Determination of the accumulation of chiral pharmaceuticals (venlafaxine and 𝘖-desmethylvenlafaxine) in rainbow darters (𝘌𝘵𝘩𝘦𝘰𝘴𝘵𝘰𝘮𝘢 𝘤𝘢𝘦𝘳𝘶𝘭𝘦𝘶𝘮)

Abstract

Pharmaceuticals are widespread contaminants of concern that enter the aquatic environment mainly via wastewater effluent. Over 50% of common pharmaceuticals are chiral, and this is notable due to the potential impact of chirality on the distribution, fate, and toxicity of these compounds. Historically, chirality has been overlooked when completing environmental risk assessments. The chiral antidepressant venlafaxine (VEN) and its major metabolite O-desmethylvenlafaxine (desVEN) are pseudo-persistent through wastewater treatment and are not removed completely, being detected in surface waters globally at levels greater than 2.0 µg/L. At these concentrations, VEN and desVEN have been shown to impact the behavior, metabolism, population structures, and other biological responses of aquatic biota in receiving environments. The possible enantioselective bioaccumulation of each enantiomer of VEN (R and S) and desVEN (R and S) needs further investigation, as they are normally treated as racemic mixtures. An optimized and unbiased extraction method for individual enantiomers (R and S) of VEN and desVEN was developed. The extraction method and all subsequent sample cleanup was evaluated for the recoveries of VEN and desVEN enantiomers and possible enantiomeric bias. Accelerated solvent extraction (ASE) using acidified acetonitrile (1% formic acid by volume) was determined to be an acceptable extraction method with regards to the recoveries and chromatography for all VEN and desVEN enantiomers (performing better in comparison to ultrasonic solvent extraction, USE). The selection and mass of a fat retainer included in extraction cells for on-line extract cleanup and subsequent sample cleanup protocols were also optimized to reduce matrix effects (e.g., ion suppression) associated with co-eluates during liquid chromatography tandem mass spectrometry (LC-MS/MS). The addition of 2.5 g of neutral aluminum oxide during ASE had no enantioselective effects on extraction but resulted in better recoveries for all enantiomers of VEN and desVEN. Subsequent extract cleanup via solid-phase extraction (SPE) using Hydrophilic-Lipophilic-Balanced (HLB) cartridges had the highest recoveries in comparison to other SPE cartridges, liquid-liquid extraction, and QuEChERS, and no enantioselective effects were observed after analysis. Fish tissue mass up to 2.4 g and a final extract volume of 0.5 mL were chosen as the best compromise for a method with satisfactory sensitivity and minimal enantiomeric bias during extraction, while avoiding detrimental ion suppression for all VEN and desVEN enantiomers (with good absolute recovery, and extraction efficiency). The method detection limits (MDLs) for the final method ranged from 0.03 - 0.05 ng/g which is lower than or comparable to VEN extraction from fish tissues reported in other methods in the literature. The validated extraction method was applied to an in-lab exposure of male rainbow darters (Etheostoma caeruleum) collected from a clean reference site in the Grand River, ON. The goal of this exposure was to determine if there was an enantioselective effect on the bioaccumulation of R-VEN and S-VEN in a sentinel small-bodied fish species exposed to a single enantiomer. The fish were exposed to 1 µg/L of R-VEN or S-VEN for up to 14 days, with samples being collected on days 0, 1, 4, and 14. S-VEN appeared to bioaccumulate in fish more than R-VEN, as S-VEN was significantly higher in fish tissue after 4 and 14 days of exposure. The metabolite S-desVEN was also found in fish exposed to S-VEN for 4 and 14 days, which suggests the metabolism of S-VEN into S-desVEN in fish, and subsequent accumulation and/or retention (there was no R- or S-desVEN detected in the water). In contrast, R-desVEN was not found in fish exposed to R-VEN at any time point. Subtle differences in bioaccumulation of VEN and desVEN enantiomers in fish were observed but further studies are needed to determine if there is an enantiomeric shift during bioaccumulation that would alter risk in wild fish.