# Three-Year Follow-Up Assessment of Anthropogenic Contamination in the Nichupe Lagoon
## Materials and Methods
### Water Sampling and Analysis  
For the NLS water quality analysis, 43 stations were selected based on previously defined spatial criteria and the hydrological influence (Figure 1). Measurements of water physicochemical parameters of dissolved oxygen (mg L\({}^{-1}\)) and salinity (ups) were measured in situ with a multiparameter sonde (YSI Professional Plus), previously calibrated according to manufacturer guidelines. To determine the trophic state of the water, surface water samples were collected at each site using a 2L Van Dorn bottle. Then, 1L of the sample was stored in dark polyethylene and transported under refrigeration (4 \({}^{\circ}\)C) for subsequent processing in the laboratory. Subsequently, it was filtered in the laboratory through 0.45 mm pore Millipore membranes for the determination of chlorophyll-a according to the method of acetone (90%) extraction [19]. The remaining 500 mL were used for analyses of dissolved nutrients (nitrite NO\({}_{2}\)\({}^{-}\); nitrate NO\({}_{3}\)\({}^{-}\); ammonium, NH\({}_{4}\)\({}^{+}\); soluble reactive phosphate, SRP; and soluble reactive silicate, SRSi) [20,21], using a spectrophotometer (Cary 60 Agilent Technologies-Agilent, Santa Clara CA, USA) equipped with UV/VIS. Caffeine was quantified following the method described by [22]; quality control for this method includes a limit of detection (LD) of 12.1 ng L\({}^{-1}\). Caffeine was extracted by solid phase extraction (SPE) and determined by gas chromatography coupled to mass spectrometry (GC-MS). For SPE, 500 mg/6 mL Strata-X cartridges (Phenomenex, 8B-S100-HCH) were used and conditioned by eluting sequentially 10 mL of ethyl acetate, 10 mL of methanol, and 10 mL of Type I water. For extraction, 1 L of sample was passed through the cartridge with a flow of 10-15 mL min\({}^{-1}\). After sampl  
Figure 1: Location of the sampling stations along the five main zones of the Nichupte Lagoon System and the main current patterns in the lagoon (represented by the dashed arrows) adapted from the numerical model by [11]. Land use is a modification of the metadata obtained from the National Biodiversity Information System, SNIB for its initials in Spanish [18]. washed with 10 mL of Type I water and dried under vacuum for approximately 120 min. Once extracted, the analyte was recovered, eluting 15 mL of ethyl acetate, and the extract was evaporated using a gentle nitrogen flow. Samples were then placed in 2 mL vials and caffeine was quantified by GC-MS operated in electron impact (EI) ionization mode and equipped with an automatic liquid sampler (Agilent Technologies 6850 Series II GC, 5975B VL MSD and 7683B ALS, respectively). Injections were carried out in splitless mode (0.5 min) at 280 \({}^{\circ}\)C. Chromatographic separation was performed using a Phenomenex Zebron ZB-5 MSi capillary column (30 mm A-0.25 mm with 0.25 \(\upmu\)m of film thickness). The carrier gas was He (ultra-pure grade) with a flow rate of 0.8 mL/min; the initial oven temperature was 60 \({}^{\circ}\)C for 1 min, then increased from 10 \({}^{\circ}\)C/min to 300 \({}^{\circ}\)C (hold time 15 min). The transfer line temperature was 280 A\(\infty\)C. Mass spectra (\(m/z\) 50-550) were recorded at a rate of five scans per second at 70 eV. Mass spectrometric analysis for quantitative determination was performed by the selected ion monitoring (SIM Mode) of two characteristic fragment ions (\(m/z\)), 194 and 109. Analytical quality control included procedural blanks and calibration curves using the analytical standard Caffeine 99% (Fluka, Switzerland). All solvents were of chromatographic grade. The limit of detection (LOD) and limit of quantification (LOQ) were calculated based on the standard deviation of the response (Sy) of the curve and the slope of the calibration curve (S), LOD = 3.3(Sy/S) and LOQ = 10(Sy/S), which were, respectively, 12.1 ng L\({}^{-1}\) and 36.7 ng L\({}^{-1}\).