2.2. Sample collection and preparation

Two WWTPs from two communities (A and B), one in western Kentucky and one in western Tennessee, were sampled for ten consecutive days every month starting March 27 to July 1st, 2020. Twenty-four-hour composite samples of raw wastewater (one aliquot every 15 min) were collected using a time-proportional autosampler and maintained at 4 °C during the collection period. WWTP-A treats an average of 4.40 million gallons per day (MGD) of primarily domestic sewage whereas WWTP-B treats an average of 1.45 MGD of sewage. All samples collected in polypropylene bottles were transported on ice to the laboratory and stored at −20 °C until further analysis.

The ammoniacal nitrogen (NH4–N) level in raw wastewater was determined as reported elsewhere (O’Rourke and Subedi, 2020; Croft et al., 2020), and used to estimate the size of the population served by the WWTPs. The concentration of ammoniacal nitrogen (ng/L), wastewater inflow (L/d), and the average per capita daily production of NH4–N (6900 mg/d) (Been et al., 2014) were utilized to determine near-accurate population. The average populations found were 16187 ± 5311 for community A and 10941 ± 3297 for community B. The average population for community A and B determined using ammoniacal nitrogen load were 16.3% lower and 3% higher, respectively, than the census-based population (USCB, 2019). The limited non-human sources of ammoniacal nitrogen could not be quantified and corrected; however, it is least affected by non-human sources compared to other conventional hydrological markers used for the estimation of population (Been et al., 2014).

The samples were equilibrated to room temperature, thoroughly mixed, centrifuged (100 mL) at 4500 rpm for 5 min, and vacuum filtered using 0.45 μm nylon membrane (47 mm diameter). The filtered samples were spiked with 500 μL of β-glucuronidase type H-2 enzyme, mixed well, and incubated at 37 °C for 2 h. The incubated samples were then allowed to cool to room temperature, spiked with a mixture of internal standards [250 ng of 8-iso-PGF2α-D₄, 187.5 ng of 5-iPF2α-VI-D₁₁, 250 ng of PGE2-D₄], and mixed thoroughly. The spiked samples were extracted using Oasis® hydrophilic-lipophilic balance (HLB) solid-phase extraction cartridges (6 c.c., 200 mg) preconditioned with 3 mL methanol followed by 3 mL of ultrapure water. Samples were extracted under a gentle vacuum at ∼1 mL/min, dried under a high vacuum for ∼3 min, and eluted with 4 mL of methanol followed by the 3 mL of 5% ammonia in methanol. The combined eluates were concentrated to ∼100 μL using a gentle flow of nitrogen gas at ambient temperature, transferred quantitatively (with multiple wash using methanol) to silanized amber glass LC-vials, adjusted final volume to 500 μL using methanol, and vortex-mixed. The final concentrate was then syringe filtered through 0.2 μm nylon filter (MilliporeSigma, Burlington, MA), and injected 1.0 μL for the analysis of target isoprostanes using ultra-performance liquid chromatography (UPLC) tandem mass spectrometry (MS/MS).

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