# Bioassays with _Allium cepa_ for the Monitoring of Toxicity in the Groundwater of Yucatan, Mexico
## 2 Methods
### Analysis of Contaminants  
Organic pollutants were analyzed based on modifications of EPA methods 3535A and 8270D [17; 18]. For the determination of organochlorine pesticides (OCP), polycyclic aromatic hydrocarbons (PAHs), and caffeine, one liter of water was collected in glass amber bottles (washed with phosphate-free laboratory detergent and rinsed with deionized water and acetone before use). Before the analysis, 0.5 g of ethylenediaminetetraacetic acid (EDTA) was added to each water sample. Then, a solid phase extraction (SPE) was performed using 500 mg/6 mL Strata-X 33 \(\upmu\)m polymeric reversed phase cartridges (Phenomenex, 8B-S100-HCH) conditioned with solvents of increasing polarity: 10 mL of ethyl acetate, 10 mL of methanol, and 10 mL of distilled water. For extraction, 1 L of the sample was passed through the cartridge with a flow of 10-15 mL/min. After passing the sample, the cartridge was washed with 10 mL of distilled water and vacuum-dried for approximately 120 min. After the extraction, analytes were recovered by eluting with 15 mL of ethyl acetate, and extracts were evaporated under a gentle nitrogen stream. Samples were placed in 2 mL vials with a PTFE/silicone septum cap. Pollutants were identified and quantified by gas chromatography/mass spectrometry (GC-MS) using a gas chromatograph coupled to a mass selective detector operated in electron impact (EI) ionization mode and equipped with an automatic liquid sampler (Agilent Technologies 7890B Series GC; 5977B MSD and 7693A Autoinjector, respectively). Samples were injected in splitless mode and the injector was at 280 \({}^{\circ}\)C. The GC was equipped with a J&W HP-5 MS capillary column (30 m \(\times\) 0.25 mm and 0.25 \(\upmu\)m of film thickness) and the carrier gas was He (ultra-pure grade) with a flow rate of 0.8 mL/min. For pesticide and caffeine analysis, the oven temperature was initially set at 50 \({}^{\circ}\)C and increased 10 \({}^{\circ}\)C/min until 180 \({}^{\circ}\)C, then 1.5 \({}^{\circ}\)C/min until 200 \({}^{\circ}\)C (hold time 2 min), and finally 6 \({}^{\circ}\)C/min until 290 \({}^{\circ}\)C (hold time 1 min) [19]; for PAH determination, oven  
Figure 1: Study area. Wells located in two transects from inland to the coast were used. One transect runs from Sierra Papacal in Mérida municipality to Chuburná in Progreso, and the other transect is from Nohuayün in Tetiz municipality to Sisal in Hunucmá.  
temperature was set at 60 \({}^{\circ}\)C, then increased 6 \({}^{\circ}\)C/min to 290 \({}^{\circ}\)C (hold time 11.67 min) [20]. The transfer line temperature was 280 \({}^{\circ}\)C. Mass spectra (\(m/z\) 50-550) were recorded at a rate of five scans per second at 70 eV. Mass spectrometric analysis was performed in selected ion monitoring (SIM) mode of two characteristic fragment ions for each compound; Supplementary Tables S2 and S3 list retention times and the ions used for identification and quantification. Nutrients (nitrate, nitrite, ammonium, silicates, and phosphate) were analyzed by spectrophotometry in microplates with methods previously described [21]; samples were collected in 35 mL under glass bottles rinsed with deionized water and nitric acid. Simple Nutrients in Seawater-whole volume from Sigma-Aldrich (QC3179-500 ML, Lot LRAA9336) was used as certified reference material to validate nutrient data.