Ix sampling sites were selected along the River Molse Nete, located

Ix sampling sites were selected along the River Molse Nete, located in Flanders (Belgium) and belonging to the basin of the River Scheldt. In addition a reference site was sampled (site 7) at the River Wimp, belonging to the same basin (Fig 1). Sampling sites 1? are situated along an existing cadmium and zincMetallothioneins in Three Freshwater Fish SpeciesTable 1. Average water quality characteristics at the different sampling sites.NO3-+ NO2–N 2.2 2.0 1.7 1.6 1.5 2.6 1.6 NA 10.0 PO43–P mg/l 0.05 0.05 0.08 0.09 0.11 0.16 0.13 NA 0.30Site 1 2 3 4 5 6 7 CQC FQCO2 mg/l 8.2 9.3 8.5 8.6 9.3 8.7 7.6 5.5-9.5 5.pH 7.2 7.2 7.1 7.2 7.3 7.3 6.8 6.5?.0 6.5?.SO42- mg/l 82 85 77 95 61 57 60 500Cl- mg/l 51 42 39 38 37 36 53 250NH4+-N mg/l 0.26 0.23 0.41 0.35 0.68 0.88 2.2 * 1.Cond. mS/ cm Cd mg/l 461 457 409 409 376 380 391 3.9 62 38 58 17 8.4 , 0.10 0.02 1.Cu mg/l 4.1 8.3 6.1 8.1 5.7 5.4 3.5 24Zn mg/l 712 5539 3853 4864 1762 922 62 30CQC: Canadian Quality Criteria for aquatic freshwater life [65] FQC: Flemish Quality Criteria [66] NA: Not Available; * pH-dependent; ND: Not Determined doi:10.1371/journal.pone.0060805.tconcentration gradient with the highest metal concentrations measured at site 2 [28,31,32]. Water characteristics such as oxygen level, pH, water Autophagy hardness and water temperature (4.6 ?6 uC) were within the same range for all sites (table 1). Water samples were collected in duplicate monthly between November and March 2001?002 at all sampling stations. To measure the total metal concentrations water samples were acidified with nitric acid (HNO3; 69 ) to a pH of 2.0 and filtered through a 0.45-mm Millipore (Bedford, MA, USA) membrane filter. All samples were Epigenetics stored in 20-ml polypropylene vials at 4 uC until analysis. Sediments were collected twice, i.e. in December and in February. Samples were taken with a ‘Petit Ponar’ grab sampler (Wildco cat.no. 1728; 235 cm2). At each site a mixed sample was taken, composed of 5 grab samples [33]. Samples were sieved with the site-water using a 500 mm-mesh sieve and stored in 500 ml polyethylene beakers at 4uC. Subsequently, supernatant was decanted carefully to prevent loss of sediment. After decantation the sediment sample was homogenised with a plastic spatula. Prior to extraction the sediment of each sampling station was centrifuged (10,000 g) to collect the pore water [34]. The remaining wet sediment was analysed on total metal content: sediments were dried at 60 uC during 48 hours and a mixture of concentrated HNO3 and HCl (4:1) was added. Eventually, samples were put in Teflon bombs and digested in a microwave oven [28,35]. At all sampling sites fish were caught within one month between December and January by electrofishing, using an Electra catch WFC7 generator producing 150 V. Three successive samplings were conducted at each site (length: 6 100 m). All fish were identified to the species level and counted. Of all specimens, forklength was measured (6 1 mm) and weight determined using a Kern 442.43 balance (6 0.1 g). From each sampling site up to 8 specimens (if present) of three fish species, i.e. gudgeon (Gobio gobio), perch (Perca fluviatilis), and roach (Rutilus rutilus), were sacrificed using an overdose of the anesthetic ethyl meta-aminobenzoate methanesulfonic acid (MS 222) and liver tissues were collected and weighed (0.001 g) in the field and immediately stored in liquid nitrogen and transported to the lab. In the lab, the liver was homogenized and separated in two parts, one pa.Ix sampling sites were selected along the River Molse Nete, located in Flanders (Belgium) and belonging to the basin of the River Scheldt. In addition a reference site was sampled (site 7) at the River Wimp, belonging to the same basin (Fig 1). Sampling sites 1? are situated along an existing cadmium and zincMetallothioneins in Three Freshwater Fish SpeciesTable 1. Average water quality characteristics at the different sampling sites.NO3-+ NO2–N 2.2 2.0 1.7 1.6 1.5 2.6 1.6 NA 10.0 PO43–P mg/l 0.05 0.05 0.08 0.09 0.11 0.16 0.13 NA 0.30Site 1 2 3 4 5 6 7 CQC FQCO2 mg/l 8.2 9.3 8.5 8.6 9.3 8.7 7.6 5.5-9.5 5.pH 7.2 7.2 7.1 7.2 7.3 7.3 6.8 6.5?.0 6.5?.SO42- mg/l 82 85 77 95 61 57 60 500Cl- mg/l 51 42 39 38 37 36 53 250NH4+-N mg/l 0.26 0.23 0.41 0.35 0.68 0.88 2.2 * 1.Cond. mS/ cm Cd mg/l 461 457 409 409 376 380 391 3.9 62 38 58 17 8.4 , 0.10 0.02 1.Cu mg/l 4.1 8.3 6.1 8.1 5.7 5.4 3.5 24Zn mg/l 712 5539 3853 4864 1762 922 62 30CQC: Canadian Quality Criteria for aquatic freshwater life [65] FQC: Flemish Quality Criteria [66] NA: Not Available; * pH-dependent; ND: Not Determined doi:10.1371/journal.pone.0060805.tconcentration gradient with the highest metal concentrations measured at site 2 [28,31,32]. Water characteristics such as oxygen level, pH, water hardness and water temperature (4.6 ?6 uC) were within the same range for all sites (table 1). Water samples were collected in duplicate monthly between November and March 2001?002 at all sampling stations. To measure the total metal concentrations water samples were acidified with nitric acid (HNO3; 69 ) to a pH of 2.0 and filtered through a 0.45-mm Millipore (Bedford, MA, USA) membrane filter. All samples were stored in 20-ml polypropylene vials at 4 uC until analysis. Sediments were collected twice, i.e. in December and in February. Samples were taken with a ‘Petit Ponar’ grab sampler (Wildco cat.no. 1728; 235 cm2). At each site a mixed sample was taken, composed of 5 grab samples [33]. Samples were sieved with the site-water using a 500 mm-mesh sieve and stored in 500 ml polyethylene beakers at 4uC. Subsequently, supernatant was decanted carefully to prevent loss of sediment. After decantation the sediment sample was homogenised with a plastic spatula. Prior to extraction the sediment of each sampling station was centrifuged (10,000 g) to collect the pore water [34]. The remaining wet sediment was analysed on total metal content: sediments were dried at 60 uC during 48 hours and a mixture of concentrated HNO3 and HCl (4:1) was added. Eventually, samples were put in Teflon bombs and digested in a microwave oven [28,35]. At all sampling sites fish were caught within one month between December and January by electrofishing, using an Electra catch WFC7 generator producing 150 V. Three successive samplings were conducted at each site (length: 6 100 m). All fish were identified to the species level and counted. Of all specimens, forklength was measured (6 1 mm) and weight determined using a Kern 442.43 balance (6 0.1 g). From each sampling site up to 8 specimens (if present) of three fish species, i.e. gudgeon (Gobio gobio), perch (Perca fluviatilis), and roach (Rutilus rutilus), were sacrificed using an overdose of the anesthetic ethyl meta-aminobenzoate methanesulfonic acid (MS 222) and liver tissues were collected and weighed (0.001 g) in the field and immediately stored in liquid nitrogen and transported to the lab. In the lab, the liver was homogenized and separated in two parts, one pa.