Analysis of atrazine and metabolites

Samples were analyzed for atrazine and metabolites using a Waters Acquity ultra-pressure liquid chromatography (UPLC) system with an autosampler and coupled with mass detection and MassLynx v.4.1 chromatography manager software (Milford, MA). The mobile phase consisted of Optima grade solvents (Thermo Fisher Scientific Inc., Waltham, MA, USA): (A) 0.2% formic acid in 40% methanol and (B) 0.2% formic acid in 40% methanol +60% acetonitrile. Separation of atrazine and metabolites was performed at 45°C on a 100 × 2.1 mm i.d., 1.7 μm Waters Acquity UPLC BEC C18 analytical column with a 2.1 × 5 mm Acquity UPLC BEH C18 1.7 um Vanguard pre-column (Waters Corp., Milford, MA) with a flow rate of 0.30 mL min⁻¹ using the following gradient: 0–1.5 min, 55% A; 1.5–2.0 min, 25% A; 2.0–3.0 min, 0% A; 3.0–4.0 min, 55% A. The autosampler was kept a 10°C to minimize atrazine degradation.

Detection and confirmation were performed using a Waters Acquity TQD (Waters Corp., Milford, MA) tandem quadrupole mass (MS-MS) detector in the Multiple Reaction Monitoring mode. The conditions of the MS-MS detector were as follow: voltages in capillary, cone, extractor, and RF lens were 0.61, 20, 3.0, and 0.1 kV, respectively; temperature of the source and desolvation were 150 and 400°C, respectively; and the gas flow of the desolvation and cone were 850 and 20 L h⁻¹, respectively. Quantification was performed using the parent and daughter ions for each compound, atrazine: 216.06 and 174.00, 2-HA 198.00 and 156.00, DIA 173.99 and 132.03, and DEA 188.02 and 146.01, respectively. Five-point calibration curves (0.02–60 μg L⁻¹) for all compounds were generated by using external standards prepared from certified solutions (Chem Services, West Chester, PA, USA). A 50-μL injection volume was used for both standards and samples. The retention times were 0.79, 0.97, 1.25, and 2.94 min for 2-HA, DIA, DEA, and atrazine, respectively. Limit of detection (LOD) for atrazine, 2HA, DIA, and DEA are 0.0072, 0.014, 0.24, and 0.0057 μg L⁻¹, respectively. These LOD's were calculated following USEPA (2010).

For all experiments, no atrazine metabolites and/or only traces were detected, thus, only outflow and inflow atrazine concentrations were used to calculate discrete removal (%) at each sampling time. Atrazine loading and cumulative removal was calculated using the inflow and outflow atrazine concentration, flow rate, and mass of biochar. All values were averaged over replication. The relationship between cumulative atrazine added to biochar and cumulative atrazine removal was tested among the three contact times to determine whether the relationships (slope and intercept) were significantly different from each other at p = 0.05. The null hypothesis was that one equation could be used to describe cumulative atrazine addition vs. cumulative atrazine removal for all three contact times. This was tested by using a series of “contrast” statements in SAS (Sas and Guide, 2003) to determine whether the slope and intercept were significantly different based on contact time.