Lead pellets in soil
Samples were taken with a soil core sampler 16 cm in diameter and 15 cm in depth. Soil samples were air-dried at room temperature, and later analyzed by radioscopy to detect the presence and location of images compatible with lead pellets, which were later confirmed by manual recovering. Radioscopy was performed on a Siemens Vertix U (500 mA–500 kV) with an exposure dose of 90 kV, 64 mA/s × 320 ms, and a distance focus-plaque of 1 m. Images were processed with a Fuji Film FCZ equipment with a digital capsule, using K-Pacs software from Image Information Systems Ltd. Fig. B1 (Appendix B, Supplementary Material), shows some examples of radioscopic images of lead pellets within soil cores.
2.2.2. Lead determination in soil
From the same sampling points in the transects used for soil cores, a second sample, 2.2 cm in diameter × 5 cm deep was taken, according to EPA Standard Procedure SOP 2012 (02/18/2000) for lead determination (i.e. 20 soil samples per site). Each single sample was divided in two aliquots, one of which was submitted for analysis while the other was conserved as backup. Samples from natural wetlands were oven-dried at 40 °C during 48 h until constant weight; all soil compositions reported are referred to dry weight. The dry samples were passed through a 2 mm stainless steel sieve and then digested with a MARS-5 Microwave Digestion System, CEM Corporation, USA, using nitric acid Pro Analysis Merck®, according to Method US EPA SW 846-3051 (power: 400 W; pressure (max.): 800 psi; temperature (max): 200 °C; time: 30 min). Lead analyses were conducted by inductively coupled plasma-atomic emission spectrophotometry (ICP-AES) (Shimadzu 9000, Shimadzu Corporation, Kyoto, Japan), following 200.7 EPA standards (U.S. Environmental Protection Agency). Standard Reference Materials were used in 2011 and 2012 to validate results from soil (RTC-Trace Elements on Fresh Water Sediment, CNS 392-050, certified value of lead: 121 mg/kg, DL: 0.5 mg/kg). Samples from rice fields were processed by graphite furnace at STPGFAA conditions according to EPA 600/R-94/111, 200.9 standards, with a Perkin Elmer AAnalyst 800 Atomic Absorption Spectrometer (Detection Limit (DL): 0.4 mg/kg).
2.2.3. Lead determination in water
Water samples were collected only from natural wetlands, following standard method 3010B (Clesceri et al., 1999). At each sampling point where surface water was found, 200 cm3 of water were collected in plastic vials previously washed with distilled water and 0.5 M HCl. Single samples were kept at pH 2 with high purity HCl (SUPRAPUR; Merck Labs). In a preliminary study, ten randomly selected unfiltered water samples were microwave-digested according to US EPA SW-3052 standard. Then, lead was quantified in aliquots of the digested and non-digested samples, filtered through 2.0 μm membranes and the results were contrasted. No significant differences were found; thus, sample digestion was not further conducted. Lead analyses were carried out on samples filtered as above with the same methods used for soil samples of natural wetlands as described in 2.2.2, at DL: 2 μg/L. Standard Reference Materials (RTC-Trace Metals by AA-2, Constant Value QCI-049, certified value of lead: 51.2 μg/L ± 1.49 μg/L/obtained value 49.9 ± 2.2 μg/L) were used.
2.2.4. Lead determination in vegetation
At each soil sampling point in natural wetlands the aboveground vegetation biomass present in a 0.5 × 0.5 m area was collected. Samples were repeatedly rinsed with collection-site water, and finally with distilled water. Then, plants were identified at family, genus or species level. When the harvested dry biomass was not enough to perform Pb determinations, plants of the same species, genus or family from two or more sampling points from each site were pooled. Plant samples were oven-dried at 60 °C during one week. Then, they were digested and analyzed as in 2.2.2, together with a Certified Reference Material. In all cases, the determinations were done with external calibration using certified standards Chem-Lab, Zedelgem B-8210, Belgium (Aquatic plant (Lagarosiphon major), Certified Reference Material BCR-060, certified lead value 64.0 mg/kg. Institute for Reference Materials and Measurements, Joint Research Centre, European Commission (Feb 08, 2011), Retieseweg 111, 2440 Geel, Belgium) (DL: 0.25 mg/kg).
Rice plants (n = 18) were only collected in the rice fields subjected to sports hunting (RF-1 and RF-2). Samples were oven-dried at 60 °C for a week. Then, they were microwave-digested in Milestone START D. Pb determinations were done by electrothermal atomization at STPF conditions. External calibration curve with certified aqueous standards was employed. A Perkin Elmer AAnalyst 800 Atomic Absorption Spectrometer was used (DL: 0.2 mg/kg).