Ângela Almeida

Presence of the pharmaceutical drug carbamazepine in coastal systems: effects on bivalves.

Carbamazepine (CBZ), an antiepileptic drug, is one of the most commonly detected pharmaceutical drugs in aquatic ecosystems, and is used as a marker of urban pollution. Since CBZ is designed to exert a biological effect, when it reaches aquatic environment high probability exist for toxic effects on non-target organisms. The present study evaluated the acute toxicity of environmentally relevant concentrations of CBZ (0.00, 0.03, 0.30, 3.00, 9.00μg/L) in the edible clams Venerupis decussata (a native species) and Venerupis philippinarum (an invasive species) collected from the Ria de Aveiro. The effects on both species were assessed through the use of a battery of biomarkers mainly related with health status and oxidative stress. Furthermore, in this work an alternative and promising tool, the direct competitive immunoassay ELISA, for the direct CBZ quantification in clams tissues, was applied. The results of the present work showed that CBZ in clams tissues increased with the exposure concentration and V. decussata gave slightly higher values than V. philippinarum. Although the clams accumulated lower levels of CBZ than the concentration of exposure, these concentrations were enough to impair the health status and induce oxidative stress. However, a different response to CBZ was observed in the two species. While in V. philippinarum the lipid peroxidation levels increased at the highest CBZ concentration (9.00μg/L), in V. decussata a significant decrease was seen. Moreover, glutathionse S-transferase activity was stimulated in V. decussata and decreased in V. philippinarum. Nevertheless, an induction of glutathione reductase, superoxide dismutase and cytochrome P450 3A4 activities was found in both species as a result of the exposure. The results indicate that, probably, V. philippinarum have a less efficient antioxidant system than V. decussata, and are therefore less capable to neutralize oxidative stress and consequently more sensitive to CBZ. The risk quotient determined for the Ria de Aveiro was higher than 1 indicating that a ecotoxicological risk is suspected. Furthermore, bioaccumulation of CBZ in clams should be taken into consideration since this chemical might be transferred along the food chain and affect non-target organisms.

The effects of carbamazepine on macroinvertebrate species: Comparing bivalves and polychaetes biochemical responses.

In the present study, the bivalve Scrobicularia plana and the polychaete Diopatra neapolitana were exposed to an increasing carbamazepine (CBZ) concentration gradient. Both species are among the most widely used bioindicators, and CBZ is one of the most commonly found drugs in the aquatic environment. After a chronic exposure (28 days), the results obtained revealed that CBZ induced biochemical alterations in both species. Our findings demonstrated that S. plana and D. neapolitana reduced the CBZ accumulation rate at higher CBZ concentrations, probably due to their capacity to decrease their feeding rates at stressful conditions. Nevertheless, this defence mechanism was not enough to prevent both species from oxidative stress. In fact, S. plana and D. neapolitana were not able to efficiently activate their antioxidant defence mechanisms which resulted in the increase of lipid peroxidation, especially at the highest CBZ concentrations. Comparing both species, it seems that S. plana was the most sensitive species since stronger biochemical alterations were observed in this species.

The impacts of pharmaceutical drugs under ocean acidification: New data on single and combined long-term effects of carbamazepine on Scrobicularia plana

Ocean acidification and increasing discharges of pharmaceutical contaminants into aquatic systems are among key and/or emerging drivers of environmental change affecting marine ecosystems. A growing body of evidence demonstrates that ocean acidification can have direct and indirect impacts on marine organisms although combined effects with other stressors, namely with pharmaceuticals, have received very little attention to date. The present study aimed to evaluate the impacts of the pharmaceutical drug Carbamazepine and pH 7.1, acting alone and in combination, on the clam Scrobicularia plana. For this, a long-term exposure (28 days)was conducted and a set of oxidative stress markers was investigated. The results obtained showed that S. plana was able to develop mechanisms to prevent oxidative damage when under low pH for a long period, presenting higher survival when exposed to this stressor compared to CBZ or the combination of CBZ with pH 7.1. Furthermore, the toxicity of CBZ on S. plana was synergistically increased under ocean acidification conditions (CBZ + pH 7.1): specimens survival was reduced and oxidative stress was enhanced when compared to single exposures. These findings add to the growing body of evidence that ocean acidification will act to increase the toxicity of CBZ to marine organisms,which has clear implications for coastal benthic ecosystems suffering chronic pollution from pharmaceutical drugs.

Long-term exposure to caffeine and carbamazepine: Impacts on the regenerative capacity of the polychaete Diopatra neapolitana.

The toxicity induced in non-target organisms by pharmaceutical drugs has been the focus of several studies. In the aquatic environment, most of the studies have been devoted to fish and bivalves, while little is known on the impacts induced in polychaetes. The present study evaluated the impacts of carbamazepine and caffeine on the regenerative capacity of Diopatra neapolitana, a polychaete species with high ecological and economic relevance. Under laboratory controlled conditions polychaetes were exposed, during 28 days, to carbamazepine (Ctl-0.0; 0.3; 3.0; 6.0; 9.0 μg/L) and caffeine (Ctl-0.0; 0.5; 3.0; 18.0 μg/L). During the experiment, at days 11, 18, 25, 32, 39 and 46 after amputation, for each specimen, the percentage of the body width regenerated was determined and the number of new segments was counted. The regenerative capacity was assessed considering the number of days needed to achieve full regeneration and the total number of new segments. The obtained results revealed that with the increase of drugs concentrations organisms regenerated less new segments and took longer to completely regenerate.

The effects of salinity changes on the Polychaete Diopatra neapolitana: Impacts on regenerative capacity and biochemical markers

Polychaetes have been identified by several authors as a group of marine invertebrates that respond rapidly to anthropogenic stressors. However, studies investigating alterations in Polychaetes affected by climate changes are scarce. Thus, the present study aimed to assess the impact of salinity changes (14, 21, 28, 35, 42g/L) on the physiological and biochemical performance of the Polychaete Diopatra neapolitana, evaluating the species regenerative ability and biochemical alterations. The results obtained demonstrated that organisms exposed to extreme salinity conditions (14, 21 and 42g/L) presented higher mortality rates, needed more days to completely regenerate the missing body region and also regenerated less chaetigers, when compared to organisms exposed to salinities 28 and 35g/L. The present study further demonstrated that D. neapolitana presented significantly lower glycogen and protein content at salinities 21 and 42g/L, which can be explained by higher energy expenditure in the physiological and biochemical processes. A marked impairment of the glutathione redox status was also recorded at salinities 21 and 42g/L. Increased antioxidant enzyme activities were observed at salinity 21g/L while LPO levels were increased at salinity 42g/L. Overall the present study demonstrated that the regenerative capacity of D. neapolitana can be used as a tool to assess environmental changes, namely salinity shifts. Moreover, stress related biomarkers revealed to be useful to evaluate the alterations in Polychaetes due to salinity changes. D. neapolitana revealed to be a good bioindicator to salinity alterations.

Biochemical impacts of Hg in Mytilus galloprovincialis under present and predicted warming scenarios

The interest in the consequences of climate change on the physiological and biochemical functioning of marine organisms is increasing, but the indirect and interactive effects resulting from warming on bioconcentration and responsiveness to pollutants are still poorly explored, particularly in terms of cellular responses. The present study investigated the impacts of Hg in Mytilus galloprovincialis under control (17°C) and warming (21°C) conditions, assessing mussels Hg bioconcentration capacity, metabolic and oxidative status after 14 and 28days of exposure. Results obtained showed greater impacts in mussels exposed for 28days in comparison to 14days of exposure. Furthermore, our findings revealed that the increase in temperature from 17 to 21°C reduced the bioconcentration of Hg by M. galloprovincialis, which may explain higher mortality rates at 17°C in comparison to 21°C. Lower Hg concentration at 21°C in mussels tissue may result from valves closure for longer periods, identified by reduced energy reserves consumption at higher temperature, which in turn might also contributed to higher oxidative stress in organisms exposed to this condition. The highest LPO levels observed in mussels exposed to higher temperatures alone indicate that warming conditions will greatly affect M. galloprovincialis. Furthermore, the present study showed that the impacts induced by the combination of Hg and warming were similar to the ones caused by increased temperature acting alone, mainly due to increased antioxidant defenses in organisms under combined effects of Hg and warming, suggesting that warming was the factor that mostly contributed to oxidative stress in mussels. Although higher mortality was observed in individuals exposed to 17°C and Hg compared to organisms exposed to Hg at 21°C, the oxidative stress induced at higher temperature may generate negative consequences on mussels reproductive and feeding capacity, growth and, consequently, on population maintenance and dynamics.

Comparison of the toxicological impacts of carbamazepine and a mixture of its photodegradation products in Scrobicularia plana.

In the aquatic environment, pharmaceutical drugs are submitted to degradation processes, where photodegradation is one of the most important mechanisms affecting the fate, persistence and toxicity of the compounds. Carbamazepine, a widely used antiepileptic, is known to suffer photodegradation in water bodies and generate photoproducts, some of them with higher potential toxicity than the parent compound. Therefore, to evaluate the toxic effects of CBZ when combined with its photoproducts, an acute exposure (96h) with the edible clam Scrobicularia plana was performed using environmental concentrations of CBZ (0.00-9.00μg/L) irradiated (and non-irradiated) with simulated solar radiation. The analysis of the irradiated CBZ solutions by mass spectrometry revealed the formation of 5 photoproducts, including acridine (a compound known to be carcinogenic). Oxidative stress results showed that the exposure to CBZ photoproducts did not increase the toxicity to clams, by comparison with the parent compound. Lipid peroxidation levels, catalase and superoxide dismutase activities were the most responsive parameters to these stressors and lipid peroxidation results appeared to show the presence of an antagonistic effect resulting from the mixture of CBZ and its photoproducts.

Hediste diversicolor as bioindicator of pharmaceutical pollution: Results from single and combined exposure to carbamazepine and caffeine

Several environmental stressors have been identified as key and/or emerging drivers of habitat change that could significantly influence marine near-shore ecosystems. These include increasing discharges of pharmaceutical contaminants into the aquatic coastal systems. Pharmaceutical drugs are often detected in aquatic environments but still information on their toxicity impacts on inhabiting species is scarce, especially when acting in combination. Furthermore, almost no information is available on the impacts of pharmaceuticals in polychaetes, often the most abundant taxon in benthic communities and commonly used as indicator species of environmental conditions. Therefore, the present study aimed to evaluate the biochemical alterations induced in the polychaete Hediste diversicolor, from a low contaminated area at the Ria de Aveiro lagoon (Portugal), by the antiepileptic drug carbamazepine (0.0 - control, 0.3, 3.0, 6.0 and 9.0μg/L) and the stimulant caffeine (0.0 - control, 0.5, 3.0, and 18.0μg/L), acting alone and in combination (0.3 CBZ+0.5 CAF and 6.0 CBZ+3.0 CAF). Glutathione S-transferases (GSTs), superoxide dismutase (SOD) and catalase (CAT) activities was determined in Hediste diversicolor from each condition. Lipid peroxidation (LPO), glutathione reduced and oxidized (GSH and GSSG), glycogen and electron transport system (ETS) were also measured. The results obtained clearly revealed that both drugs induced oxidative stress in H. diversicolor, shown by the increase on LPO levels and decrease on total glutathione and GSH/GSSG ratio with the increase of exposure concentrations. Furthermore, the present findings demonstrated that polychaetes biotransformation capacity as well as antioxidant defense mechanisms were not sufficiently efficient to fight against the excess of reactive oxygen species (ROS) leading to LPO when organisms were exposed to both drugs. Our results also demonstrated that polychaetes tended to decrease the activity of ETS when exposed to drugs, avoiding energy expenditure which may prevent them from greater damages. The present study further revealed that the impacts induced by the combination of both drugs were similar to those obtained at the highest drugs concentrations acting alone.

Ecotoxicity of the antihistaminic drug cetirizine to Ruditapes philippinarum clams

Cetirizine (CTZ) is an antihistaminic drug present in the aquatic environment, with limited information on its toxicity to organisms inhabiting this system. This study intended to evaluate the effects of CTZ on oxidative stress and energy metabolism biomarkers in the edible clam Ruditapes philippinarum after a 28days exposure to environmentally relevant CTZ concentrations (0.0, 0.3, 3.0, 6.0 and 12.0μg/L). The results obtained showed that CTZ was accumulated by clams reaching maximum concentrations (up to ~22ng/g FW) at the highest CTZ exposure concentrations (6.0 and 12.0μg/L). The bioconcentration factor (average maximum values of ~5) decreased at 12.0μg/L reflecting a reduction in clams uptake or increase of excretion capacity at this condition. The present study revealed that, in general, clams decreased the metabolic potential after exposure to CTZ (decrease in electron transport system activity), a response that led to the maintenance of glycogen content in organisms exposed to CTZ in comparison to control values. Our findings also showed that, CTZ did not exert significant levels of oxidative injury to clams. However, comparing the control with the highest exposure concentrations (6.0 and 12.0μg/L) a significant increase of the antioxidant enzyme superoxide activity (~53 and ~44%) was observed in clams exposed to CTZ. Moreover, a tendency to increase lipid peroxidation (~14 and ~9%) and carbonyl groups on proteins (~11 and ~3%) was observed in clams exposed to CTZ (6.0 and 12.0μg/L) compared to control condition. Overall the present study suggests that toxic impacts may be induced in R. philippinarum if exposed for longer periods or higher CTZ concentrations.

Toxicity associated to uptake and depuration of carbamazepine in the clam Scrobicularia plana under a chronic exposure

Carbamazepine (CBZ) is an antiepileptic drug commonly detected in aquatic systems, with toxic effects to inhabiting organisms. Limited information is known on stress response biomarkers associated to bioconcentration and depuration of CBZ in aquatic organisms. Moreover, few studies addressed if the response and recovery of organisms to a contaminant can change when they are collected in a contaminated site. This study intended to understand the bioconcentration and depuration of CBZ combined with its toxicological impact in Scrobicularia plana clams collected from two contrasting areas (MIRA, Mira channel, non-contaminated and LAR, Laranjo bay, anthropogenically impacted) from the Ria de Aveiro (Portugal). The clams were exposed for 14days to environmentally relevant CBZ concentrations (0.0, 4.0 and 8.0μg/L), followed by a 14day depuration period. CBZ concentrations in S. plana tissues were rapidly bioconcentrated during the exposure period. In the depuration period CBZ was eliminated, in some extent. The main toxic effects occurred at the highest concentration (8.0μg/L) after 14days of exposure in which the clams from LAR accumulated a higher CBZ concentration (LAR: ~10ng/g FW) than clams from MIRA (MIRA: ~7ng/g FW). LAR clams exhibited higher oxidative damage at this concentration, demonstrated by higher LPO levels over time (increase of ~1.4% relative to control) and, in comparison with MIRA clams (LAR: 17.7nmol/g FW; MIRA: 11.4nmol/g FW). After the depuration period, LAR clams recovered from the stress induced by CBZ. A decrease in LPO for LAR (decrease of ~40% in relation to the end of the exposure period) was accompanied by a decrease in CBZ tissue concentrations (decrease of ~61% relative to the end of the exposure period). MIRA clams were not oxidatively injured (low LPO levels remained unchanged after the depuration and CBZ decreased ~80% relative to the end of the exposure period).