A solid sample of diphenyl guanidine (DPG) was purchased from Sigma-Aldrich (Oakville, ON, Canada) at a reported purity of 97%. A commercial standard of 6PPD-q with a purity of 98.8% was purchased from HPC Standards (Atlanta, GA, USA). HPLC grade solvents (methanol and acetone) and formic acid (88.0%) were purchased from Fisher Scientific (Ottawa, ON, Canada). MilliQ water was produced in-house within the Water Quality Centre at Trent University. For the preparation of a solution of the oxidation biproducts of 6PPD, a solid sample of this compound (N-phenyl-N′-(1,3-dimethylbutyl)-p-phenylenediamine) was also purchased from Sigma-Aldrich, but no purity data were provided by the supplier. However, no previously reported transformation products of 6PPD (Tian et al. 2021) were detected in significant amounts in this standard.
Oxidation of 6PPD
Since there were no commercial standards available for 6PPD-q when this study was initiated, this compound was generated from 6PPD by ozonation using a modified version of the procedure described by Tian et al. (2021). As illustrated in Fig. 1, 500 mg of 6PPD pellets were ground using a ceramic mortar and pestle until a fine, dark gray powder was produced. The 6PPD powder was then spread on a fritted glass disk as a thin layer and placed in an ozone reaction chamber, which consisted of a Pyrex cylindrical fritted tube. Using a Model TOGC2 corona discharge ozone generator manufactured by Triogen O3 (East Kilgide, Scotland, U.K.), ozone was produced from pure oxygen. This ozone was then passed up through the chamber at room temperature (~ 20 °C) for 80 min at a rate of 0.5 L/min. The dose of ozone used was 20 g/Nm3 which is equivalent to 1.5 g O3 per gram of 6PPD. The ozonated 6PPD was dissolved in 50 mL of ethanol and stored in a refrigerator until analysis. Upon analysis of the solution for the concentration of 6PPD by LC-HRMS (as described below) and comparing it to the concentration of 6PPD present before ozonation, the conversion efficiency of 6PPD-q from 6PPD was estimated to be 50 ± 3.8% or 9.31 × 10−4 mol, assuming that all reacted 6PPD produced 6PPD-q and there were no other ozonated by-products produced. Therefore, the amount of 6PPD-q in the solution was estimated to be 278 mg ± 13.2 mg. Dilutions of this solution were used for analytical calibration. After the analysis of all samples was completed, a commercial standard of 6PPD-q became available. Using this commercial standard, the retention time and estimated concentration of 6PPD-q in the ozonated solution was confirmed to be 278 mg by LC-HRMS analysis with a 9-point calibration curve of the commercial standard (R2 = 0.993).
Archived extracts from surface water samples were analyzed for 6PPD-q and DPG. These extracts were prepared from surface water samples originally collected in the fall and winter of 2019 and 2020 at a provincial water quality and water level monitoring station from the Don River at Todmorden (43° 41′ 09.0″ N 79° 21′ 41.0″ W). This location in the GTA in Southern Ontario, Canada, is adjacent to a major urban highway, the Don Valley Parkway. Downstream of the monitoring station, the Don River discharges into the Toronto Harbour area of Lake Ontario.
Samples were collected as part of a water quality monitoring program by the Ontario Ministry of Environment Conservation and Parks (MECP) using composite samplers, as previously described by Johannessen et al. (2021a). In brief, composite samples were collected in the fall of 2019, as well as in the winter and early spring of 2020. The surface water samples were collected in response to significant hydrological events using an ISCO Avalanche refrigerated automated sampler (Avensys Systems, Toronto, ON, Canada) which was triggered when water levels in the Don River began to rise. On October 16–17th, 2019, “temporal” composite samples were collected at intervals throughout the rain event. These samples were collected hourly over 42 h in 300 mL aliquots and pooled in flow-weighted 3-h increments for a total of 14 temporal samples. On this date, as well as all other dates that sampling was performed (i.e., October 22–23rd, 2019, January 11–12th, 2020, March 29–30th, 2020, and March 2–4th, 2020), “event” composite samples were prepared by pooling together the samples collected over 42 h in flow-weighted proportions. In contrast to the other samples that were all collected in response to rain events, the March 2–4th, 2020 sample collection was triggered by elevated flows in the Don River caused by rapid snow melt.
Extraction was performed as described by Johannessen et al. (2021a) using Oasis hydrophilic-lipophilic balance (HLB) solid phase extraction (SPE) cartridges (6 cc, 500 mg) purchased from Waters (Milford, MA, USA). The cartridges were preconditioned with acetone, methanol, and pH neutral MilliQ water adjusted for pH with 3.0 M H2SO4. Triplicate aliquots (50 mL) of each sample were extracted using a vacuum manifold. The cartridges were eluted with 3 × 3 mL of methanol and immediately concentrated to a volume of 1 mL.
Extracts and standards were analyzed using a Q-Exactive Orbitrap high resolution mass spectrometer (HRMS) coupled to an Ultimate 3000 ultra-high pressure liquid chromatography (UPLC) system, both supplied by Thermo Fisher (Waltham, MA, USA). Samples and standards injected into the UPLC at a volume of 25 µL were separated chromatographically using a Kinetex 2.6 µm C18 column (50 × 4.6 mm) purchased from Phenomenex (Torrance, CA, USA). The flow rate was 500 µL/min and the column was maintained at room temperature. The mobile phases and their gradients were as described by Johannessen et al. (2021a, b). In brief, the binary mobile phase consisted of Solvent A, MilliQ water (pH = 7) with 0.1% of formic acid, and Solvent B, methanol with 0.1% of formic acid. The gradient was started with mobile phase B at 2%. This was increased to 99% in 12.25 min where it was held at this amount for 2.75 min before returning to the starting portions within 0.1 min. The HPLC system then equilibrated for 1 min.
The Orbitrap HRMS was operated in positive ionization mode with a heated electrospray ionization source (HESI-II). Parallel reaction monitoring (PRM) was used for data acquisition. The sheath gas flow rate and auxiliary gas flow rate were 50 AU (arbitrary units) and 15 AU, respectively. The sweep gas flow rate was 0 AU and the S-lens RF level was 50.0 AU. The spray voltage was 3.5 kV. The capillary was operated at a temperature of 320 °C and the auxiliary gas heater temperature was 300 °C. The MS2 had an AGC target of 2 × 105 and the maximum IT was 100 ms. The isolation window used was 4 m/z and the resolution of the MS2 was 70,000 at m/z 200.
The exact masses of the precursor ions monitored were m/z 269.20123 for protonated 6PPD, m/z 299.17540 for protonated 6PPD-q and m/z 212.11822 for protonated DPG. The protonated 6PPD was fragmented at a normalized collision energy of 10%, whereas the protonated 6PPD-q and DPG were fragmented at 40%. PRM allowed for the visualization of all the fragments of the precursor ions in the MS2 spectra, which was especially important for identifying 6PPD-q in the ozonated standard, since it lacked purity.
Analytes were detected using a processing method with Genesis automated integration on the XcaliburTM software (version 3.0.63). Samples were processed in a randomized manner. An 11-point calibration curve (diluted in methanol) that spanned 3 orders of magnitude (i.e., 0.488 to 500 µg/L) and had linear regression coefficients of R2 ≥ 0.99 was used to quantify the analytes. The calibrations for 6PPD and 6PPD-q were conducted separately to avoid contamination. The limits of quantification (LOQs) for each analyte were considered to be the concentration of the lowest calibration standard. The lowest calibration standard for 6PPD, 6PPD-q, and DPG was 0.488 µg/L, which corresponds to a concentration in water samples of 0.0098 µg/L.
Quality Assurance/Quality Control (QA/QC)
An archived field blank consisting of HPLC-grade deionized water was re-analyzed for the presence of 6PPD-q and DPG. This field blank was collected at the site in a polyethylene ISCO bottle and was transported, processed, transferred to PET bottles. It was stored in the same manner as the archived surface water samples. Archived laboratory blanks of MilliQ water (pH = 7) were re-analyzed, as well. In addition, methanol reagent blanks were run at the beginning, middle, and end of each sample sequence to ensure QA/QC. Furthermore, a quality control (QC) standard consisting of 6PPD-q and DPG was run at the end of the sample sequence to look for instrumental sensitivity drift.
The efficiency of extraction of each target analyte using SPE with Oasis HLB cartridges was determined by spiking 100 mL of dechlorinated tap water with the analyte at a concentration of 1 µg/L. Extractions were performed in triplicate. These spike and recovery samples then underwent the same extraction and analytical procedure as described above and the recoveries were quantified with a 10-point curve that spanned over three orders of magnitude (0.488 to 500 µg/L) and had R2 ≥ 0.97.
Precipitation data were accessed from “Toronto Historical Total Precipitation” available at toronto.weatherstats.ca/metrics/precipitation.html. Hydrometric discharge data (reported every 5 min) were accessed online from a database maintained by Environment and Climate Change Canada (i.e., wateroffice.ec.gc.ca) for Station 02HC024 on the Don River. These data, land-use information for the surrounding area, and sampling site characteristics (e.g., proximity to the highway, etc.) were described previously by Johannessen et al. (2021a, b).
Cumulative Load and Cumulative Volume Curves
Mass loads (g/event) were calculated by taking the weighted discharge from the storm event (L/hr) and multiplying these discharge data by the concentration values (g/L) and the duration of the sample collection period (hr). The runoff volume was calculated using discharge data for the receiving water site. The cumulative load and cumulative volume curves were generated as described by Peter et al. (2020).