Polychlorinated biphenyls (PCBs) were first synthesized in the late 19th century. Production spread rapidly with PCBs being used extensively in mechanical, chemical and electrical engineering among other industries. Because of their stability, resistance to degradation, and bioaccumulation, they persist over long periods of time, accumulating in plants and animals and entering the food chain [1–5]. In tissues of organisms, PCBs bioconcentrate at levels that exceed their concentrations in water, where levels may be below detectable levels. Organisms are therefore effective indicators of contamination levels because they reflect changes in the environment, and thus also in bioavailability of the substances and over long periods of time [2, 6–11].

Studies quantifying persistent organic pollutants (POPs) and metals levels in aquatic ecosystems typically focus on predators such as fish [12–21]. Fish are suitable for analyses because they are often the top predator in a food web, they accumulate a variety of contaminants, and their size is conducive to relatively rapid collection for analyses, and through human consumption they are linked with health issues [21,22]. However, the use of large fish as chemical bioindicators is of little use in shallow rivers where trophic levels are limited. In these situations, selecting a suitable indicator species is problematic.

Certain features of leeches make them potentially useful as bioindicators of water pollution [23]. Leeches are among the most numerous animals in both standing and running freshwaters. There are several advantages of using the leech as an indicator organism. Leeches of the genus Erpobdella are abundant and easily accessible in small streams. Acquiring fish of similar species and age from different locations can be problematical. Leeches are residential organisms and better reflect conditions in the place from which they have been sampled than do fish, which with exceptions, migrate and may not have grown in the area where they are captured [24]. Compared to direct testing of sediments, leeches provide more objective data since they are taken from several tens of square meters at a given site. The most frequently occurring species at the site monitored was Erpobdella octoculata (Figure 2). Erpobdella spp. have a life span of 1–2 years, they live in flowing as well as stagnant water, and may be present in bodies of water ranging from oligosaprobic to polysaprobic in character. As predators, they feed on various species of aquatic invertebrates, mainly worms and midge larvae [25].

The Skalice River was heavily contaminated in 1986 with waste waters containing an estimated 1 metric ton of polychlorinated biphenyls that leaked from a road gravel processing factory in Rožmitál pod Třemšínem [26]. The aim of the study was to evaluate the use of the leeches of the genus Erpobdella as a means of assessing polychlorinated biphenyl contamination of watercourses. Monitoring for PCBs in the Skalice River began in 1992 and continued until 2003. Samples were collected from a single site upstream and at 10 locations downstream of the source of pollution covering a distance of about 50 km.

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.

Monitoring was carried out at 11 locations on the River Skalice between years 1992 and 2003 (Table 1, Figures 3,4). 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 600–700 leeches from one site together). This composite sample was analyzed.

A 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 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 14–30 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.

GC conditions were as follows: splitless injection mode (splitless time: 2 min), injection volume: 1 μL, injector temperature: 280°C; DB-5 (5% phenyl-methylpolysiloxane) and DB-17 (50% phenyl-methylpolysiloxane) capillary columns (60 m × 0.25 mm × 0.25 μm); oven temperature program: 60°C (2 min), rate 30°C/min to 220°C, 0.5°C/min to 240°C and 2.5°C/min to 280°C (10 min). Temperature of both ECD detectors was 300°C. As a carrier gas, helium at constant flow 1.7 mL/min was used. Limits of quantification (LOQ - μg kg⁻¹ lipids) are: PCB 28 – 0.5, PCB 52 – 0.6, PCB 101 – 1.0, PCB 118 – 0.6, PCB 138 – 1.0, PCB 153 – 0.6, PCB 180 – 7.

Values of PCB congeners were not normally distributed so nonparametric tests were used. The Wilcoxon matched pairs test was used to compare PCBs between control and downstream locations. Spearman rank correlation was calculated (Daniels test) to assess decrease in PCB levels over time as well as the decrease in PCB levels with distance from the source of pollution. Data analyses were performed using Statistica software (StatSoft Inc., 2007) [28].

Benthic macroinvertebrates are moderately long-lived and are in constant contact with river sediments [8]. Contamination and toxicity of sediments will therefore affect those benthic organisms which are sensitive to them. Benthic macroinvertebrates are present, often abundantly, year-round, and since limited in mobility, reflect environmental conditions at the sampling point as well as changes over time and cumulative effects. Biological indicators can show problems otherwise missed or underestimated.

Some authors have investigated the use of suitable representatives of invertebrates as a sensors for the assessment of aquatic environment contamination with persistent organic substances [2,8]. A number have monitored the presence of leeches and concentrations of hazardous substances in their tissues to determine levels of water contamination, including organic contaminants and polychlorinated biphenyls [6,10–11,22–23,29–31]. Scrimgeour et al. [22] monitored contamination with selected persistent organic substances in the Beaver Hills Watershed (Alberta, Canada). PCB concentrations were determined in sediment samples and in leech tissue samples. In their case, environmental contamination levels were low and, in a majority of cases, below the detection limit.

Metcalfe et al. [10] assessed persistent organic compound concentrations in leech tissues and in water. Leeches were found to accumulate 16 of such compounds (two benzothiazoles, eight chlorophenols, lindane, and DDT and its derivatives), while only ten of the compounds were detected in water (benzothiazoles and chlorophenols). Accumulations of chlorinated phenolic substances in leech tissues exceeding levels in their aquatic environment have also been reported by Prahacs et al. [30]. Prahacs and Hall [11] found a high linear relationship between concentrations of chlorinated phenolic substances in water and bioconcentrations of those substances in leech tissue. This relationship is due to a slow elimination of chlorinated phenols, and it makes it possible to determine aquatic environment contamination levels from concentrations in leeches. Slow elimination of chlorophenols from, and a high degree of bioconcentration in, leech tissue has also been reported by Metcalf et al. [10] and Hall and Jacob [7]. The slow elimination increases biomonitoring sensitivity because larger amounts of hazardous substances are accumulated in leech tissue. Metcalfe et al. [29] found a high content of residues of chlorophenols in freshwater leeches when compared to fish, tadpoles, and other benthic invertebrates from an industrially polluted creek. Leeches may therefore serve as a suitable sensor-bioindicator of aquatic environment contamination with persistent organic substances.