Single and combined effects of caffeine and salicylic acid on mussel Mytilus galloprovincialis: Changes at histomorphological, molecular and biochemical levels.

Caffeine (CAF) and salicylic acid (SA) are frequently detected in waterbody, though information on their biological impact is poor. This work assesses the effects of CAF (5 ng/L to 10 µg/L) and SA (0.05 µg/L to 100 µg/L) alone and combined as CAF+SA (5 ng/L+0.05 µg/L to 10 µg/L+100 µg/L) on mussel Mytilus galloprovincialis under 12-days exposure by histomorphology of digestive gland and oxidative stress defense at molecular and biochemical levels. Besides evaluating tissue accumulation, absence of histomorphological damage and haemocyte infiltration highlighted activation of defensive mechanisms. Up-regulation of Cu/Zn-sod, Mn-sod, cat and gst combined with increased catalase and glutathione S-transferase activity were found in CAF-exposed mussels, while SA reduced ROS production and mitochondrial activity. CAF+SA exposure induced differential responses, and the integrated biomarker response (IBR) revealed more pronounced effects of SA than CAF. These results enlarge knowledge on pharmaceuticals impact on non-target organisms, emphasizing the need for proper environmental risk assessment.

Time- and dose-dependent biological effects of a sub-chronic exposure to realistic doses of salicylic acid in the gills of mussel Mytilus galloprovincialis.

Among nonsteroidal anti-inflammatory drugs (NSAIDs) commonly found in seawater and wastewater, salicylic acid (SA) represents one of the most persistent and hazardous compounds for aquatic organisms. This study was therefore designed to elucidate the biological effects of SA in mussel Mytilus galloprovincialis. During a sub-chronic exposure (12 days), mussels were exposed to five realistic concentrations of SA (C1: 0.05 µg/L; C2: 0.5 µg/L; C3: 5 µg/L; C4: 50 µg/L; C5: 100 µg/L) and gills, selected as the target organ, were collected at different time points (T3: 3 days; T5: 5 days; T12: 12 days). Exposure to SA induced no histological alterations in mussel gills, despite a relevant hemocyte infiltration was observed throughout the exposure as a defensive response to SA. Temporal modulation of glutathione S-transferase (GST), catalase (CAT), and superoxide dismutase (SOD) activities suggested the occurrence of antioxidant and detoxifying responses against SA exposure, while lipid peroxidation (LPO), except for a partial increase at T3, was prevented. Inhibition of the cholinergic system was also reported by reduced acetylcholinesterase (AChE) activity, mainly at T12. Overall, findings from this study contribute to enlarge the current knowledge on the cytotoxicity of SA, on non-target aquatic organisms, and might for the enhancement of new ecopharmacovigilance programs and optimization of the efficacy of wastewater treatment plants for mitigation of pharmaceutical pollution in coastal areas.

Comparison of cellular mechanisms induced by pharmaceutical exposure to caffeine and its combination with salicylic acid in mussel Mytilus galloprovincialis.

Urban and hospital-sourced pharmaceuticals are continuously discharged into aquatic environments, threatening biota. To date, their impact as single compounds has been widely investigated, whereas few information exists on their effects as mixtures. We assessed the time-dependent biological impact induced by environmental concentrations of caffeine alone (CAF; 5 ng/L to 10 µg/L) and its combination with salicylic acid (CAF+SA; 5 ng/L+0.05 µg/L to 10 µg/L+100 µg/L) on gills of mussel Mytilus galloprovincialis during a 12-day exposure. Although no histological alteration was observed in mussel gills, haemocyte infiltration was noticed at T12 following CAF+SA exposure, as confirmed by flow cytometry with increased hyalinocytes. Both the treatments induced lipid peroxidation and cholinergic neurotoxicity, which the antioxidant system was unable to counteract. We have highlighted the biological risks posed by pharmaceuticals on biota under environmental scenarios, contributing to the enhancement of ecopharmacovigilance programmes and amelioration of the efficacy of wastewater treatment plants.

Histological endpoints and oxidative stress transcriptional responses in the Mediterranean mussel Mytilus galloprovincialis exposed to realistic doses of salicylic acid.

Despite the availability of analytic data, little is known about the toxicity of salicylic acid (SA) on aquatic non-target organisms. The present study aimed at evaluating the impact of SA through a short-term exposure of the Mediterranean mussel Mytilus galloprovincialis to five environmentally relevant concentrations of SA. A set of suitable biomarkers was applied at selected time-points on mussel digestive glands, including histological observations and expression of oxidative stress related genes. The obtained results showed a conspicuous hemocytic infiltration among mussel digestive tubules, as confirmed also by a flow cytometric approach that revealed an increase of halinocytes and granulocytes. Interestingly, a significant dose and time dependent decrease in the expression levels of oxidative stress related genes was found in mussels exposed to SA except for the glutathione S-transferase gene that was significantly up-regulated in a time-dependent manner confirming its important role against oxidant species and in the metabolism of pharmaceuticals.

A multi-biomarker approach for the early assessment of the toxicity of hospital wastewater using the freshwater organism Daphnia magna.

Hospital wastewater (HWW) contains different hazardous substances resulting from a combination of medical and non-medical activities of hospitals, including pharmaceutical residues. These substances may represent a threat to the aquatic environment if they do not follow specific treatment processes. Therefore, we aimed to investigate the effects of the untreated effluent collected from a general hospital in Mahdia City (Tunisia) on neonatal stages of the freshwater crustacean Daphnia magna. Test organisms were exposed to three proportions (3.12%, 6.25%, and 12.5% v/v) of HWW. After 48 h of exposure, a battery of biomarkers was measured, including the quantification of antioxidant enzymes [catalase (CAT) and total and selenium-dependent glutathione peroxidase (total GPx; Se-GPx)], phase II biotransformation isoenzymes glutathione-S-transferases (GSTs), cyclooxygenases (COX) involved in the regulation of the inflammatory process, and total cholinesterases (ChEs) activities. Lipid peroxidation (LPO) was measured to estimate oxidative damage. The here-obtained results showed significant decreases of CAT and GSTs activities and also on LPO content in daphnids, whereas Se-GPx activity was significantly increased in a dose-dependent manner. Impairment of cholinesterasic and COX activities were also observed, with a significant decrease of ChEs and an increase of COX enzymatic activities. Considering these findings, HWW was capable of inducing an imbalance of the antioxidant defense system, but without resulting in oxidative damage in test organisms, suggesting that peroxidases and alternative detoxifying pathways were able to prevent the oxidant potential of several drugs, which were found in the tested effluents. In general, this study demonstrated the toxicity of hospital effluents, measured in terms of the potential impairment of key pathways, namely neurotransmission, antioxidant defense, and inflammatory homeostasis of crustaceans.

In vivo toxicities of the hospital effluent in Mahdia Tunisia.

Hospital effluent (HE) is one of the most important sources of pharmaceuticals released into the environment. This kind of pollution is a recognized problem for both human health and aquatic life. Consequently, in the present study, we assessed the effects of untreated hospital effluent on mice via biochemical and histopathological determinations. Female mice were given free access to water bottles containing untreated HE at different dilutions for 21 days. Then clinical biochemistry and histopathology evaluation were conducted. Serum biochemistry analysis showed the presence of significant increase in cholesterol, triglycerides, glycaemia and total bilirubin. However, phosphatase alkaline and urea activities have been significantly decreased compared to the control group. No significant variation was observed for the rest of the studied parameters (high-density lipoproteins; low-density lipoproteins and uric acid). Additionally, multiple alterations, including cellular necrosis, leucocyte infiltration and congestion, were observed in different tissues of mice exposed to the tested HE.

Occurrence of 40 pharmaceutically active compounds in hospital and urban wastewaters and their contribution to Mahdia coastal seawater contamination.

In the present study, the occurrence of 40 pharmaceuticals belonging to several therapeutic groups was investigated for the first time in hospital effluent, wastewater treatment plant influent and effluent, and seawater in Mahdia, Tunisia. Forty-six samples were collected within a 6-month sampling period. Pharmaceuticals were analyzed using solid-phase extraction followed by ultra-performance liquid chromatography-triple quadrupole mass spectrometry. Thirty-three out of the forty target compounds were detected over a wide concentration of ranges, from nanograms per liter to micrograms per liter, depending on the type of sample. Maximum values were detected for caffeine at 902 µgL-1 in hospital wastewater. This compound, as well as salicylic acid, sulfadiazine, and sulfamethizole, were detected in all samples. The average concentration of total pharmaceuticals in hospital wastewater (340 µgL-1) was higher than those detected in influent and effluent wastewater and seawater (275.11 and 0.2 µgL-1, respectively). Risk quotients (RQs) were also estimated to provide a preliminary environmental risk assessment and results revealed that sulfadiazine, sulfamethoxazole, and fluoxetine could pose medium/high risk to the tested aquatic organisms for maximum measured concentrations in wastewater (including hospital and WWTP samples). Although the measured environmental concentrations (MECs) detected in seawater samples might not pose a toxic effect to the aquatic organisms (except for salicylic acid, sulfamethoxazole and fluoxetine), further researches are needed due to the continuous release of wastewater in the environment and the limited efficiency of wastewater treatment processes.