated as manifested by the increase of antioxidants activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and glutathione-s-transferase (GST), lipid peroxidation (LPO) level and 8-hydroxy-2'-deoxyguanosine (8-OHdG) content. The neurotoxicity was also strengthened by Bap toxicity manifested as inhibited acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) activities. In addition, the global DNA methylation level was investigated, and a significant DNA hypomethylation was observed in Bap exposed the blood clam. The correlation analysis showed that the global DNA methylation was negatively correlated with antioxidants (SOD, CAT and POD) activities, but positively correlated choline enzymes (AChE and ChAT) activities. These results collectively suggested that acute Bap exposure can cause damage in gills structures in the blood clam possibly by generating oxidative stress and neurotoxicity, and the global DNA methylation was inhibited to increase the transcriptional expression level of antioxidants genes and consequently elevate antioxidants activities against Bap toxicity. These results are hoped to shed some new light on the study of ecotoxicology effect of PAHs on marine bivalves.

Possible roles of gonadotropin-releasing hormone (GnRH) and melatonin in the control of gonadal development of clam Ruditapes philippinarum.

Gonadotropin-releasing Hormone (GnRH) is a key reproductive endocrine regulator, and melatonin is considered as a potent candidate in the regulation of photoperiod-related reproductive endocrinology. Nevertheless, their function during gonadal development of molluscs has not been uncovered yet. In the present study, RNAi of GnRH and melatonin injection were conducted on marine bivalve manila clam Ruditapes philippinarum. Tissue section showed that gonadal development was significantly inhibited in male clams injected with GnRH dsRNA for 21 days. For GnRH RNAi treatment group, the expression levels of steroid synthetic enzyme genes 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17ß-hydroxysteroid dehydrogenase (17ß-HSD), cytochrome P450 (CYP3A) and melatonin receptor homolog (MTNR) gene were significantly down-regulated in female clams while significantly up-regulated in male clams. In melatonin injection group, the expression of GnRH was significantly inhibited and the expression of 3ß-HSD, 17ß-HSD, CYP3A and MTNR genes also increased which was in line with the GnRH dsRNA injection group in male clams. These results suggest that melatonin may affect GnRH expression and both have effects on gonadal development of bivalves. This study provides evidence for understanding the effects of melatonin and GnRH on reproductive endocrinology and gonadal development in bivalve molluscs.

DCOIT unbalances the antioxidant defense system in juvenile and adults of the marine bivalve Amarilladesma mactroides (Mollusca: Bivalvia).

DCOIT is a co-biocide that is part of the formulation of the commercial antifouling Sea-Nine 211® and although it is "safe to use", negative effects have been reported on the antioxidant defense system of non-target organisms. Therefore, the objective of this research was to verify and compare the response of antioxidant enzymes of juveniles and adults of Amarilladesma mactroides exposed to DCOIT. The animals were exposed to solvent control (DMSO 0.01%) and DCOIT (measured concentration 0.01 mg/L and 0.13 mg/L) for 96 h, then gills, digestive gland and mantle were collected for analysis of the enzymatic activity of glutathione S-transferase (GST), superoxide dismutase (SOD) and catalase (CAT). The results revealed that adults, in relation to juveniles, have low basal activity of GST and SOD enzymes in the gills and digestive gland and high basal activity of SOD and CAT in the mantle. DCOIT did not alter GST activity in the gills of any life stage, while both concentrations decreased SOD and CAT in adults. In the digestive gland, it was observed that DCOIT (0.13 mg/L) decreased the GST activity in adults and CAT in juveniles, and both concentrations of the co-biocide decreased the SOD and CAT in adults. In the mantle, DCOIT (0.13 mg/L) increased CAT in juveniles. We conclude that juveniles have greater basal activity of antioxidant enzymes than adults and, in addition, DCOIT negatively affected the adults of A. mactroides, mainly decreasing the activity of GST, SOD and CAT in the gills and digestive gland of these organisms.