Mild Effects of Sunscreen Agents on a Marine Flatfish: Oxidative Stress, Energetic Profiles, Neurotoxicity and Behaviour in Response to Titanium Dioxide Nanoparticles and Oxybenzone.

UV filters are potentially harmful to marine organisms. Given their worldwide dissemination and the scarcity of studies on marine fish, we evaluated the toxicity of an organic (oxybenzone) and an inorganic (titanium dioxide nanoparticles) UV filter, individually and in a binary mixture, in the turbot (Scophthalmus maximus). Fish were intraperitoneally injected and a multi-level assessment was carried out 3 and 7 days later. Oxybenzone and titanium dioxide nanoparticles induced mild effects on turbot, both isolated and in mixture. Neither oxidative stress (intestine, liver and kidney) nor neurotoxicity (brain) was found. However, liver metabolic function was altered after 7 days, suggesting the impairment of the aerobic metabolism. An increased motility rate in oxybenzone treatment was the only behavioural alteration (day 7). The intestine and liver were preferentially targeted, while kidney and brain were unaffected. Both infra- and supra-additive interactions were perceived, with a toxicodynamic nature, resulting either in favourable or unfavourable toxicological outcomes, which were markedly dependent on the organ, parameter and post-injection time. The combined exposure to the UV filters did not show a consistent increment in toxicity in comparison with the isolated exposures, which is an ecologically relevant finding providing key information towards the formulation of environmentally safe sunscreen products.

The role of contamination history and gender on the genotoxic responses of the crayfish Procambarus clarkii to a penoxsulam-based herbicide.

The responses of non-target organisms to pesticide exposure are still poorly explored in what concerns the development of adjustments favouring population success. Owing to the vital role of DNA integrity, it is important to identify genome-maintenance skills and their determinant factors. Thus, the major aims of the present study were: (i) to assess the genotoxicity of the penoxsulam-based herbicide (Viper®) to the crayfish Procambarus clarkii; (ii) to understand the influence of gender and contamination history in the genotoxic responses following exposure to this herbicide; (iii) to investigate the damage mechanisms involved in putative adjustments shown by P. clarkii. Two populations were tested, one from a reference site and the other from a historically contaminated site. Specimens from both populations were exposed to Viper®, considering environmentally relevant penoxsulam concentrations (20 and 40 µg L-1) and to a model genotoxicant (EMS). Comet assay was adopted to assess the genetic damage in gills. The results disclosed the genotoxicity of the herbicide to crayfish (a non-target organism). Additionally, organisms exposed to the highest concentration of penoxsulam signalized the influence of factor "population" towards the genotoxic pressure (measured as effective DNA breaks): P2 males from the historically impacted population displayed a significantly higher susceptibly (by up to 53.98%) when compared to control, while the homologous group from the reference population presented levels similar to its respective control. When DNA lesion-repair enzymes were considered, DNA oxidation patterns suggested an increased ability of this gender (39.75% lower than negative control) to deal with this particular type of damage, namely considering pyrimidines oxidation. It is worth remarking that the influence of the exposure history on the protection/vulnerability to the penoxsulam-based herbicide was only evident in males, despite depending on the type of DNA damage: when the non-specific damage was considered, organisms from the impacted population seemed to be more vulnerable while regarding to the oxidative damage, males from the impacted population appeared to be more protected than organisms that have never been exposed to penoxsulam. Overall, the influence of factors "gender" and "contamination history" was demonstrated as well as its dependence on DNA damage type was evident. EMS groups did not present the differences between populations, reinforcing the agent-specific adjustment hypothesis.These findings highlighted the importance of considering differential physiological backgrounds in ecogenotoxicological analysis, hence favouring the elaboration of more plausible and holistic approaches integrating the environmental risk assessment of pesticides.

Propensity to metal accumulation and oxidative stress responses of two benthic species (Cerastoderma edule and Nephtys hombergii): are tolerance processes limiting their responsiveness?

The chronic exposure of benthic organisms to metals in sediments can lead to the development of tolerance mechanisms, thus diminishing their responsiveness. This study aims to evaluate the accumulation profiles of V, Cr, Co, Ni, As, Cd, Pb and Hg and antioxidant system responses of two benthic organisms (Cerastoderma edule, Bivalvia; Nephtys hombergii, Polychaeta). This approach will provide clarifications about the ability of each species to signalise metal contamination. Organisms of both species were collected at the Tagus estuary, in two sites with distinct contamination degrees (ALC, slightly contaminated; BAR, highly contaminated). Accordingly, C. edule accumulated higher concentrations of As, Pb and Hg at BAR compared to ALC. However, antioxidant responses of C. edule were almost unaltered at BAR and no peroxidative damage occurred, suggesting adjustment mechanisms to the presence of metals. In contrast, N. hombergii showed a minor propensity to metal accumulation, only signalising spatial differences for As and Pb and accumulating lower concentrations of metals than C. edule. The differences in metal accumulation observed between species might be due to their distinctive foraging behaviour and/or the ability of N. hombergii to minimise the metal uptake. Despite that, the accumulation of As and Pb was on the basis of the polychaete antioxidant defences inhibition at BAR, including CAT, SOD, GR and GPx. The integrated biomarker response index (IBRv2) confirmed that N. hombergii was more affected by metal exposure than C. edule. In the light of current findings, in field-based studies, the information of C. edule as a bioindicator should be complemented by that provided by another benthic species, since tolerance mechanisms to metals can hinder a correct diagnosis of sediment contamination and of the system's health. Overall, the present study contributed to improve the lack of fundamental knowledge of two widespread and common estuarine species, providing insights of the metal accumulation profiles under a scenario of chronic contamination. Finally, this work provided useful information that can be applied in the interpretation of future environmental monitoring studies.

Evidences of DNA and chromosomal damage induced by the mancozeb-based fungicide Mancozan® in fish (Anguilla anguilla L.).

The formulation Mancozan®, containing mancozeb as active ingredient, is among the most widely used fungicides. Although mancozeb has been detected in surface waters, studies addressing the genotoxic risk to fish arising from the use of this formulation, testing environmentally realistic concentrations, are absent from the literature. Hence, this work aimed to investigate the DNA and chromosome damaging potential of Mancozan® (0.29 and 2.9µgL-1) in the European eel (Anguilla anguilla L.), after a short-term exposure (3days), through the adoption of the comet and the erythrocytic nuclear abnormality (ENA) assays. In addition, it was intended to elucidate the subjacent damage mechanisms, improving the comet assay with the adoption of the endonucleases formamidopyrimidine DNA glycosylase (FPG) and endonuclease III (EndoIII), which detect oxidized bases. The highest Mancozan® concentration was able to affect the DNA integrity (comet assay), while the adoption of endonucleases pointed out an oxidative cause to the damage. Regarding the chromosomal damage (ENA assay), both concentrations displayed significant effects, revealing the clastogenic and/or aneugenic properties of Mancozan®. Furthermore, the two genotoxic endpoints were significantly correlated. Overall, the results revealed a genetic hazard to fish inhabiting aquatic systems contaminated by Mancozan® and strongly recommend the development of biomonitoring and regulatory policies regarding the utilization of this agrochemical.

Genotoxicity evaluation of the herbicide Garlon(®) and its active ingredient (triclopyr) in fish (Anguilla anguilla L.) using the comet assay.

Triclopyr-based herbicides are broadly used worldwide for site preparation and forest vegetation management. Thus, following application, these agrochemicals can inadvertently reach the aquatic ecosystems. Garlon(®) is one of the most popular commercial denominations of this group of herbicides, considered as highly toxic to fish, even by its manufacturer. Although DNA is frequently regarded as a target of pesticide toxicity, the genotoxic potential of Garlon(®) to fish remains completely unknown. Hence, the main goal of this study was to evaluate the genotoxicity of Garlon(®) and its active ingredient (triclopyr), clarifying the underlying mechanisms. Therefore, the comet assay, implemented as the standard procedure, with an extra step involving DNA lesion-specific repair enzymes (formamidopyrimidine DNA glycosylase and endonuclease III), was used to identify DNA damage in blood cells of Anguilla anguilla L. Short-term exposures (1 and 3 days) to Garlon(®) and triclopyr were carried out, adopting environmentally realistic concentrations (67.6 and 270.5 µg L(-1) Garlon(®) and 30 and 120 µg L(-1) triclopyr). The results concerning the nonspecific DNA damage proved the risk of the herbicide Garlon(®) and its active ingredient triclopyr in both tested concentrations and exposure lengths. In addition, the higher genotoxic potential of the formulation, in comparison with the active ingredient, was demonstrated. When the additional breaks corresponding to net enzyme-sensitive sites were considered, none of the conditions revealed significant levels of oxidative damage. This identification of the genotoxic properties of triclopyr-based herbicides to fish highlights the need to develop less hazardous formulations, as well as the adoption of mitigation measures related to the application of these agrochemicals in the framework of forestry and agriculture sustainable management.

Assessment of chromosomal damage induced by a deltamethrin-based insecticide in fish (Anguilla anguilla L.) - a follow-up study upon exposure and post-exposure periods.

The pyrethroid insecticide Decis®, containing deltamethrin as active ingredient, is among the most popular broad-spectrum biocides, with wide application in agriculture and home pest control. The occurrence of deltamethrin in the aquatic environment is well-established, but the possible genotoxic effects of Decis® in non-target organisms, namely fish, remain unknown. Hence, this work aimed to evaluate the cytogenetic damaging potential of Decis® in European eel (Anguilla anguilla L.), adopting the erythrocytic nuclear abnormalities (ENAs) assay. In addition, it was intended to investigate the damage progression in the post-exposure period. The frequency of immature erythrocytes (IE) was also determined to provide indirect information on the erythrocyte catabolism and erythropoiesis rate. Fish were exposed to 17.5 and 35 µg L(-1) of Decis® (equivalent to 0.05 and 0.1 µg L(-1) of deltamethrin, respectively) during 1 and 3 days. Thereafter, fish were transferred to clean water and kept for 1, 7 and 14 days. The results demonstrated a clear potential to induce chromosomal damage following 3 days exposure, depicted in an ENA frequency increase for both Decis® concentrations. The transient nature of this cytogenetic damage was also demonstrated, as ENA frequency returned to the control level 1 and 7 days after cessation of the exposure, respectively for the higher and the lower Decis® concentration. Moreover, this response pattern suggested a rapid metabolization and elimination of the formulation constituents by A. anguilla, combined with an increased erythrocyte turnover in fish exposed to the higher Decis® concentration, as pointed out by the IE frequency rise. Overall, the demonstrated genotoxic properties of Decis® pointed out increased risk factors to fish exposed to this insecticide.

Biopesticide Turex®'s cytotoxicity, genotoxicity and cell cycle arrest on HepG2 cell line.

Population growth leads to the need for more efficient techniques and compounds in agriculture, such as pesticides, to deal with the ever-growing demand. Pesticides may end up in the environment, often compromising the ecosystem affecting all organisms including humans. Therefore, the consequences of exposure to these compounds to biota and humans needs to be assessed. Bearing this in mind, the aim of this study was to examine the in vitro cytotoxicity and genotoxicity attributed to exposure to the biopesticide Turex® utilizing the liver cell line HepG2. Cells were incubated with biopesticide Turex® at 250, 500, 1000, 1500 or 2000 µg/L in both non-activated and activated forms for 24 and 48 h. Subsequent effects on cell viability were assessed using the MTT. The influence on cell cycle dynamics was determined by flow cytometry, while DNA damage was measured by the comet assay. Data demonstrated that activated Turex® induced cytotoxicity and DNA damage after 48 h in HepG2 cell line. The cell cycle progression was not markedly affected by Turex® at any concentration or duration of exposure. In conclusion, data demonstrated the potential adverse effects attributed to exposure to biopesticide Turex® in human cell line HepG2. Consequently, this type of biopesticide needs to be further investigated to determine the potential adverse in vivo effects on various non-target organisms.

Shipping can be achieved but cargo arrives with damage - titanium dioxide nanoparticles affect the DNA of Pacific oyster sperm.

Titanium dioxide nanoparticles (TiO2 NP) were reported to be reprotoxic in humans and fish. However, the effects of these NP on the reproduction of marine bivalves, namely oysters, remain unknown. Thus, a short-term (1 h) direct exposure of sperm of the Pacific oyster (Crassostrea gigas) to two TiO2 NP concentrations (1 and 10 mg.L-1) was performed, and sperm motility, antioxidant responses, and DNA integrity were evaluated. Although no changes occurred in sperm motility and the activities of the antioxidants, the genetic damage indicator increased at both concentrations, demonstrating that TiO2 NP affects the DNA integrity of oyster sperm. Although DNA transfer can happen, it does not fulfill its biological mission since the transferred DNA is not intact and may compromise the reproduction and recruitment of the oysters. This vulnerability of C. gigas sperm towards TiO2 NP highlights the importance of studying the effects of NPs exposure to broadcast spawners.

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.

Silver nanoparticles and silver ions indistinguishably decrease sperm motility in Pacific oysters (Magallana gigas) after short-term direct exposure.

The present study aimed to evaluate the reprotoxicity of environmental (0.25 µg.L-1) and supra-environmental (25 µg.L-1 and 250 µg.L-1) levels of silver nanoparticles (Ag NP) on the Pacific oyster (Magallana gigas), by determining sperm quality. For that, we evaluated sperm motility, mitochondrial function and oxidative stress. To determine whether the Ag toxicity was related to the NP or its dissociation into Ag ions (Ag+), we tested the same concentrations of Ag+. We observed no dose-dependent responses for Ag NP and Ag+, and both impaired sperm motility indistinctly without affecting mitochondrial function or inducing membrane damage. We hypothesize that the toxicity of Ag NP is mainly due to adhesion to the sperm membrane. Blockade of membrane ion channels may also be a mechanism by which Ag NP and Ag+ induce toxicity. The presence of Ag in the marine ecosystem is of environmental concern as it may affect reproduction in oysters.

Measuring DNA modifications with the comet assay: a compendium of protocols.

The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA-DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.

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.

Evaluation of DNA damage induced by environmental exposure to mercury in Liza aurata using the comet assay.

Mercury (Hg) is one of the major aquatic contaminants even though emissions have been reduced over the years. Despite the relative abundance of investigations carried out on Hg toxicity, there is a scarcity of studies on its DNA damaging effects in fish under realistic exposure conditions. This study assessed the Hg genotoxicity in Golden grey mullets (Liza aurata) at Laranjo basin, a particularly contaminated area of Ria de Aveiro (Portugal) well known for its Hg contamination gradient. (1) Fish were seasonally caught at Laranjo basin and at a reference site (S. Jacinto), and (2) animals from the reference site were transplanted and caged (at bottom and surface), for 3 days, in two different locations within Laranjo basin. Using the comet assay, blood was analyzed for genetic damage and apoptotic cell frequency. The seasonal survey showed greater DNA damage in the Hg-contaminated area for all sampling seasons excluding winter. The temporal variation pattern of DNA lesions was: summer approximately autumn > winter > spring. Fish caged at Laranjo also exhibited greater DNA damage than those caged at the reference site, highlighting the importance of gill uptake on the toxicity of this metal. No increased susceptibility to apoptosis was detected in either wild or caged fish, indicating that mercury damages DNA of blood cells by a nonapoptotic mechanism. Both L. aurata and the comet assay proved to be sensitive and suitable for genotoxicity biomonitoring in mercury-contaminated coastal systems.

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.

Marine macroalgae as a dietary source of genoprotection in gilthead seabream (Sparus aurata) against endogenous and exogenous challenges.

DNA integrity and stability are essential to organisms' health and survival. However, it has been neglected in what concerns to fish farming, disregarding the potential impact of endogenous/ exogenous factors. As marine macroalgae constitute a source of natural compounds with a large spectrum of biological activities, this study, situated in the interface of nutritional-genetic research and development of algae practical applications, aimed to evaluate the genoprotective properties of a macroalgae-enriched diet (total percentage of 5%, incorporating equal percentages of Ulva rigida, Gracilaria gracilis and Fucus vesiculosus) in gilthead seabream (Sparus aurata). Protection was assessed in relation to a basal genome integrity and against an exogenous genotoxic challenge (cyclophosphamide; CP). Fish were reared for 30 days with the supplemented diet, being then injected with CP and sampled at days 3 and 10 post-injection (p.i.). To evaluate whether the favorable effects remain after the end of supplementation, a fish subgroup previously fed with algae-enriched diet was submitted to a diet reversion at day 3 p.i., being thereafter fed with the standard diet. Genetic damage was evaluated through the erythrocytic nuclear abnormalities (ENA) and comet assays and complemented by the assessment of the antioxidant system. Results pointed out that algae-enriched feed exhibits anti-genotoxic properties, mostly expressed in relation to the exogenous pressure, manifest in relation to DNA strand breaks and chromosomal lesions, also reducing oxidative DNA damage. Nonetheless, blood antioxidants were only punctually altered by the supplemented diet (e.g. catalase and glutathione-S-transferase). Analyzing the effect persistence, it was perceived that 7 days without algae uptake was enough to partially reduce the protection efficacy. Overall, these findings are promising towards the benefits of macroalgae inclusion in fish diet, and thus, to invigorate mariculture activity and the commercial use of algae, also providing new insights on the DNA protection mechanisms.

Silver (nano)materials cause genotoxicity in Enchytraeus crypticus, as determined by the comet assay.

Enchytraeids have been used in standard ecotoxicity testing for approximately 20 yr. Since adopting the standard test for survival and reproduction, a number of additional tools have been developed, including transcriptomics and enzymatic biomarkers. So far, a genotoxicity tool and endpoint have not been used; hence, the goals of the present study included optimization of the in vivo alkaline comet assay in Enchytraeus crypticus. Further, the effect of silver nanomaterial (Ag NM300K, dispersed, 15 nm) was tested and compared with silver nitrate. Hydrogen peroxide was used as a positive control. The various steps were optimized. The fully detailed standard operating procedure is presented. Silver materials caused genotoxicity, this being differentiated for the nano and non-nano forms. Silver nitrate caused genotoxicity after 3 d of exposure in a dose-related manner, although after 7 d the effects were either reduced or repaired. Ag NM300K caused higher genotoxicity after 7 d for the lowest concentration, highlighting a potential nonmonotonic dose-response effect. Overall, the comet assay showed the power to discriminate effects between materials and also toxicity at low relevant doses. Environ Toxicol Chem 2018;37:184-191. © 2017 SETAC.

Metals(loids) targeting fish eyes and brain in a contaminated estuary - Uncovering neurosensory (un)susceptibility through bioaccumulation, antioxidant and morphometric profiles.

This study examined the susceptibility of fish (Liza aurata) eyes and brain to metals(loids) contamination under realistic exposure conditions. A multidimensional approach was applied to fish caught at a chronically contaminated site (BAR) and at a reference site of the Tagus estuary (Portugal), which comprised metals(loids) accumulation in eyes and brain together with a battery of enzymatic and non-enzymatic antioxidants, as well as brain morphometry (i.e. cell density). Trace element levels in the blood, gills, liver and kidney allowed interpretations on their preferential pathway(s) to the eyes and brain. Metals(loids) accumulation pointed out the elevated vulnerability of the fish eyes at BAR, probably related with the direct waterborne uptake. Pb uptake in L. aurata eyes could be associated both with water and indirect pathways. At the most contaminated site, metals(loids) were on the basis of pro-oxidant conditions in the ocular tissues, while no indication of toxicity was recorded in the brain. Overall, the results disclosed a differential bioaccumulation among fish organs, suggesting that, in the L. aurata population studied, metal organotropism underlie the lower susceptibility of the brain comparing to the eyes. However, mechanisms remain little understood and further work is needed.

Insights into neurosensory toxicity of mercury in fish eyes stemming from tissue burdens, oxidative stress and synaptic transmission profiles.

This study aims to contribute to fill a knowledge gap related with Hg effects in fish eyes. As a pioneering strategy, Hg bioaccumulation in eye wall of the wild grey mullet (Liza aurata) was assessed, together with oxidative stress and synaptic transmission profiles. This approach was complemented by the characterisation of environmental contamination (both in water and sediment). Sampling was conducted in winter and summer in two sites of a Portuguese coastal lagoon (Aveiro lagoon): Largo do Laranjo (LAR) - located in an Hg contaminated/confined area; São Jacinto (SJ) - closer to the lagoon inlet and selected as reference site. Levels of total Hg (tHg), inorganic Hg (iHg) and methylmercury (MeHg) in eye wall were higher at LAR than SJ, both in winter and summer, reflecting the environmental contamination patterns. Moreover, fish caught at LAR in winter showed a significant decrease of catalase and superoxide dismutase activities, in line with the occurrence of peroxidative damage. A different spatial pattern was recorded in summer, being characterised by the increment of glutathione peroxidase and glutathione reductase activities at LAR, as well as total glutathione content, preventing the occurrence of lipid peroxidation. Also in summer, a significant decrease of acetylcholinesterase activity was recorded in fish eyes at LAR, pointed out Hg as an anticholinergic agent. Besides Hg, water salinity had probably an indirect effect on spatial and winter-summer variation patterns of AChE. Current data pointed out that Hg (in iHg and MeHg forms) could exert ocular toxicity both by the promotion of oxidative stress and by the interference with neurotransmission processes.