Solid-phase microextraction (SPME) is a promising technique for determining organic contaminants within biotic systems. Existing in vivo SPME-kinetic calibration (SPME-KC) approaches are unwieldy due to the necessity of predetermining a distribution coefficient for the analyte of interest in the tissue and the preloading of a calibrating compound to the fiber. In this study, a rapid and convenient SPME alternative calibration method for in vivo analysis, termed SPME-sampling rate (SPME-SR) calibration, was developed and validated under both laboratory and field conditions to eliminate such presampling requirements. Briefly, the SPME probe is inserted into tissue, in this study fish dorsal-epaxial muscle, for 20 min allowing the concentrations of target analytes in the fish muscle to be determined by the extracted amount of analyte and the predetermined sampling rates. Atrazine, carbamazepine, and fluoxetine were detected nonlethally in the low ppb levels within fish muscle, with both laboratory and field-derived results obtained by in vivo SPME-KC comparable (within a factor of 1.27) to those obtained by lethal sampling followed by tissue liquid extraction. The technique described in this study represents an important advance which broadens the application of SPME in vivo sampling technology.
The space-resolved solid-phase microextraction (SR-SPME) technique was employed to study the tissue-specific bioconcentration of pharmaceuticals in live fish. The segmented design of the SPME fibers allowed for the simultaneous determination of pharmaceutical residues in fish dorsal-epaxial muscle and adipose tissue with a single SPME fiber. The miniaturized fiber endowed the technique with high spatial resolution allowing for quantification of analytes within adjacent, relatively small tissues of immature rainbow trout. The pre-equilibrium sampling and kinetic calibration approach yielded efficient and accurate quantitation of pharmaceuticals in fish tissue. The ability of the SPME method to repeatedly sample the same fish circumvents problems arising from interanimal variation, thus improving the precision of generated bioconcentration kinetic profiles. In vivo monitoring with SR-SPME was validated with in vitro liquid extraction of tissue samples using methanol. Of the nine compounds evaluated, five (atrazine, gemfibrozil, carbamazepine, ibuprofen, and fluoxetine) bioconcentrated in adipose and muscle tissue over the eight exposure days. Although the accumulation of analytes in both tissues was positively correlated, each compound partitioned with differing affinities as modified by their hydrophobicity and unique molecular structure. Water samples analyzed using the SPME technique yielded results similar to those determined by solid-phase extraction (SPE); however, SPME was more rapid and operationally much simpler. This study illustrates the application conditions for in situ SR-SPME while demonstrating the potential of these miniaturized SPME fibers for simultaneous in vivo repeated sampling of multiple tissues.
Intersex in fish downstream of municipal wastewater treatment plants (MWWTPs) is a global concern. Consistent high rates of intersex in male rainbow darter (Etheostoma caeruleum) have been reported for several years in the Grand River, in southern Ontario, Canada, in close proximity to two MWWTPs. The larger MWWTP (Kitchener) recently underwent upgrades that included the conversion from a carbonaceous activated sludge to nitrifying activated sludge treatment process. This created a unique opportunity to assess whether upgrades designed to improve effluent quality could also remediate the intersex previously observed in wild fish. Multiple years (2007-2012) of intersex data on male rainbow darter collected before the upgrades at sites associated with the MWWTP outfall were compared with intersex data collected in postupgrade years (2013-2015). These upgrades resulted in a reduction from 70 to 100% intersex incidence (preupgrade) to <10% in postupgrade years. Although the cause of intersex remains unknown, indicators of effluent quality including nutrients, pharmaceuticals, and estrogenicity improved in the effluent after the upgrades. This study demonstrated that investment in MWWTP upgrades improved effluent quality and was associated with an immediate change in biological responses in the receiving environment. This is an important finding considering the tremendous cost of wastewater infrastructure.
Municipal wastewater effluent (MWWE) and its constituents, such as chemicals of emerging concern, pose a potential threat to the sustainability of fish populations by disrupting key endocrine functions in aquatic organisms. While studies have demonstrated changes in biological markers of exposure of aquatic organisms to groups of chemicals of emerging concern, the variability of these markers over time has not been sufficiently described in wild fish species. The aim of this study was to assess the spatial and temporal variability of biological markers in response to MWWE exposure and to test the consistency of these responses between seasons and among years. Rainbow darter (Etheostoma caeruleum) were collected in spring and fall seasons over a 5-year period in the Grand River, Ontario, Canada. In addition to surface water chemistry (nutrients and selected pharmaceuticals), measures were taken across levels of biological organization in rainbow darter. The measurements of hormone production, gonad development, and intersex severity were temporally consistent and suggested impaired reproduction in male fish collected downstream of MWWE outfalls. In contrast, ovarian development and hormone production in females appeared to be influenced more by urbanization than MWWE. Measures of gene expression and somatic indices were highly variable between sites and years, respectively, and were inconclusive in terms of the impacts of MWWE overall. Robust biomonitoring programs must consider these factors in both the design and interpretation of results, especially when spatial and temporal sampling of biological endpoints is limited. Assessing the effects of contaminants and other stressors on fish in watersheds would be greatly enhanced by an approach that considers natural variability in the endpoints being measured.
The variability and extent of the intersex condition (oocytes in testes, or testis-ova) was documented in fish along an urban gradient in the Grand River, Ontario, Canada, that included major wastewater treatment plant outfalls. A method for rapid enumeration of testis-ova was developed and applied that increased the capacity to quantify intersex prevalence and severity. Male rainbow darters (Etheostoma caeruleum) sampled downstream of the first major wastewater outfall (Waterloo) had a significant increase, relative to 4 upstream reference sites, in the mean proportion of fish with at least 1 testis-oocyte per lobe of testes (9-20% proportion with ≤ 1 testis-oocyte/lobe vs 32-53% and >1.4 testis-oocyte/lobe). A much higher mean incidence of intersex proportion and degree was observed immediately downstream of the second wastewater outfall (Kitchener; 73-100% and 8-70 testis-oocyte/lobe); but only 6.3 km downstream of the Kitchener outfall, the occurrence of intersex dropped to those of the reference sites. In contrast, downstream of a tertiary treated wastewater outfall on a small tributary, intersex was similar to reference sites. Estrogenicity, measured using a yeast estrogen screen, followed a similar pattern, increasing from 0.81 ± 0.02 ng/L 17b-estradiol equivalents (EEq) (Guelph), to 4.32 ± 0.07 ng/L (Waterloo), and 16.99 ± 0.40 ng/L (Kitchener). Female rainbow darter downstream of the Kitchener outfall showed significant decreases in gonadosomatic index and liver somatic index, and increases in condition factor (k) relative to corresponding reference sites. The prevalence of intersex and alterations in somatic indices suggest that exposure to municipal wastewater effluent discharges can impact endocrine function, energy use, and energy storage in wild fish.
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