Multi- and transgenerational effects of environmental chemicals on mollusks: An underexplored experimental design in aquatic (eco)toxicological studies.

(Eco)toxicological studies frequently evaluate the effects of chemicals in one life stage of organisms, but the use of these outcomes can only partially estimate populational effects. In this regard, multi- and/or transgenerational studies should be performed in order to provide information on contaminant effects in a populational functioning context. The present review aimed to summarize and critically evaluate the current knowledge regarding multi- and/or transgenerational effects of traditional and emerging environmental chemicals on mollusks. Results showed that these kinds of studies were performed in aquatic mollusks (bivalve and gastropod), being Gastropoda the mollusk Class most frequently studied. Additionally, freshwater species and multigenerational studies were more common for this class. For the Bivalvia class, only marine species were evaluated, and transgenerational exposure was more commonly assessed. The effects were reported for 15 species, highlighting the marine bivalves Crassostrea gigas and Saccostrea glomerata, and the freshwater gastropod Lymnaea stagnalis. Multi- and transgenerational effects were described for 8 environmental chemical groups, mainly metals, pesticides, and pharmaceuticals. In general, multi- and transgenerational exposure induced biometric, developmental, and reproductive impairments in mollusks, indicating that environmental chemicals might lead to generational impairments, reduced population growth and reproductive capacity, and decreased fitness. The current study indicated that bivalves and gastropods are suitable organism models to assess the multi- and transgenerational adverse effects induced by traditional and emerging environmental chemicals.

Mollusc shell shape as pollution biomarkers: Which is the best biological model?

Alterations in mollusc shells have been proposed contamination biomarkers. We used geometric morphometrics analyses associated with analytical determinations of contaminants to select suitable biological models among species widely distributed on coastal zones. The study was carried out using Lottia subrugosa (herbivore limpet), Crassostrea brasiliana (filter-feeder bivalve), and Stramonita brasiliensis (carnivore gastropod) obtained along a marked contamination gradient at Santos Estuarine System (Brazil). L. subrugosa and S. brasiliensis presented distinct shapes along the gradient, while no significant differences in shell form were seen for C. brasiliana. Indeed, limpets and snails presented morphometric parameters consistent with measured contamination levels hazardous substances. Based on cross-validation models, the reliability of morphometric responses was over 75% for the herbivore and carnivore species. In addition, for S. brasiliensis, a 95.2% confidence was detected in most contaminated sites. Therefore, shell alterations on carnivorous gastropods should be further investigated, seeking to be effectively employed as pollution biomarkers.

Contamination gradient affects differently carbonic anhydrase activity of mollusks depending on their feeding habits.

Aquatic organisms that inhabit coastal areas are often exposed to several contaminants. It is known that the bioaccumulation of contaminants can be amplified according to the species feeding habits and contaminant properties. As a consequence, species can experience different effects to contaminant exposure even if they inhabit the same area. The present study aimed to investigate the activities of carbonic anhydrase (CA), Ca2+-ATPase, and Mg2+-ATPase in different tissues (soft tissue, mantle, and gill) of three mollusk species (Lottia subrugosa, Stramonita brasiliensis, and Crassostrea brasiliana) with different feeding habits (herbivore, carnivore, and filter-feeder, respectively) which were sampled within a known contamination gradient at Santos Estuarine System (Southeastern Brazil). From the three enzymes tested, only CA was affected by the presence of contaminants within the contamination gradient evaluated. In general, the CA activity from the three species were lower in contaminated sites when compared to the reference site. The contrasting CA activity response observed in S. brasiliensis compared to L. subrugosa and C. brasiliana could be related to the tissue-specificity of this enzyme activity and species feeding habits (filter-feeders can accumulate more contaminants than herbivores and even carnivores). Results indicated that C. brasiliana mantle is the most suitable tissue for the use of CA analysis as a biomarker.

Shell form and enzymatic alterations in Lottia subrugosa (Gastropoda, Lotiidae) transplanted to a contaminated site.

Studies have shown that shell morphology and enzymatic activities in mollusks are affected by contaminants exposure. However, the correlation between enzymatic activities and the biomineralization process are not fully understood. The present study used a transplant bioassay and field sampling to evaluate shell measurements and the activities of carbonic anhydrase, Ca2+-ATPase, and Mg2+-ATPase in Lottia subrugosa sampled in Brazilian sites under different contamination levels. Results showed that, in general, shells from the reference site (Palmas) were more rounded than the ones from the contaminated site (Balsa). Effects in enzymatic activities in specimens from transplant bioassay were attributed to the known high contaminant levels present at Balsa. While the lack of enzymatic activity alterations during field sampling was attributed to physiological adaptation to contaminants exposure. Enzymatic activities were not correlated to shell biometric parameters in field sampling, indicating that these enzymes were not related to shell alterations detected in the present study.

Mollusk shell alterations resulting from coastal contamination and other environmental factors.

Effects of contamination on aquatic organisms have been investigated and employed as biomarkers in environmental quality assessment for years. A commonly referenced aquatic organism, mollusks represent a group of major interest in toxicological studies. Both gastropods and bivalves have external mineral shells that protects their soft tissue from predation and desiccation. These structures are composed of an organic matrix and an inorganic matrix, both of which are affected by environmental changes, including exposure to hazardous chemicals. This literature review evaluates studies that propose mollusk shell alterations as biomarkers of aquatic system quality. The studies included herein show that changes to natural variables such as salinity, temperature, food availability, hydrodynamics, desiccation, predatory pressure, and substrate type may influence the form, structure, and composition of mollusk shells. However, in the spatial and temporal studies performed in coastal waters around the world, shells of organisms sampled from multi-impacted areas were found to differ in the form and composition of both organic and inorganic matrices relative to shells from less contaminated areas. Though these findings are useful, the toxicological studies were often performed in the field and were not able to attribute shell alterations to a specific molecule. It is known that the organic matrix of shells regulates the biomineralization process; proteomic analyses of shells may therefore elucidate how different contaminants affect shell biomineralization. Further research using approaches that allow a clearer distinction between shell alterations caused by natural variations and those caused by anthropogenic influence, as well as studies to identify which molecule is responsible for such alterations or to determine the ecological implications of shell alterations, are needed before any responses can be applied universally.

Historical shell form variation in Lottia subrugosa from southeast Brazilian coast: Possible responses to anthropogenic pressures.

Mollusk shells can provide important information regarding environmental parameters. It is known that shell morphology is affected by both natural and anthropogenic factors. However, few studies have investigated alterations in shell morphology over a historical perspective and considering chemical contamination and climate changes. The present study assessed shell form (shape and size) variations of limpet (Lottia subrugosa) shells sampled from 1950 to 1981 (past) in comparison with organisms obtained in 2018 (present). Differences between shells from the past and present (2018) were detected, being shell weight and height the two most important affected parameters. The differences observed were attributed to the possible increase in contamination over the years due to human population growth and to climate change. Additionally, when shells from the past were evaluated according to the decade they were sampled, results indicate that it was necessary an interval of 40 years to shell form be altered within populations.

Behavioural and brain biomarker responses in yellowfin bream (Acanthopagrus australis) after inorganic mercury ingestion.

To assess the effects of inorganic mercury ingestion on fish general behaviour and brain biochemical markers (acetylcholinesterase - AChE; lipid peroxidation - LPO; glutathione S-transferase - GST; catalase - CAT), juvenile yellowfin bream (Acanthopagrus australis) were fed mercury dosed food (low = 0.7 mg kg-1, medium = 2.4 mg kg-1 and high = 6 mg kg-1) or undosed food (control = 0.2 mg kg-1) for up to 16 days (5 replicates per treatment). Behavioural indicators, LPO levels and GST activity significantly changed overtime, but were not affected by mercury concentration. In contrast, CAT activity was higher in exposed fish in comparison to controls after 4 days, but not after 8 and 16 days. An additional depuration treatment was evaluated and fish from this treatment were less active and had significantly lower LPO levels and CAT activity than fish constantly exposed to the medium treatment. Overall, results from the present study indicate that a diet containing inorganic mercury impaired yellowfin bream growth and initially affected fish brain CAT activity, however fish were able to recover even with continuous exposure.

Ingestion of inorganic mercury by juvenile black tiger prawns (Penaeus monodon) alters biochemical markers.

There is a lack of information regarding the effects on biochemical markers in invertebrates diet-exposed to inorganic mercury. In the present study, juvenile black tiger prawn (Penaeus monodon) were fed with food dosed with mercuric chloride (low: 0.2 mg kg-1; medium: 0.77 mg kg-1; high: 1.41 mg kg-1; higher: 2.52 mg kg-1) and control (0.03 mg kg-1) over 12 days. At the end of exposure periods, ventral nerve cord, compound eyes and muscle were dissected for biochemical marker analyses (acetylcholinesterase - AChE; lipoperoxidation - LPO; glutathione S-transferase - GST; catalase - CAT). Prawn muscle showed an increase in total mercury concentration over time for low and high treatments, but did not show an accumulation in comparison to controls. AChE activity tended to decrease over time in all tissues. CAT activity increased over time in controls and lower dose treatments but was suppressed in the higher treatment relative to controls on day 12; indicating that inorganic mercury is impacting the normal stress response by reducing the capacity to degrade hydrogen peroxide. In contrast, no effect was observed in LPO and GST activity. A depuration treatment was performed and compared to medium treatment; only AChE and GST activities from muscle showed significant difference, with AChE activity from depuration treatment lower than medium treatment, while the opposite was observed in GST activity. Overall, the low mercury accumulation observed over 12 days of exposure may have limited the biochemical stress responses, which could also have limited the detection of differences in the depuration treatment relative to medium treatment.

Effects of oral exposure to inorganic mercury on the feeding behaviour and biochemical markers in yellowfin bream (Acanthopagrus australis).

Mercury is a known toxic metal, but studies on the effects of inorganic mercury ingestion in aquatic organisms are scarce. The present study aimed to investigate changes in feeding behaviour and biomarkers (lipid peroxidation, acetylcholinesterase, glutathione S-transferase and catalase activities) of yellowfin bream (Acanthopagrus australis) after ingestion of inorganic mercury (control: 0.2 mg kg-1, low: 0.7 mg kg-1, medium: 2.4 mg kg-1 and high: 6 mg kg-1) over 16 days. After 4 days, exposed fish attempted feeding more often, and showed a significantly lower eating success than controls. However, these differences became less notable with longer exposure periods. Most biochemical markers varied over time, regardless of mercury treatment. However, biomarker responses to mercury were also observed, mostly with increased exposure period and were dependant on the tissue analysed. This study showed that fish can recover from initial feeding behaviour effects of inorganic mercury, but showed delayed response in tissue biomarkers.

Behavioural and biochemical alterations in Penaeus monodon post-larvae diet-exposed to inorganic mercury.

Mercury is a metal naturally present in the environment with concentrations in aquatic systems increasing annually due to human activities. This represents a great concern mainly due to its high toxicity to organisms and consequences for human health. Most studies regarding the toxic effect of mercury have focussed on freshwater species using water as the exposure and uptake pathway. In contrast, the present study investigated the effects of dietary exposure of mercury to the marine crustacean Penaeus monodon post-larvae during 96 h to evaluate changes in behaviour (swimming activity and risk taken) and in biochemical biomarkers [acetylcholinesterase (AChE) and glutathione S-transferase (GST)]. Results showed a decrease in swimming activity with an increase in mercury exposure, but no changes were observed regarding the behavioural response 'risk taken'. Prawns from medium (0.56 µg g-1) and high (1.18 µg g-1) treatments had their GST activity reduced in relation to the beginning of experiment (time 0), while AChE activity was increased in the low (0.15 µg g-1) treatment in relation to time 0. In the present study, behaviour analysis were clearer than biochemical biomarkers and results might indicate P. monodon populations from a mercury contaminated environment might be at risk, since the behavioural alterations observed increases the risk of predation.

Toxic effects of the herbicide Roundup in the guppy Poecilia vivipara acclimated to fresh water.

Although it is believed that glyphosate-based herbicides are relatively nontoxic to humans, its broad use in agriculture and consequent contamination of aquatic systems is a concern. In the present study, reproductive (sperm quality) and biochemical parameters (acetylcholinesterase and glutathione S-transferase activity, lipoperoxidation, and antioxidant capacity against peroxyl radicals) were evaluated in adult guppies (Poecilia vivipara) acclimated to fresh water and exposed (96 h) to environmentally realistic concentrations of glyphosate (130 and 700 µg L(-1)) as the commercial formulation Roundup. Male guppies exposed to Roundup showed a poorer sperm quality, measured as reduced plasmatic membrane integrity, mitochondrial functionality, DNA integrity, motility, motility period and concentration of spermatic cells, than those kept under control condition (no Roundup addition to the water). Most of the spermatic parameters analyzed showed strong association to each other, which may help to understand the mechanisms underlying the observed reduction in sperm quality. Exposure to Roundup did not alter the biochemical parameters analyzed, though differences between genders were observed and deserve further investigations. Findings from the present study suggest that exposure to environmentally relevant concentrations of Roundup may negatively affect at long-term the reproduction of P. vivipara, with consequent changes in fish populations inhabiting environments contaminated with the herbicide.