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Carbamazepine is a psychiatric pharmaceutical widely detected in aquatic environments. Due to its generalized occurrence and environmental persistence it might be considered as an anthropogenic pollution indicator. In this research, a previously developed enzyme-linked immunosorbent assay (ELISA), based on a commercial monoclonal antibody, was applied to the quantification of carbamazepine in ground, surface and wastewaters and results were validated by liquid chromatographytandem mass spectrometry (LCMS/MS).
The performance of the applied ELISA methodology was tested in the presence of high concentrations of sodium chloride and dissolved organic matter. The method was not significantly affected by matrix effects, being adequate for the quantification of carbamazepine in environmental samples, even without sample pre-treatment. This method allows the quantification of carbamazepine in the range of 0.0310 µg L-1, with a relative error lower than 30%. Due to a pH dependent cross-reactivity with cetirizine, an antihistaminic drug, the assay also enabled the quantification of cetirizine in the samples.
The application of the developed method to the quantification of carbamazepine was performed by using environmental samples with very different matrices, collected in the geographical area of Ria de Aveiro, an estuarine system located in the North of Portugal. Carbamazepine was detected in all analyzed wastewater samples and in one surface water with concentrations between 0.1 and 0.7 µg L-1. Validation with LCMS/MS revealed that results obtained by ELISA are 228% overestimated, which was considered highly satisfactory due to the absence of sample pre-treatments.
Caffeine is known to be one of the most consumed psychoactive drugs. For this reason, caffeine is continuously released into the environment with potential impacts on inhabiting organisms. The current study evaluated the biochemical alterations induced in the clam species Ruditapes philippinarum after exposure for 28 days to caffeine (0.5, 3.0 and 18.0 mg/L). The results obtained showed that, with the increasing caffeine concentrations, an increase in clams defense mechanisms (such as antioxidant and biotransformation enzymes activity) was induced which was accompanied by an increase in protein content. Nevertheless, although an increase on defense mechanisms was observed, clams were not able to prevent cells from lipid peroxidation that increased with the increase of caffeine concentration. Furthermore, with the increase of exposure concentrations, clams increased their metabolic activity (measured by electron transport activity), reducing their energy reserves (glycogen content), to fight against oxidative stress. Overall, the present study demonstrated that caffeine may impact bivalves, even at environmentally relevant concentrations, inducing oxidative stress in organisms. The present study is an important contribution to address knowledge gaps regarding the impacts of long-term exposures to pharmaceuticals since most of the studies assessed the effects after acute exposures, most of them up to 96 h.
The negative effects induced in marine organisms by Climate Change related abiotic factors consequences, namely ocean warming, are well-known. However, few works studied the combined impacts of ocean warming and contaminants, as pharmaceutical drugs. Carbamazepine (CBZ) and cetirizine (CTZ) occur in the marine environment, showing negative effects in marine organisms. This study aimed to evaluate the impacts of Ocean warming on the effects of CBZ and CTZ, when acting individually and combined (drug vs drug), in the edible clam Ruditapes philippinarum. For that, drugs concentration, bioconcentration factors and biochemical parameters, related with clam’s metabolic capacity and oxidative stress, were evaluated after 28 days exposure to environmentally relevant scenarios of these stressors. The results showed limited impacts of the drugs (single and combined) at control and warming condition. Indeed, it appeared that warming improved the oxidative status of contaminated clams (higher reduced to oxidized glutathione ratio, lower lipid peroxidation and Protein carbonylation levels), especially when both drugs were combined. This may result from clam’s defence mechanisms activation and reduced metabolic capacity that, respectively, increased elimination and limited production of reactive oxygen species. At low stress levels, defence mechanisms were not activated which resulted into oxidative stress. The present findings highlighted that under higher stress levels clams may be able to activate defence strategies that were sufficient to avoid cellular damages and loss of redox homeostasis.
Nevertheless, low concentrations were tested in the present study and the observed responses may greatly Change under increased pollution levels or temperatures. Further research on this topic is needed since marine heat waves are increasing in frequency and intensity and pollution levels of some pharmaceuticals are also increasing in coastal systems.
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 28 days exposure to environmentally relevant CTZ concentrations (0.0, 0.3, 3.0, 6.0 and 12.0 mu g/L). The results obtained showed that CTZ was accumulated by clams reaching maximum concentrations (up to similar to 22 ng/g FW) at the highest CTZ exposure concentrations (6.0 and 12.0 mu g/L). The bioconcentration factor (average maximum values of similar to 5) decreased at 12.0 mu g/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 12.0 mu g/L) a significant increase of the antioxidant enzyme superoxide activity (similar to 53 and similar to 44%) was observed in clams exposed to CTZ. Moreover, a tendency to increase lipid peroxidation (similar to 14 and similar to 9%) and carbonyl groups on proteins (similar to 11 and similar to 3%) was observed in clams exposed to CTZ (6.0 and 12.0 mu g/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.
Several works evaluated the toxicity of pharmaceutical drugs and climate related changes in invertebrates but few explored the combined effects of both stressors, namely considering their mode of action (MoA). Carbamazepine (CBZ) and cetirizine (CTZ) are pharmaceutical drugs detected in the environment and the toxicity derived from the combined effects of these drugs with ocean acidification (OA) is poorly explored. Thus, the present study investigated the biochemical parameters related to an oxidative stress response and the transcription of genes related to the MoA of CBZ (1.0 mg/L) and CTZ (0.6 mg/L) in the clam Ruditapes philippinarum chronically exposed (28 days) to control (7.8) and low (7.5) pH conditions. The results obtained showed that despite the clams accumulated both drugs, at low pH the clams exposed to CTZ decreased drug concentration and BCF values (CTZ uptake: 2.0 ± 0.5 ng/g fresh weight; BCF: 3.8 ± 0.9) in comparison with clams exposed to control pH (CTZ uptake: 2.9 ± 0.3 ng/g fresh weight; BCF: 5.5 ± 0.6). No oxidative stress was induced by the exposure to CBZ or CTZ at each pH level, but the transcription of several genes related with the MoA (neurotransmission, immunity and biomineralization) was altered by low pH, drug exposure and the combination of both stressors. At both pH conditions, CBZ increased the transcription of GABA receptor gene (neurotransmission) and CTZ led to a decrease of Perlucin gene (biomineralization) transcription. The transcription of MyD88 gene (immunity) decreased at low pH (7.5) combined with drug exposure (CBZ or CTZ). Thus, it was highlighted that the interaction of drug exposure and low pH conditions can change bivalves’ sensitivity to drugs or alter drugs toxicity.
In the aquatic environment, organisms are exposed to complex mixtures of contaminants which may alter the toxicity profile of each compound, compared to its toxicity alone. Pharmaceutical drugs (e.g. carbamazepine (CBZ) and cetirizine (CTZ)) and metals (e.g. cadmium (Cd)) are among those contaminants that co-occur in the environment. However, most studies concerning their toxicity towards aquatic species are based on single exposure experiments. Thus, the present study aimed to evaluate single and combined effects of Cd and CBZ or CTZ (single conditions: Cd, CTZ, CBZ; combined conditions: CTZ+Cd, CBZ+Cd) on biomarkers related to oxidative stress and energy metabolism in the edible clam Ruditapes philippinarum, by exposing the organisms for 28 days to environmentally relevant concentrations of these contaminants. The biomarkers studied were: i) the electron transport system activity, protein and glycogen contents (indicators of organisms’ metabolic status and energy reserves); ii) lipid peroxidation and the ratio between reduced and oxidized glutathione (indicators of oxidative stress); iii) superoxide dismutase and catalase activities (enzymes indicators of antioxidant defence) and iv) activity of glutathione S-transferases (family of enzymes indicators of biotransformation capacity). Results obtained showed that the uptake of Cd and CBZ was not affected by the combined presence of the contaminants.
However, for CTZ, the uptake was higher in the presence than in the absence of Cd. Concerning toxicity data, in general, the combined exposures (CTZ+Cd, CBZ+Cd) had lower biological effects than the contaminants alone. Nevertheless, our data showed that despite the low concentrations tested, they were enough to exert biological effects that differed between single and combined treatments, evidencing the need to conduct more co-exposure studies to increase the environmental relevance of the gathered data.
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 9.0 μg/L) and the stimulant caffeine (0.0 - control, 0.5, 3.0, and 18.0 μg/L), acting alone and in combination (0.3 CBZ + 0.5 CAF and 6.0 CBZ + 3.0 CAF). Glutathione Stransferases (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 ETSwhen exposed to drugs, avoiding energy expenditurewhich may prevent them fromgreater damages. The present study further revealed that the impacts induced by the combination of both drugswere similar to those obtained at the highest drugs concentrations acting alone.
In the present study, the bivalve Scrobicularia plana, collected from two contrasting areas (pristine location and mercury contaminated area), was selected to assess the biochemical alterations imposed by pH decrease, carbamazepine (an antiepileptic) and the combined effect of both stressors. The effects on oxidative stress related biomarkers after 96 h exposure revealed that pH decrease and carbamazepine induced alterations on clams, with greater impacts on individuals from the contaminated area which presented higher mortality, higher lipid peroxidation and higher glutathione S-transferase activity. These results emphasize the risk of extrapolating results from one area to another, since the same species inhabiting different areas may be affected differently when exposed to the same stressors. Furthermore, the results obtained showed that, when combined, the impact of pH decrease and carbamazepine was lower than each stressor acting alone, which could be related to the defence mechanism of valves closure when bivalves are under higher stressful conditions.
In the last decade studies have reported the presence of several pharmaceutical drugs in aquatic environments worldwide and an increasing effort has been done to understand the impacts induced on wildlife. Among the most abundant drugs in the environment is caffeine, which has been reported as an effective chemical anthropogenic marker. However, as for the majority of pharmaceuticals, scarce information is available on the adverse effects of caffeine on marine benthic organisms, namely polychaetes which are the most abundant group of organisms in several aquatic ecossystems. Thus, the present study aimed to evaluate the biochemical alterations induced by environmentally relevant concentrations of caffeine on the polychaete species Diopatra neapolitana and Arenicola marina. The results obtained demonstrated that after 28 days exposure oxidative stress was induced in both species, especially noticed in A. marina, resulting from the incapacity of antioxidant and biotransformation enzymes to prevent cells from lipid peroxidation. The present study further revealed that D. neapolitana used glycogen and proteins as energy to develop defense mechanisms while in A. marina these reserves were maintained independently on the exposure concentration, reinforcing the low capacity of this species to fight against oxidative stress.
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 mg/L) and caffeine (Ctl-0.0; 0.5; 3.0; 18.0 mg/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.