The following hypotheses were tested:For each contaminated site and PCB congener: the content of PCB in fresh leech tissue is higher at the monitored site than at the control site.For each site and PCB congener: the content of PCB in fresh leech tissue decreased at the site during the time period.For each year and PCB congener: the content of PCB in fresh leech tissue decreased with distance from the source of pollution.2.?Material and methodsMonitoring was carried out at 11 locations on the River Skalice between years 1992 and 2003 (Table 1, Figures 3,,4).4). Sample site 1 was a control site located two km above the pollution source. Leeches of the genus Erpobdella (prevailing species E. octoculata) were collected every year except 2002. These collections were performed once a year in June.

Due to the low abundance of leeches it was not possible to collect samples every year from each site. Leeches were dried slightly on filter paper, packed into microtone bags, immediately chilled, and frozen to ?20?C. One sample from each site weighed 50 g (corresponding to a composite sample of 600�C700 leeches from one site together). This composite sample was analyzed.Figure 3.Collection of leeches in the Skalice River.Table 1.Sample sites on the River SkaliceA homogenous leech sample weighing 30 g was mixed with 100 g of anhydrous sodium sulphate to form a flowing powder and then extracted for 8 hours in a Soxhlet apparatus with 340 mL of a hexane-dichloromethane (1:1, v/v) solvent mixture.

The crude extract was carefully evaporated and dissolved in 10 mL of a cyclohexane-ethyl acetate mixture (1:1, v/v) containing PCB 112 (this congener is not present either in commercial mixtures or environmental samples), employed as an internal standard.A clean-up of crude extracts was carried out by an Carfilzomib automatic gel permeation chromatographic system (GPC) employing S-X3 Bio Beads. As a mobile phase, cyclohexane-ethyl acetate (1:1, v/v) was used at a flow rate 0.6 mL/min and the fraction corresponding to the elution volume of 14�C30 mL was collected. The eluate was evaporated by a rotary vacuum evaporator at 40��C and the residual solvent was carefully removed by a gentle stream of nitrogen. The residue was then dissolved in 1 mL of isooctane and treated with concentrated sulphuric acid to eliminate any co-extracted residues. An aliquot of the upper isooctane layer was transferred into a glass vial for following GC analysis.An HP 5890 Ser. II, gas chromatograph (Agilent Technologies, USA) equipped with an electronic pressure control (EPC), a split/injector, two parallel 63Ni electron capture detectors (ECDs) and two parallel columns possessing a different selectivity (DB-5 and DB-17, both J&W Scientific, USA) were employed for all analyses of PCBs and OCPs.