Evaluation of ketoprofen toxicity in two freshwater species: Effects on biochemical, physiological and population endpoints.

Among the most used non-steroidal anti-inflammatory drugs (NSAIDs), ketoprofen (KTF) assumes an important position. Nevertheless, its ecotoxicological effects in non-target organisms are poorly characterized, despite its use and frequency of occurrence in aquatic matrices. Thus, the aim of this study was to evaluate the possible toxicological effects of KTF contamination, in two freshwater species, Lemna minor and Daphnia magna, by measuring biochemical, physiological and population parameters. To attain this objective, both species were exposed to KTF at the same concentrations (0, 0.24, 1.2, 6 and 30 µg/L). L. minor plants were exposed during 4 d to these levels of KTF, and the enzymatic activity (catalase (CAT), glutathione S-transferases (GSTs) and carbonic anhydrase (CA)), and pigments content (chlorophylls a, b and total and carotenoids) were analyzed to evaluate the toxicity of this drug. D. magna was acutely and chronically exposed to KTF, and enzymatic activities (CAT, GSTs and cyclooxygenase (COX)), the feeding rates, and reproduction traits were assessed. In L.minor, KTF provoked alterations in all enzyme activities, however, it was not capable of causing any alteration in any pigment levels. On the other hand, KTF also provoked alterations in all enzymatic activities in D. magna, but did not affect feeding rates and life-history parameters. In conclusion, exposure to KTF, provoked biochemical alterations in both species. However, these alterations were not reflected into deleterious effects on physiological and populational traits of L. minor and D. magna.

Can salicylic acid modulate biochemical, physiological and population alterations in a macrophyte species under chemical stress by diclofenac?

Salicylic acid (SA) is a pharmaceutical drug that may exert toxic effects by its own; however, simultaneous exposure of plants to SA and to other substances, often results in the significant changes in the patterns of toxic response/resistance to these other sources of chemical stress. Thus, the aim of this work was to investigate the capacity of SA of modulating Lemna minor responses co-exposed to the pharmaceutical drug, diclofenac - DCF. To attain this objective, L. minor was exposed for 7 days, to DCF alone, and to combinations of DCF with SA. After exposure, biochemical, physiological and population endpoints were analyzed as follows: catalase (CAT) and glutathione S-transferases (GSTs) activities, pigments content (chlorophyll a (Chl a), b (Chl b) and total (TChl), carotenoids (Car) and [Chl a]/[Chl b] and [TChl]/[Car] ratios), and growth specific rate, fresh weight and root length. Single exposures to DCF were capable of causing effects in all analyzed endpoints. However, co-exposure of DCF with SA partially reverted these effects. Finally, we may suggest that SA is capable to prevent the toxicity of DCF in macrophytes, by modulating the toxic response of exposed plants.

Effects of diclofenac and salicylic acid exposure on Lemna minor: Is time a factor?

The global occurrence of pharmaceuticals in the aquatic environment has been considered a particularly concerning problem with unknown consequences. Non-steroidal anti-inflammatory drugs (NSAIDs) including diclofenac (DCF) and salicylic acid (SA), are among the most frequently prescribed drugs in the world, being consequently commonly found in the aquatic environment. Prolonged experiments (with duration of exposure that surpass those recommended by already established testing guidelines) are important to obtain ecologically relevant data to address the issue of NSAIDs ecotoxicity, because by being more realistically (namely in terms of levels and durations of exposure), such tests may indicate realistic challenges posed to aquatic organisms. Among the most common test species that are used for assessing environmental quality, plants play a leading role. Lemna species are among the most important plants used for ecotoxicity testing. Therefore, the aim of this study was to evaluate the temporal effect of a prolonged exposure of DCF and SA on Lemna minor. To attain this purpose, L. minor plants were chronically exposed to 0, 4, 20, and 100 µg/L of both pharmaceuticals, and samplings were performed at 6, 10 and 14 days of exposure. The analyzed endpoints were: levels of chlorophyll a, b and total, carotenoids; and enzymatic biomarkers, such as catalase, ascorbate peroxidase and glutathione-S-transferases. Diclofenac was responsible for alterations in all analyzed parameters in different intervals of exposure. Salicylic acid exposure was not capable of causing alterations on pigment contents of L. minor, however, enzymatic biomarkers were altered at all sampling intervals. Thus, it is possible to conclude that both pharmaceuticals can cause damage on the tested macrophyte species, biochemical parameters being more sensitive than physiological ones. Additional prolonged experiments are required to understand the chronic effects of different pharmaceuticals in the aquatic environment, especially in plants.

Evaluation of pharmaceutical toxic effects of non-standard endpoints on the macrophyte species Lemna minor and Lemna gibba.

In the last years the environmental presence of pharmaceuticals has gained increasing attention. Research data show that these compounds can cause toxicological effects in different species of fish, mollusks and macroinvertebrates. However, the literature is scarce in terms of ecotoxicity data especially focusing on plants as test organisms. Ecotoxicological plant-based tests following the standard OEDC guideline 221 (OECD, 2006) are strongly restricted due to the recommended end-points: growth and yield of plants. It is necessary to develop and validate alternative macrophyte-based tests (non-standard endpoints), more sensible and providing additional information about the chemical contamination effects in plants. To attain this purpose, species from the Lemna genus were selected. Thus, the aim of this study was to analyze the toxic effects of pharmaceuticals in non-standard endpoints on two macrophyte species, Lemna minor and Lemna gibba. To this purpose an acute assay (96 h) was performed with L. minor and L. gibba exposed to chlorpromazine (CPZ), paracetamol (APAP), and diclofenac (DCF), in the following concentration ranges: 0 to 20 µg/L, 0 to 125 µg/L, and 0 to 100 µg/L, respectively. The analyzed endpoints were: levels of chlorophyll a and b, total chlorophyll, carotenoids, anthocyanins; chlorophyll fluorescence; and catalase activity. In general, higher concentrations of the tested pharmaceuticals caused significant effects on both Lemna species in terms of the different endpoints analyzed. In conclusion, acute exposures to CPZ, APAP, and DCF differently affected the defensive system of the tested species; among chlorophylls, chlorophyll b content was more affected, but pharmaceutical exposure was not able to cause alterations on chlorophyll fluorescence.