Distribution of antidepressants and their metabolites in brook trout exposed to municipal wastewaters before and after ozone treatment – Evidence of biological effects
Research highlights
► We have developed a reliable analytical method for the quantification by LC–MS/MS of trace amounts of antidepressants in small fish tissues homogenate extracts. ► Following exposure to municipal wastewater, we found significant amounts of antidepressants bioaccumulated in brook trout tissue—in decreasing order: liver > brain > muscle. ► We have proposed the Na/K-ATPase pump activity as biomarker of exposure to antidepressants. ► We observed a significant correlated relationship between the biomarker response in brain synaptosomes and the brain tissue concentrations of certain antidepressants.
Introduction
Over the last decade, pharmaceutical and personal-care products (PPCPs) have been recognized as a major source of pollution for the aquatic environment (Halling-Sørensen et al., 1998, Daughton and Ternes, 1999). Their occurrence in most municipal effluent seems to indicate that conventional wastewater treatments have only a limited ability to remove such substances from sewage. In the 1990s, it was discovered that some active drugs have the capability to interfere with ecosystems in concentrations as low as a few nanograms per liter (Halling-Sørensen et al., 1998). Until now, different pharmaceutically active compounds (PhACs) have been detected in the environment: analgesics, antibiotics, antiepileptics, antidepressants, synthetic and natural hormones (Jones et al., 2006, Lishman et al., 2006). Antidepressant drugs are among the PhACs likely to be found in Canada (IMS Health Canada, 2006), United States (Schultz et al., 2010), and other countries around the world (Calisto and Esteves, 2009). The commonly prescribed antidepressants from the class of selective serotonin reuptake inhibitors (SSRIs) have become the most successful class of marketed antidepressants, with the number of new prescriptions written for these drugs now exceeding that of tricyclic antidepressants (DeVane, 1999).
To date, most analytical methods for determining the presence of antidepressants and their respective metabolites in environmental matrixes have been developed for surface waters and wastewater effluents (Lishman et al., 2006, Lajeunesse et al., 2008, Schultz and Furlong, 2008, Metcalfe et al., 2010), sediments (Schultz et al., 2010), and sewage sludge (Radjenović et al., 2009). Due to the complexity of samples, these analyses typically employ liquid chromatography–tandem mass spectrometry (LC–MS/MS). Since the occurrence of antidepressants in municipal wastewater could lead to physiological and behavioural effects on aquatic organisms (Fong, 1998, Fong, 2001, Calisto and Esteves, 2009), it is critical that these compounds be measured in organisms using reliable analytical methods. Even so, only a few analytical methods have been reported on antidepressants in aquatic organisms (Brooks et al., 2005, Chu and Metcalfe, 2007, Ramirez et al., 2007, Metcalfe et al., 2010, Schultz et al., 2010). Unfortunately, the required mass of tissues often reached up to 2 g, making difficult the detection of SSRIs and their metabolites with small organisms or limited quantity of target tissues.
As antidepressants are present at relatively low levels in the environment (e.g. ng L−1), risk for acute toxic effects is unlikely, but chronic environmental toxic effects cannot be excluded (Péry et al., 2008, Carlsson et al., 2006). The potential ecotoxicological impacts of neuroactive drugs on aquatic organisms are not well understood at the present time. Moreover, the lack of information about the fate and long-term subtle effects of these compounds and/or their metabolites in aquatic organisms makes accurate risk assessment difficult (Gagné and Blaise, 2004, Stanley et al., 2007, Paterson and Metcalfe, 2008). Hence, the measurement of the Na/K-dependent ATPase pump activity in synapsomes represents a more specific biomarker of the effects of SSRIs (Kanoh et al., 1998). The serotonin reuptake pump (or transporter) exchanges one K ion per molecule of serotonin. The loss of K ions outside the synapse button is counterbalanced by the Na/K-ATPase pump which maintains Na and K ions respectively outside and inside of the membrane. This process requires energy (e.g. ATP → ADP + inorganic phosphate) that can be easily measured (Kakko et al., 2003). Thus, the presence of SSRIs decreases the serotonin-dependent hydrolysis of ATP in neuron synaptosomes by the competitive inhibition of the transporters. At present, the modulation of Na/K-dependent ATPase activity in nerve synaptosomes by the SSRIs in fish is largely unknown. Despite its possible usefulness, this promising methodology alone cannot bring sufficient information about the exact concentration of drug needed to elicit biological effects in fish. Therefore, a combination of a biomarker of effects and a tissue SSRIs analysis would strengthen the link between the presence of this class of contaminants in target tissues and the resulting early biological effects.
In this study, we report the development of a reliable LC–MS/MS method for the trace detection of antidepressants in tissues of brook trout exposed to treated (ozone 15 mg L−1) and non-disinfected effluent at the Montreal wastewater treatment plant (WWTP). The developed method was applied to determine the concentrations and distribution of antidepressant in liver, brain, and muscle tissues. Based on bioaccumulation results, a new efficient biomarker (Na/K/serotonin-dependent ATPase activity in neurons synaptosomal membranes) is proposed to track the early biological effects of SSRIs.
Section snippets
Chemicals and standards
Certified standard (>98% purity grade) fluoxetine (FLU), norfluoxetine (NFLU), paroxetine (PAR), sertraline (SER), and (S)-citalopram (CIT) were provided by Toronto Research Chemical Inc. (North York, Ontario, Canada). Desmethylsertraline (DSER), venlafaxine (VEN), O-desmethylvenlafaxine (DVEN), and bupropion-d9 were obtained from Nanjing Jinglong PharmaTech (Nanjing, China). Amitriptyline (AMI) and nortriptyline (NTRI), HEPES, sodium chloride, sodium hydroxide, ethylenediaminetetraacetic acid
LC–MS/MS method validation
Recovery tests were completed on spiked reference brook trout tissues. Additional validation tests were also performed on two different freshwater mussel tissues. The percent recovery of each antidepressant (e.g. 25 ng of antidepressant in 200 mg of tissue) was calculated as follows: the ratio of the spiked sample (area analyte/area ISTD) divided by the ratio of the standard previously prepared by spiking set amounts of analytes in the extracted matrix. Satisfactory results of recovery were
Conclusion
A sensitive and rugged LC–MS/MS method has been developed for the simultaneous analysis of antidepressants along with their respective N-desmethyl metabolites in limited fish tissues extracts samples. The results reported supports the observation that antidepressant may accumulate in fish living in an effluent-dominated stream. In the present study, most drug residues partitioned in liver and brain tissues depicting the possibility of distribution in specific tissues. The higher concentrations
Acknowledgements
The authors thank the city of Montreal for providing access to the mesocosms. Sophie Trépanier from Environment Canada is also acknowledged for her meticulous efforts in the experimental setup of the mesocosms. Additional thanks are extended to Keltie Purcell from Environment Canada for editing the manuscript. This work was supported by the Chemical Management Plan – Health Canada, the St. Lawrence Action Plan and the Canadian Foundation for Innovation.
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