Toxicological effects resulting from co-exposure to nanomaterials and to a ß-blocker pharmaceutical drug in the non-target macrophyte species Lemna minor.

The wide use of carbon-based materials for various purposes leads to their discharge in the aquatic systems, and simultaneous occurrence with other environmental contaminants, such as pharmaceutical drugs. This co-occurrence can adversely affect exposed aquatic organisms. Up to now, few studies have considered the simultaneous toxicity of nanomaterials, and organic contaminants, including pharmaceutical drugs, towards aquatic plants. Thus, this study aimed to assess the toxic effects of the co-exposure of propranolol (PRO), and nanomaterials based on cellulose nanocrystal, and graphene oxide in the aquatic macrophyte Lemna minor. The observed effects included reduction of growth rate in 13% in co-exposure 1 (nanomaterials + PRO 5 µg L-1), and 52-64% in co-exposure 2 (nanomaterials + PRO 51.3 mg L-1), fresh weight reduction of 94-97% in co-exposure 2 compared to control group, and increased pigment production caused by co-exposure treatments. The analysis of PCA showed that co-exposure 1 (nanomaterials + PRO 5 µg L-1) positively affected growth, and fresh weight, and co-exposure 2 positively affected pigments content. The results suggested that the presence of nanomaterials enhanced the overall toxicity of PRO, exerting deleterious effects in the freshwater plant L. minor, suggesting that this higher toxicity resulting from co-exposure was a consequence of the interaction between nanomaterials and PRO.

Evaluation of parental and transgenerational effects of clotrimazole in Daphnia magna - A multi-parametric approach.

Azole antifungals inhibit the cytochrome P450 complex, decreasing the production of ergosterol in fungi, and compromising the biosynthesis of ecdysteroids in crustaceans, which are hormones regulating reproduction and ecdysis. The azole antifungal clotrimazole (CLO) raises environmental concerns due its toxicity. This work evaluated the effects on the number of moults, feeding rate, growth, reproduction, transgenerational reproductive effects on two different generations (F0, parental generation; and F1, organisms born from F0), and energetic balance in Daphnia magna. Neonates (<24 h) were exposed to sublethal concentrations (0, 2.7, and 3.4 mg/L) of CLO, to assess its effects on the moulting process. Neonates were also exposed to environmentally realistic concentrations of CLO (0, 30, 150, 750, and 3750 ng/L) for 24 and 96 h, to assess adverse effects on their feeding behaviour. Effects on energy reserves (fatty acids, glycogen, and protein levels) were also measured in animals exposed to CLO. A reproduction test was carried out to evaluate the amount and size of neonates from F0 and F1 generations. CLO exposure decreased the number of moults, and the size of organisms, but did not alter the feeding pattern of 5 days old individuals. However, neonates (<24 h) exposed to CLO had a significant decrease in their feeding pattern. CLO decreased the fatty acids content in exposed animals, but did not change glycogen and protein. CLO also decreased the size of adult daphnids from the third brood, born from animals exposed in F0; in F1 animals, the size of neonates from the third brood was decreased. This study evidenced the toxic effects caused by CLO on growth, feeding and reproduction of D. magna. Nevertheless, it is not possible to conclude whether the effects are due to the inhibition of cytochrome P450 enzymes, or to unspecific effects caused by general toxic stress and decreased nutrition.

Ecotoxicological effects of the azole antifungal agent clotrimazole on the macrophyte species Lemna minor and Lemna gibba.

Pharmaceuticals are a large and diverse group of compounds used to treat, prevent and diagnose disease. Among these, a group that has been recently detected in the aquatic environment is that of the azole compounds, commonly used as antifungals. Clotrimazole (CLO) is a nonbiodegradable persistent azole compound, with broad-spectrum antifungal activity for which virtually no toxicological data are available, especially towards aquatic plants. The few existent data point to a documented interference with cytochrome P450 system of exposed organisms. Therefore, the aim of this paper was to evaluate the ecotoxicological effects of the fungicide CLO on two aquatic macrophyte species, namely, Lemna minor and Lemna gibba. To attain this purpose, an acute assay (96 h) was performed with both species being exposed to CLO, in a concentration range of 0 to 5 µg L-1. The analyzed endpoints were levels of chlorophyll a and b, total, carotenoids, catalase (CAT) and glutathione -s-transferases activities (GSTs). In general, CLO exposure caused some minor alterations in L. minor and L. gibba pigment contents. Antioxidant enzymes exhibited a different pattern in both species, since the highest concentrations of CLO caused an increase on CAT activity, and a decrease on GSTs activity in L. minor, and the opposite in L. gibba, reflected by a decrease on CAT activity and an increase on GSTs activity in all tested concentrations. These results demonstrate that CLO exposure resulted in potential deleterious effects on macrophytes, namely with the involvement of the antioxidant defense mechanisms that were likely deployed to cope with pro-oxidative conditions established by CLO.

Daphnia magna responses to fish kairomone and chlorpromazine exposures.

To avoid being preyed, organisms must be able to identify predatory threats by sensing molecules released by predators (kairomones), and to employ effective strategies to prevent detection by predators. Furthermore, in the wild, organisms are also exposed to chemicals that may alter their behavioral traits, such as neuroactive pharmaceuticals. Considering the co-occurrence of both types of chemicals, their possible interaction needs to be studied. To address this topic, the aim of this study was to verify the effects of fish kairomone (FK - a chemical associated to putative predation by fish) and chlorpromazine (CPZ - neuroactive pharmaceutical drug, environmental contaminant), isolated and in combination, in different functional endpoints of Daphnia magna, such as oxygen consumption, feeding rate, behavior and reproduction. Among these endpoints, oxygen consumption was only affected by the combination of compounds (FK + CPZ). On the other hand, feeding rate was affected by all treatments, being lower than control. For life history traits and phototactic behavior, the effects of FK predominated over the ones caused by CPZ exposure, incrementing the reproductive output of females, leading to greater population growth rates and increasing negative phototactic behaviour.

Multi-parametric analysis of ciprofloxacin toxicity at ecologically relevant levels: Short- and long-term effects on Daphnia magna.

The increased presence of emergent compounds, such as pharmaceuticals drugs, in the aquatic compartment has been acknowledged as an evolving environmental issue whose consequences are not yet fully characterized. Specific classes of pharmaceutical drugs, such as fluoroquinolone antibiotics, can exert toxic effects to non-target species with ecological significance, since these compounds are environmentally stable and persistent, and may interact with some of the key physiologic processes of organisms. Despite such characteristics, knowledge about the effects of these drugs is still scarce, especially to non-target organisms. The present study aimed to evaluate the effects of chronic and acute exposures of the cladoceran Daphnia magna to the fluoroquinolone antibiotic ciprofloxacin. Putative toxic effects were assessed, following acute and chronic exposures to ecologically relevant concentrations of ciprofloxacin, through enzymatic (cholinesterase - ChEs, catalase - CAT, glutathione S-transferases - GSTs) and non-enzymatic (thiobarbituric acid reactive substances - TBARS, glycogen - Gly) biomarkers. In addition, we also determined behavioural (swimming distance - SD) and morphological (body length of the first brood - BL1B) endpoints in animals exposed to this drug. Ciprofloxacin acute exposure resulted in increased CAT and ChEs activities, and inhibited GSTs activity. After chronic exposure, ChEs activity was significantly inhibited, while GSTs activity was significantly enhanced. TBARS levels were only increased at higher concentrations of ciprofloxacin. CAT activity and Gly content did not evidence a clear and significant pattern of variation. SD was slightly inhibited during dark cycles. BL1B presented a significant decrease for animals subjected to an intermediate concentration. Results showed that even ecologically relevant concentrations of ciprofloxacin may cause oxidative stress in individuals of D. magna. The present study showed important data that corroborate the occurrence of significant biochemical alterations in key features of an aquatic organism when exposed to relevant levels of a widely used antibiotic, establishing essential links between environmental exposure to this specific drug and putative toxic challenges that may result in irreversible changes and damages, especially at the individual level. However, changes in the size of neonates suggest that population alterations are likely to occur under real scenarios of chronic contamination by this drug.