Hitting two targets with one shot on pesticide genotoxicity assessment - Identifying risk while unveiling ex vivo approach as a throughput tool in gill-breathing animals.

Pesticides in the environment often compromise the ecosystem, thus requiring reliable approaches to assess their effects. Commonly used approaches, such as in vivo, come with several disadvantages, namely in the light of the 3 R's policy. Seeking for accurate and ethical approaches, this study intended to validate the ex vivo technique as an alternative, and to assess the genotoxicity of chemically-based pesticides and a biopesticide. The ex vivo approach was applied to gill cells of Procambarus clarkii for 2, 4 and 8 h. Cell viability and DNA integrity were evaluated to determine the applicability of this approach. Crayfish gill cells only showed to be suitable for exposures of 2 h. Accordingly, genotoxicity was evaluated in gill cells exposed, for 2 h, to environmentally relevant concentrations of the chemically-based pesticides dimethoate (20 µg L-1), imazalil (160 µg L-1) and penoxsulam (23 µg L-1), as well as to the bioinsecticide Turex® (25, 50, 100, 200 and 400 µg L-1). Every chemically-based pesticide demonstrated to be genotoxic, despite not inducing oxidative DNA damage. On the other hand, Turex® showed no genotoxic effects. Overall, the ex vivo approach demonstrated to be possible and practical to implement, improving the number of outcomes with a lower number of organisms. The findings from the screening test suggest that biological pesticides may pose a lower risk to non-target organisms compared to chemically-based pesticides.

Exploring the Antioxidant and Genoprotective Potential of Salicornia ramosissima Incorporation in the Diet of the European Seabass (Dicentrarchus labrax).

The identification of novel feed materials as a source of functional ingredients is a topical priority in the finfish aquaculture sector. Due to the agrotechnical practices associated and phytochemical profiling, halophytes emerge as a new source of feedstuff for aquafeeds, with the potential to boost productivity and environmental sustainability. Therefore, the present study aimed to assess the potential of Salicornia ramosissima incorporation (2.5, 5, and 10%), for 2 months, in the diet of juvenile European seabass, seeking antioxidant (in the liver, gills, and blood) and genoprotective (DNA and chromosomal integrity in blood) benefits. Halophyte inclusion showed no impairments on growth performance. Moreover, a tissue-specific antioxidant improvement was apparent, namely through the GSH-related defense subsystem, but revealing multiple and complex mechanisms. A genotoxic trigger (regarded as a pro-genoprotective mechanism) was identified in the first month of supplementation. A clear protection of DNA integrity was detected in the second month, for all the supplementation levels (and the most prominent melioration at 10%). Overall, these results pointed out a functionality of S. ramosissima-supplemented diets and a promising way to improve aquaculture practices, also unraveling a complementary novel, low-value raw material, and a path to its valorization.

Improving knowledge on genotoxicity dynamics in somatic and germ cells of crayfish (Procambarus clarkii).

The harmful effects of pesticides can be extended beyond the exposure time scale. Appraisals combining exposure and long-term post-exposure periods appear as an unavoidable approach in pesticide risk assessment, thus allowing a better understanding of the real impact of agrochemicals in non-target organisms. This study aimed to evaluate the progression of genetic damage in somatic and germ tissues of the crayfish Procambarus clarkii, also seeking for gender-specificities, following exposure (7 days) to penoxsulam (23 µg L-1 ) and a post-exposure (70 days) period. The same approach was applied to the model genotoxicant ethyl methanesulfonate (EMS; 5 mg L-1 ) as a complementary mean to improve knowledge on genotoxicity dynamics (induction vs. recovery). Penoxsulam induced DNA damage in all tested tissues, disclosing tissue- and gender-specificities, where females showed to be more vulnerable than males in the gills, while males demonstrated higher susceptibility in what concerns internal organs, that is, hepatopancreas and gonad. Crayfish were unable to recover from the DNA damage induced by EMS in gills and hepatopancreas (both genders) as well as in spermatozoa. The genotoxicity in the hepatopancreas was only perceptible in the post-exposure period. Oxidative DNA lesions were identified in hepatopancreas and spermatozoa of EMS-exposed crayfish. The spermatozoa proved to be the most vulnerable cell type. It became clear that the characterization of the genotoxic hazard of a given agent must integrate a complete set of information, addressing different types of DNA damage, tissue- and gender-specificities, as well as a long-term appraisal of temporal progression of damage.

Intergenerational Patterns of DNA Methylation in Procambarus clarkii Following Exposure to Genotoxicants: A Conjugation in Past Simple or Past Continuous?

Epigenome is susceptible to modulation by environmental pressures-namely, through alterations in global DNA methylation, impacting the organism condition and, ultimately, reverberating on the phenotype of the subsequent generations. Hence, an intergenerational study was conducted, aiming to clarify the influence of genotoxicants on global DNA methylation of the crayfish Procambarus clarkii. Two subsequent generations were exposed to the herbicide penoxsulam (Px; 23 µg·L-1) and to the genotoxicant model ethyl methanesulfonate (EMS; 5 mg·L-1). Px did not induce changes in DNA methylation of adult crayfish (F0). However, the hypomethylation occurring in unexposed F1 juveniles demonstrated that the history of exposure per se can modulate epigenome. In F1 descendants of the Px-exposed group, methylome (hypermethylated) was more affected in males than in females. EMS-induced hypomethylation in adult females (F0), also showed gender specificity. In addition, hypomethylation was also observed in the unexposed F1 crayfish, indicating an intergenerational epigenetic effect. The modulatory role of past exposure to penoxsulam or to EMS also showed a dependency on the crayfish developmental stage. Overall, this research revealed that indirect experiences (events occurring in a predecessor generation) can have an impact even greater than direct experiences (present events) on the epigenetic dynamics.

Invasive clams (Ruditapes philippinarum) are better equipped to deal with harmful algal blooms toxins than native species (R. decussatus): evidence of species-specific toxicokinetics and DNA vulnerability.

This study aims to assess and compare the kinetics (accumulation/elimination) of the marine biotoxins okadaic acid (OA) and dinophysistoxin-1 (DTX1), between native (Ruditapes decussatus) and invasive (Ruditapes philippinarum) clam species, and their genotoxic effects and DNA recover capacity after, exposure to toxic dinoflagellates Prorocentrum lima. Clams were fed with P. lima for 5 days and then to non-toxic algae (post-exposure) during other 5 days. Toxin concentrations determined in clams by LC-MS/MS were related with DNA damage and repair assessment through the comet and base excision repair (BER) assays, respectively. Differential accumulation patterns were observed between the invasive and native species. The invasive species consistently and progressively accumulated the toxins during the first 24 h of exposure, while the native clams showed drastic variations in the toxin accumulation. Nevertheless, at the end of a 5 days of exposure period, the native clams presented higher toxin concentrations, nearly reaching the legal regulatory limit for human consumption. In addition, native clams were vastly affected by OA and DTX1, presenting an increment in the DNA damage since the first day, with a correspondent increase in the repair activity. On the other hand, invasive clams were not affected by the dinoflagellate toxins, exhibiting only some signs of the challenge, namely an increase in the DNA repair mechanisms in the post-exposure period. Invasive clams R. philippinarum are better adapted to cope with harmful algal blooms and OA-group toxins than native species. These results may increase farming interest and may lead to new introductions of the invasive clams. In sympatry sites, exposure to OA-group toxins may unbalance clams species biomass and distribution as exposure to toxic dinoflagellates affects the native clams from cellular to a population level, representing a significant threat to development and maintenance of R. decussatus populations.

DNA damage and oxidative stress responses of mussels Mytilus galloprovincialis to paralytic shellfish toxins under warming and acidification conditions - Elucidation on the organ-specificity.

Commonly affected by changes in climate and environmental conditions, coastal areas are very dynamic environments where shellfish play an important ecological role. In this study, the oxidative stress and genotoxic responses of mussels (Mytilus galloprovincialis) exposed to paralytic shellfish toxin (PST) - producing dinoflagellates Gymnodinium catenatum were evaluated under i) current conditions (CC: 19 °C; pH 8.0), ii) warming (W: 24 °C; pH 8.0), iii) acidification (A:19 °C; pH 7.6) and iv) combined effect of warming and acidification (WA: 24 °C; pH 7.6). Mussels were fed with G. catenatum for 5 days, and to a non-toxic diet during the following 10 days. A battery of oxidative stress biomarkers and comet assay was performed at the peak of toxin accumulation and at the end of the post-exposure phase. Under CC, gills and hepatopancreas displayed different responses/vulnerabilities and mechanisms to cope with PST. While gills presented a tendency for lipid peroxidation (LPO) and genetic damage (expressed by the Genetic Damage Indicator - GDI), hepatopancreas seems to better cope with the toxins, as no LPO was observed. However, the mechanisms involved in hepatopancreas protection were not enough to maintain DNA integrity. The absence of LPO, and the antioxidant system low responsiveness, suggests DNA damage was not oxidative. When exposed to toxic algae under W, toxin-modulated antioxidant responses were observed in both gills and hepatopancreas. Simultaneous exposure to the stressors highlighted gills susceptibility with a synergistic interaction increasing DNA damage. Exposure to toxic algae under A led to genotoxicity potentiation in both organs. The combined effect of WA did not cause relevant interactions in gills antioxidant responses, but stressors interactions impacted LPO and GDI. Antioxidant responses and LPO pointed out to be modulated by the environmental conditions in hepatopancreas, while GDI results support the dominance of toxin-triggered process. Overall, these results reveal that simultaneous exposure to warming, acidification and PSTs impairs mussel DNA integrity, compromising the genetic information due to the synergetic effects. Finally, this study highlights the increasing ecological risk of harmful algal blooms to Mytilus galloprovinciallis populations.

Crystallinity and microchemistry of Nassarius reticulatus (Caenogastropoda) statoliths: towards their structure stability and homogeneity.

Gastropod statoliths are spherical biocarbonates formed during their lifespan. The stability and homogeneity of these structures' mineral matrix was characterised along their radiuses, using Nassarius reticulatus as a model. Generally, they were proved to be bimineralic. Two of the three CaCO3 crystalline polymorphs occurring in biocarbonates - aragonite and calcite - coexist along statolith radiuses, aragonite being unequivocally the most abundant phase. The presence of a diffuse organic matrix was also perceived by the detection of a weak Raman band between 2800 and 3000 cm(-1) consistently observed along radiuses. Beyond the apparent stability and homogeneity, different crystalline orientations were disclosed by Raman spectroscopy. A change in the intensity pattern of the features related to the lattice and bending modes of aragonite between different radiuses give new insights for a possible spherulitic-like growth of these structures. As expected from the relative homogeneity of both mineral and organic signals, there was no pattern on the distribution of Ca, O, Na and S along radiuses. However, a higher concentration of Sr occurs in growth rings (known as winter tags), corroborating the already described negative correlation between the concentration of this element in statoliths and temperature. Despite the apparent stability and homogeneity of the matrix during its lifespan, the periodic distribution of Sr potentially influences a dissimilar incorporation of trace elements in increments and growth rings. Since gastropod statolith elemental fingerprinting was recently suggested as a new tool to monitor marine environmental changes, the pressing need for further studies on the incorporation of traces in these structures is highlighted.