Accumulation, Depuration, and Biological Effects of Polystyrene Microplastic Spheres and Adsorbed Cadmium and Benzo(a)pyrene on the Mussel Mytilus galloprovincialis

Filter feeders are target species for microplastic (MP) pollution, as particles can accumulate in the digestive system, disturbing feeding processes and becoming internalized in tissues. MPs may also carry pathogens or pollutants present in the environment. This work assessed the influence of polystyrene (PS) MP size and concentration on accumulation and depuration time and the role of MPs as vectors for metallic (Cd) and organic (benzo(a)pyrene, BaP) pollutants. One-day exposure to pristine MPs induced a concentration-dependent accumulation in the digestive gland (in the stomach and duct lumen), and after 3-day depuration, 45 µm MPs appeared between gill filaments, while 4.5 µm MPs also occurred within gill filaments. After 3-day exposure to contaminated 4.5 µm MPs, mussels showed increased BaP levels whilst Cd accumulation did not occur. Here, PS showed higher affinity to BaP than to Cd. Three-day exposure to pristine or contaminated MPs did not provoke significant alterations in antioxidant and peroxisomal enzyme activities in the gills and digestive gland nor in lysosomal membrane stability. Exposure to dissolved contaminants and to MP-BaP caused histological alterations in the digestive gland. In conclusion, these short-term studies suggest that MPs are ingested and internalized in a size-dependent manner and act as carriers of the persistent organic pollutant BaP.

Does Thermal Stress Modulate the Biochemical and Physiological Responses of Ruditapes Decussatus Exposed to the Progestin Levonorgestrel?

The combined effects of the abiotic factors such as temperature and synthetic progestins on the biochemical and physiological performances of marine clam Ruditapes decssatus under sub-chronic conditions are poorly studied. Here, we investigated the effects of levonorgestrel (1000 ng/l) alone or combined with increasing temperature of 20, 24 and 28°C on the biochemical and physiological responses and the gonad histology of the clam (Ruditapes decussatus) for 28 days. Our results revealed that, in clams treated with levonorgestrel (LNG) alone, there was an impairment of the defense against oxidative stress related to the inductions of CAT, GST and PSH, a reduction in the lysosomal membrane stability, a physiological disturbance via an increase in the level of estradiol, a decrease in the number of gonad oocytes and a reduction in vitellogenin like protein. Furthermore, exposure to heat stress of 20°C, 24°C and 28°C, alone or in combination with LNG, enhanced SOD antioxidant activity which was ineffective in preventing lipoperoxidation and altered clam’s physiological processes of reproduction via dramatic reduction of gonad oocyte numbers and of vitellogenin like protein reserves. Overall, these findings can be taken as a warning sign of temperature and levonorgestrel progestin effect that can lead to effective changes in the structure and function of target vital organs, such as the gonads of the clam Ruditapes decussatus, and this may have implications for the whole organism and populations.

Interactive Immunomodulation in the Mediterranean Mussel Mytilus galloprovincialis Under Thermal Stress and Cadmium Exposure

Marine bivalves are frequently exposed to multiple co-occurring challenges such as temperature extremes and anthropogenic pollution. These stressors can elicit negative effects on several biological pathways, including antioxidant and neuroendocrine-immune (NEI) systems, leading to immune disorders and altered immunocytes functionality. Since interactive mechanisms of action and resulting outcomes are still scarcely explored, we examined the single and combined effects of increased temperature (+5°C) and cadmium (20 μg/L) in the Mediterranean mussel Mytilus galloprovincialis. Analyzed parameters included cholinergic system in gills and hemolymph (acetylcholinesterase activity, AChE), total oxyradical scavenging capacity in gills and key functional processes in hemocytes, including lysosomal membrane stability, hemocytes subpopulations ratio, phagocytosis capacity, and onset of genotoxic damage. Results highlighted interactive inhibition of AChE activity along to a concomitant increased total oxyradical scavenging capacity, confirming neuroendocrine-immune system (NEI) disturbance and oxidative pressure. In hemocytes, lysosomal membrane stability and granulocytes:hyalinocytes ratio revealed additive effects of stressors, while a consistent reduction of phagocytosis was caused by temperature stress, with a slightly antagonistic effect of cadmium. Pearson’s correlation statistics provided either positive or negative relationships between investigated parameters and stressors, allowing to hypothesize putative mechanism of immune system functional alterations. The overall results suggest that the occurrence of short-term events of increased temperature and concomitant metal exposure could elicit interactive and negative effects on immune system efficiency of marine organisms.

Assessing the Impact of Chrysene-Sorbed Polystyrene Microplastics on Different Life Stages of the Mediterranean Mussel Mytilus galloprovincialis

The sorption of organic pollutants to marine plastic litter may pose risks to marine organisms, notably for what concerns their intake and transfer through microplastic (MP) ingestion. This study investigated the effects of polystyrene MP loaded with chrysene (CHR) on early-stage and physiological endpoints measured in the Mediterranean mussel Mytilus galloprovincialis. The same concentrations of virgin microplastics (MP) and MP loaded with 10.8 µg CHR/mg (CHR-MP) were administered to mussel gametes/embryos (25 × 103 items/mL) and adults (5⋅× 103 items/L); further treatments included 0.1 mg/L of freely dissolved CHR and a second CHR concentration corresponding to that vehiculated by CHR-MP during exposure (3.78 µg/L and 0.73 ng/L for gamete/embryos and adults, respectively). None of the treatments affected gamete fertilization, while 0.1 mg/L CHR induced embryotoxicity. In adults, CHR-MP and MP similarly affected lysosomal membrane stability and neutral lipids and induced slight effects on oxidative stress endpoints. CHR affected tested endpoints only at 0.1 mg/L, with lysosomal, oxidative stress and neurotoxicity biomarkers generally showing greater alterations than those induced by CHR-MP and MP. This study shows that the CHR sorption on MP does not alter the impact of virgin MP on mussels and may pose limited risks compared to other routes of exposure.

First Evidence of In Vitro Effects of C6O4—A Substitute of PFOA—On Haemocytes of the Clam Ruditapes philippinarum

Alternative chemicals to per- and poly-fluoroalkyl substances have recently been introduced in various industrial processes. 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 replacement for perfluorooctanoic acid (PFOA). From an ecotoxicological point of view, in vitro assays are useful tools for assessing the negative effects and understanding the mechanisms of action of chemicals at the cellular level. Here, we present the results of an in vitro study in which the effects of C6O4 were evaluated—for the first time—on haemocytes of the clam Ruditapes philippinarum. Cells were exposed to three concentrations of C6O4 (0.05, 0.5, 5 μg/mL) and the effects on haemocyte viability, haemocyte morphology, differential haemocyte count, lysosomal membrane stability, superoxide anion production, acid phosphatase, and β-glucuronidase activities, as well as on the percentage of micronuclei and chromosomal aberrations were evaluated. The results demonstrated that C6O4 significantly affected haemocyte morphology, lysosomal membrane stability, hydrolytic enzyme activity, and superoxide anion production, and promoted chromosomal aberrations. To the best of our knowledge, this is the first study revealing the in vitro effects of C6O4, a substitute for PFOA, on haemocytes from a bivalve species.

Interplay Between Nanoplastics and the Immune System of the Mediterranean Sea Urchin Paracentrotus lividus

The present study highlights for the first time the interplay between model nanoplastics, such as the carboxyl-modified polystyrene nanoparticles (PS-COOH, 60 nm) NPs and the coelomocytes of the sea urchin Paracentrotus lividus, a benthic grazer widely distributed in Mediterranean coastal area, upon acute in vitro exposure (4 h) (5 and 25 μg mL–1). Insight into PS-COOH trafficking (uptake and clearance) and effects on immune cell functions (i.e., cell viability, lysosomal membrane stability, and phagocytosis) are provided. Dynamic Light Scattering analysis reveals that PS NP suspensions in CF undergo a quick agglomeration, more pronounced for PS-COOH (608.3 ± 43 nm) compared to PS-NH2 (329.2 ± 5 nm). However, both PS NPs are still found as nano-scale agglomerates in CF after 4 h of exposure, as shown by the polydispersity index > 0.3 associated with the presence of different PS NP size populations in the CF. The observed changes in ζ-potential upon suspension in CF (–11.1 ± 3 mV and –12.1 ± 4 mV for PS-COOH and PS-NH2, respectively) confirm the formation of a bio-corona on both PS NPs. Optical fluorescence microscopy and fluorimetric analyses using fluorescently labeled PS-COOH (60 nm) reveal a fast uptake of PS-COOH primarily by phagocytes within 1 h of exposure. Upon transfer to PS NP-free CF, a significant decrease in fluorescence signal is observed, suggesting a fast cell clearance. No effect on cell viability is observed after 4 h of exposure to PS-COOH, however a significant decrease in lysosomal membrane stability (23.7 ± 4.8%) and phagocytic capacity (63.43 ± 3.4%) is observed at the highest concentration tested. Similarly, a significant reduction in cell viability, lysosomal membrane stability and phagocytosis is found upon exposure to PS-NH2 (25 μg mL–1), which confirms the important role of surface charges in triggering immunotoxicity. Overall, our results show that, although being quickly internalized, PS-COOH can be easily eliminated by the coelomocytes but may still be able to trigger an immune response upon long-term exposure scenarios. Taking into account that sediments along Mediterranean coasts are a sink for micro- and nanoplastics, the latter can reach concentrations able to exceed toxicity-thresholds for marine benthic species.

Antioxidant defense system responses, lysosomal membrane stability and DNA damage in earthworms (Eisenia fetida) exposed to perfluorooctanoic acid: an integrated biomarker approach to evaluating toxicity

The integrated biomarker response (IBR) index was calculated to evaluate the integrated toxicological effects of PFOA on earthworm Eisenia fetida.